The control of glucose concentration is a crucial factor in clinical diagnosis and the food industry. Electrochemical biosensors based on reduced graphene oxide (rGO) and conducting polymers have a high potential for practical application. A novel thermal reduction protocol of graphene oxide (GO) in the presence of malonic acid was applied for the synthesis of rGO. The rGO was characterized by scanning electron microscopy, X-ray diffraction analysis, Fourier-transform infrared spectroscopy, and Raman spectroscopy. rGO in combination with polyaniline (PANI), Nafion, and glucose oxidase (GOx) was used to develop an amperometric glucose biosensor. A graphite rod (GR) electrode premodified with a dispersion of PANI nanostructures and rGO, Nafion, and GOx was proposed as the working electrode of the biosensor. The optimal ratio of PANI and rGO in the dispersion used as a matrix for GOx immobilization was equal to 1:10. The developed glucose biosensor was characterized by a wide linear range (from 0.5 to 50 mM), low limit of detection (0.089 mM), good selectivity, reproducibility, and stability. Therefore, the developed biosensor is suitable for glucose determination in human serum. The PANI nanostructure and rGO dispersion is a promising material for the construction of electrochemical glucose biosensors. It is expected that the global market of medical devices will reach $342.9 billion by 2021 .2-hybridized carbon atoms with out-of-plane π bonds [2 gases [Graphene is a carbon material with high electrical conductivity, resulting from sp π bonds . Despite π bonds ,8. Nowad π bonds . However π bonds . Further[2 gases . The eff[2 gases . There i[2 gases . However[2 gases , the emp[2 gases ,15.Polyaniline (PANI) is a conducting polymer with unique physical properties ,17,18. PA new method of thermal reduction of GO in the presence of malonic acid was developed for the production of rGO. The applicability of the rGO and PANI nanostructure dispersion as a matrix for GOx immobilization in the design of a glucose biosensor was evaluated. To achieve the best performance of the biosensor, the PANI and rGO ratio in the dispersion used for the premodification of a graphite rod (GR) working electrode was optimized by evaluating the analytical parameters of the glucose biosensor.Polyaniline nanostructures were synthesized using interfacial polymerization according to the protocol reported by Abdolahi et al. . Aniline2S2O8  (5.0 g) and P2O5  (5.0 g) in 98 wt% sulfuric acid  (24.0 mL) medium. Then, the dry, pre-oxidized graphite was subjected to oxidation, following the methodology reported by Hummers et al. [GO was prepared according to the protocol described in the work of Yan et al. . In a tys et al. . GO prod−1) at 600 °C for 30 min. Finally, the thermally reduced mixture of GO and malonic acid was washed with deionized water and dried in air. The synthesized black product was denoted as rGO.GO and malonic acid  in a weight ratio of 1:3 were ground in an agate mortar for 10 min. Afterward, the prepared solid mixture was annealed in a horizontal tube furnace  under argon  flow   at magnifications of 20,000 and 50,000.The elemental composition and purity were confirmed by energy-dispersive X-ray (EDX) analysis by scanning electron microscopy  equipped with an EDX detector.θ range from 5° to 60° using CuKαλ = 1.5406 Å radiation. Measurements were carried out at 30 kV and 15 mA with a step size of 0.010° and a dwell time of 1.0 s. The interlayer distance d for graphite, GO, and rGO was evaluated using Bragg’s equation,n is a positive integer, λ is the wavelength of the X-ray beam, d is the distance between the adjacent interlayers, and θ is the diffraction angle.X-ray diffraction (XRD) analysis was performed using a MiniFlex II diffractometer . The diffractograms were recorded in the 2−1. Infrared spectra were recorded at a 4 cm−1 resolution and using the KBr pellet technique in transmission mode.Fourier-transform infrared (FTIR) spectroscopy measurements were obtained by an FTIR spectrometer  in the range of 700–4000 cmRaman spectra were recorded using an inVia Raman spectrometer  equipped with a thermoelectrically cooled (−70 °C) CCD camera. The He-Ne gas laser was provided an excitation beam at 532 nm with power restricted to 1 mW. The integration time was 100 s.Material dispersions used for further GR electrode modification were prepared in i-propanol prior to use. One milligram of PANI nanostructures was dissolved in 100 μL of i-propanol solution containing 0.5% Nafion . Prior to use, the dispersion was stirred vigorously in an ultrasonic bath for 15 min. The rGO solution was prepared in the same manner as the PANI dispersion. Then, the PANI and rGO dispersions were mixed and added to the ultrasonic bath for an additional 15 min to prepare PANI and rGO mixtures of the required  ratios.2 was cut into 4 cm length pieces. The working surface of each electrode was alternately hand-polished with different types  of emery paper, washed with distilled water, and dried at room temperature. The lateral surface of the GR electrodes was isolated with a silicone tube to prevent contact with the solution.GR  with a working surface area of 0.71 cmAspergillus niger (EC.1.1.3.4.) of 360 U mg−1 enzymatic activity was used for biosensor design. Four different types of GR electrodes were prepared: modified just with GOx (GR/GOx), modified with PANI nanostructures and GOx (GR/PANI/GOx), modified with rGO and GOx (GR/rGO/GOx), and modified with a PANI and rGO mixture and GOx (GR/PANI:rGO/GOx). Shortly, 6 μL of the required dispersion was deposited on the electrode surface. After evaporation of the solvent, 3 μL of 40 mg mL−1 GOx solution was added on the surface and the evaporation procedure was repeated. The modified electrode was hung over a 25% glutaraldehyde  solution for 15 min and rinsed with distilled water.GOx  from 3COONa , 5 mM of Na2HPO4 × 12 H2O , 5 mM of NaH2PO4 × H2O , and 0.1 M of KCl . The glucose solution was kept for 24 h before use in order to reach an equilibrium between glucose isomers.Forty milligrams per milliliter of GOx and 1 M of D-(+)-glucose stock solutions were prepared in an acetate-phosphate-buffered saline (A-PBS) solution (pH 6.0) containing 5 mM of CH3 M KCl electrode  as a reference electrode; and a platinum wire as an auxiliary electrode. All experiments were performed at a constant +0.3 V potential in A-PBS solution (pH 6.0) with 6 mM of N-methylphenazonium methyl sulfate (PMS) . The solution was mixed with a magnetic stirrer at 300 rpm during anodic current response measurements. After a steady current was reached (stable baseline), a corresponding amount of glucose was added into the electrochemical cell. The anodic current change (ΔI) after addition of glucose was monitored as an analytical signal of the developed biosensor. During GOx-catalyzed oxidation of glucose, the transfer of electrons from the enzyme redox center to the electrode occurs via the PMS present in the solution  with NOVA software using a three-electrode system consisting of a modified GR electrode, which was employed as a working electrode; an Ag/AgCl solution . Additioy = ax/(b + x). The maximal current (ΔmaxI) generated under saturated substrate conditions was the a parameter, while the apparent Michaelis constant (Mapp.K) was the b parameter of the hyperbolic function.Michaelis–Menten kinetics was used for the evaluation of the biocatalytic performance of immobilized GOx. The obtained results were approximated using the hyperbolic function maxI and Mapp.K calculation. Calibration curve parameters, such as slope and correlation coefficient, were evaluated. The limit of detection (LOD) as the lowest concentration of the analyte that gives an analytical signal greater than the background value plus 3δ was also estimated using the statistic software OriginPro.OriginPro software (version 2017) was used for the ΔDuring interfacial polymerization, PANI nanostructures are formed at the interface of two immiscible solutions. After the induction period, which normally takes up to 30 s, PANI nanostructures migrate to the aqueous phase, which is above the interface -1,2. TheThe morphology of the synthesized materials was evaluated using the SEM imaging technique . Samples1:10 nanocomposite  is almost eight times higher than for GO (1.95), indicating effective thermal reduction of GO. The elements C, S, N, and Cl were identified in PANI. The presence of Cl and S displays the successful attachment of the doping species onto the polymer. EXD results of PANI:rGO1:10 show 93.27, 5.56, 1.04, and 0.13 at% of carbon, oxygen, nitrogen, and chlorine, respectively. These data confirm the successful formation of a PANI:rGO1:10 composite.The obtained results of elemental analysis are listed in θ = 26.60° and 2θ = 54.70°, which correspond to the planes (002) and (004) of a hexagonal lattice, respectively. According to Bragg’s equation (Equation (1)), the interlayer spacing of the (002) plane reflection equals 0.335 nm, which is a representative d-spacing of stacked graphene sheets in graphite. The diffraction pattern of GO exhibits two peaks at 2θ = 11.28° and 2θ = 42.38°, corresponding to the reflections of (001) and (100), respectively. The XRD pattern of GO is similar to the XRD data that have been reported by Bandara [001 of GO was expanded to 0.784 nm due to the presence of oxygen-containing functional groups between the graphene layers. It reveals the successful oxidation of graphite. The XRD results of the rGO sample show that during the thermal reduction of GO with an additive, the (001) peak disappears, while the (002) peak shifts to the position typical for graphite (2θ = 26.33°), with an interlayer distance of 0.338 nm. This can be attributed to the removal of water and oxygen-containing groups and a partial restoration of the sp2 carbon network. Furthermore, after the thermal reduction of GO, the peaks in the XRD patterns become broader. This results from a decrease in the crystallite size. Moreover, the diffraction peaks centered at approximately 2θ = 26.33° are asymmetric because of the mixed polycrystalline structure of graphitic and disordered domains. The presence of the (002) and (100) planes confirms that the structure of rGO is between the structures of graphite and GO.The structural changes in GO after annealing were investigated by XRD analysis. The recorded diffraction patterns of graphite, GO, and rGO are presented in −1 are attributed to the symmetric and asymmetric stretching modes of C–H, which are characteristic of –CH2– and –CH3 groups [−1 indicates the vibrations of hydroxyl groups or intercalated water molecules. The peak at 1633 cm−1 corresponds to the skeletal vibrations of C=C bonds [−1 may be related to the stretching modes of the tertiary alcohol (C–OH) and phenol (C–O) groups, respectively [−1 are changed due to the attachment of hydroxyl groups to the graphene sheets. The new band at 1730 cm−1 is associated with the stretching mode of C=O corresponding to the carboxyl group. Moreover, the existence of epoxy groups (C–O–C) was confirmed from the absorption bands in the range of 1300–1200 cm−1 [−1 indicates the C=C vibrations attributed to the aromatic carbon rings [2 structure should enhance the electrical conductivity of rGO [−1 show methylene and ethylene groups on the basal plane and edges of graphene, describing the defective parts of the structure, such as sp3 defects, C vacancies, and dislocations. The improved electrical conductivity and more chemically reactive defective sites in the graphene layer make rGO a favored graphene material for the preparation of electrodes for biosensors [Oxygen-containing functional groups of the synthesized products were identified by FTIR spectroscopy. FTIR spectra of graphite, GO, and rGO are compared in 3 groups . The bro=C bonds . The absectively . The FTIectively . The pro200 cm−1 . After ton rings . This cay of rGO . The higosensors .−1 arises due to the vibrations of sp2-hybridized carbon atoms. The mode appearing at 1580 cm−1 is the D band, which is related to the structural defects, such as grain boundaries, C vacancies, sp3 defects, and armchair and zigzag edges of the carbon material [−1) because of oxygen-containing functional groups and sp3 carbon atoms in the structure. The G mode in the spectrum of rGO is shifted to a lower wavenumber (1591 cm−1) due to the reduction of graphene oxide, which results in the partial restoration of the π-conjugated carbon network. The intensity ratio of D and G bands (ID/IG) could be used to investigate the degree of disorder in the graphene structure [ID/IG is 0.26 for graphite. It is noticed that the intensity ratio increases to 0.89 and 1.14 for GO and rGO, respectively. The high ID/IG ratio in GO confirms that its lattice is distorted and has a large number of sp3-like defects caused by the oxidation process. Thus, the process of oxidation and reduction leads to an increase in structural defects in the products due to the formation of sp2 carbon domains in samples. The broader D and G bands of GO and rGO reveal the lower crystallinity of the samples, which confirms XRD data [Raman spectroscopy is a fast and nondestructive characterization method that gives useful information about the lattice structure, structural disorders, crystallization, and defects of carbon materials . Recordematerial . Its inttructure ,41. The XRD data .The dispersions of PANI and rGO in i-propanol containing 0.5% of Nafion were used for the fabrication of GR/PANI/GOx and GR/rGO/GOx electrodes. GOx was adsorbed on the premodified working GR electrode and cross-linked by glutaraldehyde vapor. The effect of rGO and PANI dispersions on the amperometric response of biosensors based on GR/PANI/GOx or GR/rGO/GOx electrodes in the presence of glucose was evaluated and compared with the response of biosensors based on GR/GOx electrodes. The calibration plots are presented in −1 for GR/GOx to 0.92 μA mM−1 for GR/PANI/GOx and 0.69 μA mM−1 for GR/rGO/GOx. However, the linear range of detection for biosensors based both on PANI- and rGO-premodified electrodes became wider. Biosensors based on GR/GOx electrodes had a linear range of detection from 0.5 to 10 mM (R = 0.9980), and the linear ranges of detection in the case of GR/PANI/GOx and GR/rGO/GOx electrodes were 1–25 mM (R = 0.9908) and 3–25 mM (R = 0.9839), respectively. Premodification of the electrodes with PANI nanostructures and rGO also led to an increase in the MappK. value from 12.0 ± 1.9 mM (GR/GOx) to 77.7 ± 9.5 mM and 30.5 ± 4.9 mM, respectively, which also testifies to a wider linear range of biosensors [maxI value of the biosensor based on a GR/rGO/GOx (78.7 ± 5.2 μA) electrode in comparison to a GR/PANI/GOx electrode (44.8 ± 2.7 μA) could be related to better electron transfer through the matrix. An increase in the standard deviation (SD) in the case of the biosensor based on the GR/rGO/GOx electrode indicated decreased reproducibility, wherein SD values calculated for biosensors based on GR/PANI/GOx and GR/GOx were comparable. Possible explanations may be found in the better suitability of PANI nanostructures for the role of the matrix for enzyme immobilization, taking into account the hydrophobic rGO nature.It is obvious that premodification of the electrodes with a PANI or rGO dispersion affected the electrochemical performance of the fabricated biosensors. Due to the employment of a PANI or rGO dispersion in the construction of the biosensor, the sensitivity decreased from 2.0 μA mMosensors . A higheTo improve the glucose biosensor’s performance, another premodification of the GR electrode strategy was implemented by using both PANI and rGO dispersions together. The effect of the PANI and rGO dispersion ratio on the analytical signal of the developed biosensors was studied to select the optimal composition of PANI and rGO. The calibration plots are presented in 5:1/GOx electrode was equal to 1.0 μA mM−1, while the linear range of detection was from 3 to 100 mM. A gradual increase in the rGO amount in the dispersion allowed improving the analytical characteristics of the developed biosensors. It is obvious that the electrode premodified with dispersions of PANI and rGO mixed in a ratio equal to 1:10 showed the best performance. The linear range of detection of the GR/PANI:rGO1:10/GOx biosensor became wider in comparison with the above-described biosensors (R = 0.9958). The sensitivity of the biosensor based on the GR/PANI:rGO1:10/GOx electrode was equal to 2.8 μA mM−1, and the LOD was estimated to be 0.089 mM. The limit of quantification (LOQ) was calculated as 0.30 mM at a signal-to-noise ratio of 10 standard deviations. The developed biosensor possessed good reproducibility, since the relative standard deviation (RSD) calculated from three independent measurements for 3, 10, and 100 mM of glucose was, respectively, equal to 4.66%, 4.71%, and 4.48%. Moreover, the PANI:rGO dispersion matrix due to the increase in the diffusion limitation had a significant influence on the MappK. value. It was 12.0 ±1.9 mM for the biosensor based on the GR/GOx electrode, while for the biosensor based on the GR/PANI:rGO1:10/GOx electrode, MappK. was equal to 74.4 ± 9.0 mM.It was found that a change in the composition of the dispersion used for the premodifcation of the working electrode strongly affects the analytical performance of the biosensor. A biosensor based on an electrode premodified with PANI and rGO dispersions mixed in a ratio equal to 5:1 was characterized by quite a low current response. The sensitivity of the biosensor based on the GR/PANI:rGOosensors , and it It should be noted that a further increase in the PANI and rGO ratio up to 1:15 had a negative impact on biosensor performance. This clearly illustrates that a PANI-to-rGO ratio of 1:10 is optimal for the premodification of the working electrode. This observation suggests that a PANI nanostructure matrix environment encourages the maintenance of enzyme activity during measurements. The hydrophobic rGO nature should not be forgotten in this context. An excessive amount of rGO in the matrix could be the reason for GOx molecules’ conformational alterations affecting the stability and catalytic activity of the enzyme . On the 1:10/GOx electrodes in comparison to other biosensors intended for glucose concentration detection.The analytical parameters of the designed biosensor and other glucose biosensors based on GOx are summarized in 1:10/GOx electrode was evaluated in human serum diluted 10 times with A-PBS solution. Electrochemical measurements were performed after the addition of 5 mM of glucose to the serum. The obtained results demonstrate good accuracy. The glucose concentration calculated as a mean value of three independent experiments was equal to 4.99 ± 0.11 mM.The performance of an amperometric glucose biosensor based on the GR/PANI:rGO1:10/GOx electrode was kept above A-PBS solution (pH 6.0) in a closed vessel at 4 °C. A significant decrease in the measured current response by 32% was registered after 1 day. However, on the 8th day, the current response was still higher than 50% of the initial analytical signal.Stability is an important parameter of the glucose biosensor, which refers to the precision of the same glucose concentration measurements over a period of time under continuous monitoring. The current response generated after the addition of 25 mM of glucose to A-PBS solution (pH 6.0) with 6 mM of PMS was monitored over an 8-day period A. The fa1:10/GOx electrode was studied. Such concentrations were chosen since the typical AA and UA concentrations in the blood serum for a healthy adult are in the range of 0.040–0.141 and 0.149–0.446 mM, respectively [Some electroactive species coexisting in a real sample of human serum could influence the current response of the electrochemical biosensors . The effectively . Serum dectively , which s1:10/GOx electrode is selective to glucose in comparison to other tested carbohydrates.The selectivity of the developed biosensor was evaluated by adding aliquots of five carbohydrates. In this experiment, 5 mM of glucose, 5 mM of fructose, 5 mM of mannose, 5 mM of galactose, 5 mM of sucrose, and again 5 mM of glucose were added one after the other to the electrochemical cell, while waiting for the reaching a saturation current response after previous sugar addition. It is obvious  that theThe study demonstrated the methods that are suitable for the synthesis of rGO thermally reduced at a relative low temperature of 600 °C in the presence of malonic acid. A dispersion of synthesized rGO with PANI nanostructures can be used for the premodification of the working electrode and as a matrix for GOx immobilization, improving the electrochemical performance of the glucose biosensor. Moreover, a PANI and rGO ratio equal to 1:10 was selected and found to be optimal for working electrode premodification and allowed us to achieve the best analytical parameters of the biosensor. The developed biosensor is suitable for determining the glucose concentration in human serum. Additionally, due to the broad linear range of detection, the designed biosensor can be applied to samples with high glucose concentrations or in which the glucose concentration could vary considerably. Moreover, such an analytical system may be appropriate for the determination of the typical glucose concentration in undiluted or diluted blood samples. We believe that while PANI acts as a matrix for enzyme immobilization, the rGO presence in the matrix facilitates electron transfer from the GOx redox center to the electrode in the presence of a soluble redox mediator and increases the current response of the biosensor. Although the premodified working electrode was used for immobilization of GOx and further determination of glucose, it could be easily adapted to other biosensors based on a wide variety of enzymes.


A well-dispersed phase of exfoliated graphene oxide (GO) nanosheets was initially prepared in water. This was concentrated by centrifugation and was mixed with a liquid epoxy resin. The remaining water was removed by evaporation, leaving a GO dispersion in epoxy resin. A stoichiometric amount of an anhydride curing agent was added to this epoxy-resin mixture containing the GO nanosheets, which was then cured at 90 °C for 1 h followed by 160 °C for 2 h. A second thermal treatment step of 200 °C for 30 min was then undertaken to reduce further the GO in situ in the epoxy nanocomposite. An examination of the morphology of such nanocomposites containing reduced graphene oxide (rGO) revealed that a very good dispersion of rGO was achieved throughout the epoxy polymer. Various thermal and mechanical properties of the epoxy nanocomposites were measured, and the most noteworthy finding was a remarkable increase in the thermal conductivity when relatively very low contents of rGO were present. For example, a value of 0.25 W/mK was measured at 30 °C for the nanocomposite with merely 0.06 weight percentage (wt%) of rGO present, which represents an increase of ~40% compared with that of the unmodified epoxy polymer. This value represents one of the largest increases in the thermal conductivity per wt% of added rGO yet reported. These observations have been attributed to the excellent dispersion of rGO achieved in these nanocomposites made via this facile production method. The present results show that it is now possible to tune the properties of an epoxy polymer with a simple and viable method of GO addition. This disrupts electron and phonon transport, leading to poor electrical and thermal properties, respectively, of the GO so prepared, although it has the potential to increase the ease of dispersion of the GO when used as a filler in the production of polymer nanocomposites. The application of the graphene-based polymer nanocomposites requires that the OCFGs are reduced, to give reduced graphene oxide (rGO), in order to partially or fully obtain the excellent properties of graphene. Various reduction strategies for the GO are well known and have been reported in the literature [The starting material for the production of graphene-based polymer nanocomposites is frequently graphene oxide (GO), which is usually prepared either via an electrochemical method  or a cheterature –7. For eterature , 9. Howeterature –14. ThisConsidering the work reported in the literature relevant to such a facile production method, Yang et al.  investigIn this paper, we present a facile and effective route to prepare graphene–epoxy polymer nanocomposites with a remarkable increase in the thermal conductivity of the epoxy polymer at relatively very low contents of rGO. The ability of the OCFGs to impart a high degree of compatibility of the GO with epoxy resin is first exploited by mixing an aqueous dispersion of GO with the epoxy resin. Next, the remaining water is removed by evaporation, leaving a GO dispersion in the epoxy resin, which is cured with an anhydride curing agent. The final step is to reduce the GO in situ in the epoxy polymeric matrix at 200 °C. The result is a good dispersion of rGO in the crosslinked epoxy polymer. The advantages of this facile production route, compared to previously reported routes, include: (a) the relatively high content of GO in the aqueous suspension used means that the processing time is considerably shorter; (b) it does not involve the use of hazardous GO reducing agents such as hydrazine; (c) in situ reduction of the GO after curing of the epoxy polymer eliminates the extra processing step of reducing the GO prior to incorporation into the matrix; (d) the difficulties associated with dispersing powdery rGO into the epoxy resin are avoided; and (e) the relatively high temperature of 200 °C used to reduce the GO ensures an effective in situ reduction of the GO without degrading the chosen epoxy polymer. The thermal and mechanical properties of the epoxy nanocomposites so produced are reported in the present paper, together with the remarkable thermal conductivities at relatively very low contents of rGO.A standard diglycidyl ether of bis-phenol A (DGEBA) liquid epoxy resin  having an epoxide equivalent weight (EEW) of 185 g/eq. was used. The curing agent was an accelerated methylhexahydrophthalic acid anhydride  having an anhydride equivalent weight (AEW) of 170 g/eq.2SO4:0.3 L H3PO4) was added to 24 g of natural graphite flakes  under vigorous stirring, followed by the addition of 144 g of KMnO4  in small portions. The reaction mixture was then kept at 50 °C under vigorous stirring for 18 h. The mixture was cooled to room temperature, and the oxidation reactions were stopped by a drop-wise addition of 1.72 L of 2 wt% aqueous H2O2. The GO suspension was washed by repeated centrifugation and re-dispersion in distilled water, using a Sorvall LYNX 6000 Superspeed Centrifuge . This washing procedure was repeated until the pH of the supernatant matched that of the used distilled water, which typically occurred after 16 washing cycles. Typically two low-speed (<1000 rpm) centrifugation cycles were then performed to remove un-exfoliated graphite particles.Graphene oxide was prepared via a modified Tour et al.  synthesi3 and was cured at 90 °C for 1 h and then post-cured at 160 °C for 2 h to produce bulk nanocomposite plates. A further thermal treatment step of 200 °C for 30 min was then undertaken to ensure an effective in situ thermal reduction of the GO in the crosslinked epoxy polymer nanocomposite [A concentrated aqueous suspension of GO 13 mg/mL) was mixed with the epoxy resin by stirring first manually and then mechanically at 500 rpm using a radial-flow impeller for 30 min, as depicted in Fig. 3 mg/mL womposite , 17. TheThe unmodified epoxy polymer control was prepared in the same way as outlined above. However, in this case, the concentrated aqueous GO suspension was replaced with deionized water whose volume was equivalent to that of the aqueous rGO suspension used for the preparation of the nanocomposite with 0.06 wt% rGO (i.e. the highest rGO content used).Field-emission gun scanning electron microscopy (FEG-SEM) was used to image the dispersion of the rGO by examination of fractured samples of the nanocomposites. A Leo 1525  microscope was used, with an accelerating voltage of 5 kV. The samples were sputter-coated with a thin film of chromium prior to examination. Transmission electron microscopy (TEM) was carried out using a 2000FX microscope  employing an accelerating voltage of 200 kV. Thin slices (about 70 nm in thickness) were cut for TEM using a PowerTome XL ultramicrotome . Atomic force microscopy (AFM) was carried out to determine the morphology of the GO using a MultiMode scanning probe microscope . The microscope was equipped with a NanoScope IV controller and an E scanner. The concentrated aqueous GO suspension (13 mg/mL) was diluted to 0.1 mg/mL using deionized water. The diluted GO suspension was then dip-coated onto a mica sheet  and allowed to dry. Height and phase images were captured by AFM at a resolution of 512 pixels × 512 pixels and a scan speed of 1 Hz, using silicon probes in a tapping mode of operation.Fourier transform infrared spectroscopy (FTIR) was carried out on freeze-dried GO and thermally-reduced GO samples using a Spectrum 100 FTIR Spectrometer . Aqueous GO suspensions, produced as outlined above, were freeze-dried using a Powerdry LL1500 freeze dryer  to obtain the dry GO powder used for FTIR and TGA. The freeze-dried GO was thermally reduced according to the cure plus reduction schedule adopted for the production of the nanocomposites .3 pieces cut from the bulk nanocomposite plates. A displacement rate of 1 mm/min was used, and the strain was measured using a clip-on extensometer . Tensile modulus values were calculated between strains of 0.05 and 0.25%. The tensile properties were averaged from the results obtained from a minimum of five specimens.Uniaxial tensile tests were conducted at room temperature using a universal testing machine . Dumbbell specimens having a gauge length of 25 mm were machined from 75 × 13.5 × 3 mm3 were cut from the bulk nanocomposite plates. The samples were subjected to a temperature sweep from 30 to 200 °C at a heating rate of 2 °C/min in dual-cantilever mode at a frequency of 1 Hz using an oscillation strain of 0.05%. The glass transition temperature, Tg, of each sample was taken at the maximum of the tan δ curve. The number average molecular weight between crosslinks, Mnc, was calculated from:q is the front factor, ρ is the density of the epoxy determined at room temperature , T is the temperature and Er is the rubbery storage modulus determined at a temperature of T = 453 K (180 °C). Since the density of the epoxy was determined at room temperature, Pearson and Yee [q, of 0.725.The thermo-mechanical properties of the nanocomposites were determined by dynamic mechanical thermal analysis (DMTA) using a Q800 analyser . Rectangular samples with dimensions of 60 × 10 × 3 mm.2 g/cm3 ), R is t = 453 K 0 °C. Sin−9 mbar. The spectra were acquired using a MXR1 monochromated Al Kα X-ray source (hυ = 1486.6 eV). An X-ray spot of ~400 μm radius was employed. High-resolution, core-level C1s spectra were acquired using a pass energy of 20 eV. The GO sample spectra were charge-referenced against the C1s peak at 284.4 eV to correct for charging effects during acquisition. Quantitative surface chemical analyses were calculated from the high-resolution, core-level spectra following the removal of a nonlinear (Shirley) background. The manufacturer’s Avantage software was used which incorporates the appropriate sensitivity factors and corrects for the electron energy analyser transmission function.X-ray photoelectron spectroscopy (XPS) analysis of the GO and rGO was performed using a Theta Probe spectrometer , operated at a base pressure of 1 × 10θ range of 5°–60°, using a step size of 0.02° and 20 s per step.X-ray diffraction (XRD) patterns of the samples were acquired using an X’Pert PRO diffractometer . The diffractometer was equipped with a Cu Kα radiation source. A generator voltage of 40 kV and tube current of 40 mA were used for all measurements over a 2Thermogravimetric analysis (TGA) was carried out on the nanocomposites and freeze-dried GO samples using a TGA/DSC 1 . The samples were analysed using a heating rate of 10 °C/min over a temperature range of 30–800 °C in either an air or a nitrogen atmosphere.The thermal conductivities of the nanocomposites were determined by the laser flash technique using an LFA-427 Nanoflash . The samples were coated with a thin layer of graphite prior to testing to increase absorption and transmission of infrared radiation energy. Three shots each were made on 10 mm × 10 mm samples, 2 mm thick, over a temperature range of 30–60 at 10 °C intervals. A laser voltage of 450 V and a pulse width of 0.8 ms were used. The thermal diffusivity of each sample was measured and converted to the thermal conductivity using the specific heat capacity estimated from that of a graphite control sample whose thermal diffusivity had been determined using the same set of conditions.Preparation of the aqueous suspension of GO via the modified Hummers method leads to intercalation of OCFGs between the GO sheets formed during the oxidation of the graphite. This leads to an increase in the interlayer separation and hence a weakening of the attractive forces between the GO sheets. Moreover, the presence of the OCFGs imparts a high degree of compatibility of the GO with water and subsequently with the epoxy resin. These factors make it relatively easy to exfoliate and disperse the GO, with a minimum energy input, in the aqueous suspension. To illustrate this, a concentrated aqueous GO suspension of a content of 13 mg/mL was diluted to 0.1 mg/mL by adding deionized water, and the resulting suspension was vigorously shaken. The GO suspension was then dip-coated onto a mica sheet, dried and examined using AFM. Figure 2 and H2O [Reduction of the GO was carried out in situ in the cured epoxy polymer; however, to establish suitable reduction conditions, it was necessary to carry out preliminary tests on freeze-dried (powdery) GO. TGA was performed on this powdery GO in a nitrogen atmosphere, and the resulting thermogram is shown in Fig.  and H2O . A furth and H2O , and thi and H2O  demonstr and H2O , as seenOnce the reduction temperature of 200 °C was selected, the effect of the thermal treatment time on GO was investigated to allow a suitable reduction time to be chosen. Freeze-dried (powdery) GO samples were reduced at 200 °C for different lengths of time ranging from 10 min to 6 h, and Fig. The thermograms obtained in air for the rGO samples are presented in Fig. There is little variation in the relative amounts of the carbon atoms bonded to other carbon atoms or to OCFGs as the thermal treatment time is increased. Although Jeong et al.  have pre2 and H2O [Based on the results of the above experiments, to reduce the GO without degrading the epoxy polymer, the reduction of the GO in the cured epoxy polymer nanocomposite was carried out in situ using a thermal treatment schedule of 200 °C for 30 min. Figure  and H2O , see Fig and H2O , giving −1. The peaks at 1734, 1636, 1621.5 and 1223.5 cm−1 correspond to C=O, HO–C=O, C=C and C–O–C stretching, respectively, while those at 1351 and 1047 cm−1 correspond to C–OH stretching in the basal aromatic plane and at the edge of the GO, respectively [−1 corresponding to HO–C=O disappears, while the C–OH and C–O–C peaks originally at 1351 and 1223.5 cm−1, respectively, in GO are replaced by a broad peak of cyclic moieties in rGO. It can be seen, however, that the peak at 1047 cm−1 which corresponds to the stretching of C–OH attached to the edge of the GO sheet is unchanged. It is believed that these hydroxyl groups which are attached to the edge of the GO sheet are very stable and will only dissociate at temperatures in excess of 650 °C [−1. This is indicative of an increase in the recovery of sp2-hybridized carbon domains after the reduction of the GO.Figure θ = 26.6° in GNP is shifted to 2θ = 10.5° in GO after chemical oxidation. However, the (002) peak is partially restored to 2θ = 24.4° in rGO as many of the OCFGs are removed during thermal reduction. The corresponding interlayer spacing, as measured, decreases from 0.84 nm in GO to 0.36 nm in rGO as the OCFGs intercalated between individual sheets are removed [2 or CO during reduction [The position of the (002) diffraction peak in XRD can usually be correlated to the size of the diffraction grating . The reduction in chemical heterogeneity as the GO is reduced can be seen very clearly by the position of the diffraction peaks in the XRD patterns of GO and rGO shown in Fig. Figure For the rGO/epoxy polymer nanocomposites, where the GO is reduced in situ, it is instructive to first consider how the rGO is dispersed in the polymer matrix. The size and dispersion of the rGO sheets can be imaged using FEG-SEM on fractured samples. For convenience, the fracture surfaces of samples broken during tensile tests were used. (Note that the results of the tensile tests are discussed separately below). The unmodified epoxy is a homogeneous polymer as expected, see Fig. The images shown in Fig. The microstructure of the nanocomposite containing 0.06 wt% rGO appears different to that of the lower contents, see Fig. The morphology of the nanocomposites revealed a good dispersion of rGO. This has been attributed to the compatibility between the GO and the epoxy, as well as to the increased interlayer spacing between sheets due to the intercalated OCFGs which is expected to weaken the van der Waals and π–π interactions between individual sheets. Although increased agglomeration of rGO was observed in the nanocomposite with 0.06 wt%, isolated rGO sheets are visible which illustrate the good dispersion inherent in these nanocomposites.E′) and the damping coefficient (tan δ) for each of the nanocomposites. Selected values of the storage modulus plus the glass transition temperature and height of the tan δ peak are shown in Table Eg′, increases from 2.59 GPa for the unmodified epoxy to 2.74 GPa for the nanocomposite with 0.06 wt% of rGO (i.e. an increase of 5.8%). In comparison, Chandrasekaran et al. [Eg′ increase of only 5.1% for a GNP/epoxy nanocomposite containing nearly double the filler content (i.e. 0.1 wt% of GNP), at 25 °C. The notable increase in Eg′ observed in the present work is due to the high modulus of the rGO and the processing route adopted. The latter takes advantage of the relatively large flake size due to the minimal mechanical energy input during processing, the compatibility between the GO and the epoxy as well as the presence of intercalated OCFGs which lead to increased interlayer separation. These factors enable a good dispersion of the GO as seen in Fig. Eg′, as reported in the literature, may be the wavy topology of rGO and the imperfections in the rGO lattice resulting from the sp3-hybridized domains which allow for better mechanical interlocking between the rGO and the polymer matrix [Eg′ [Eg′ for the unmodified epoxy and the nanocomposite with 0.06 wt% of rGO. A value of 420 GPa is obtained for the effective modulus of the rGO, which compares well with literature values [The thermo-mechanical properties of the nanocomposites were examined using dynamic mechanical thermal analysis (DMTA). Figure n et al.  reportedr matrix . Furtherix [Eg′ . Indeed,e values .Figure 9Tg of the nanocomposites decreases with increasing rGO content, for example, the Tg decreased by 57.8 °C (i.e. 57%) with 0.06 wt% of rGO, as shown in Fig. δ peak, see Table Mnc, between crosslinks estimated from Eq. Mnc clearly increases, so the epoxy matrix becomes less highly crosslinked, with increasing rGO content. This is attributed to the fact that in situ reduction of the GO leads to the opening and/or elimination of OCFGs which may lead to the formation of covalent bonds between the GO and the epoxy matrix as mentioned previously. Furthermore, GO has been reported to promote the homopolymerization of epoxy resin [The glass transition temperature of the unmodified epoxy was measured to be 158.6 °C, see Table xy resin . These fxy resin , 34, 35.xy resin , 37.W350) is shown in Table W350 with increasing rGO content can be observed, except for the nanocomposite containing 0.02 wt% rGO. The onset temperature of thermal degradation, Ton, also decreased with increasing rGO content from 287.4 °C for the unmodified epoxy to 207.1 °C for the 0.06 wt% nanocomposite (i.e. a decrease of 80.3 °C), in accordance with the trend in Tg shown in Fig. Ton and W350 for the 0.01 and 0.02 wt% rGO nanocomposites may be attributed to experimental variation. The maximum degradation temperature, Tmax, and the temperature of 50% mass loss, T50%, both remained largely unchanged, see Table Ton and W350 are indicative of decreasing thermal stability with increasing rGO content, although increases in thermal stability with increasing rGO content have also been reported in the literature [The thermal stability of the nanocomposites in air was investigated by thermogravimetric analysis (TGA). Thermograms and derivative thermograms (DTGs) are shown in Fig. terature , 38.FiguAs discussed earlier, some OCFGs remain attached to the rGO sheets after reduction, as shown in Fig. The reaction between pendant OCFGs on the GO surface and epoxy resin is well known. Yang et al.  have shoEg′, and a value of 580 GPa is obtained for the effective modulus of the rGO, which compares well with the 420 GPa calculated from the values of Eg′ and with literature values [Figure e values . The notThe tensile strength and elongation at break of the unmodified epoxy and of the rGO/epoxy nanocomposites are shown in Fig. 2 and H2O, which are by-products of low-temperature reduction of the GO, can be released during the curing of the epoxy polymer [Tg observed from 133.5 °C for the 0.05 wt% nanocomposite to 100.8 °C for the 0.06 wt% nanocomposite  owing to intercalated OCFGs, as well as the compatibility between the OCFGs and the matrix epoxy to achieve a good dispersion of rGO in the nanocomposite via in situ processing. The good dispersion of GO in the aqueous media was transferred to a DGEBA epoxy resin with minimal mechanical energy input. This was followed by in situ reduction of the GO to rGO at 200 °C, which eliminated a substantial amount of the OCFGs.Tg and in the resistance of the nanocomposites to thermal oxidation, the storage and tensile moduli were increased significantly. This was attributed to the excellent rGO dispersion and strong interfacial adhesion between the rGO and the epoxy matrix. The presence of defects caused by the low-temperature reduction of the OCFGs led to a reduction in the tensile strength and elongation at break. The observed thermal conductivity of 0.25 W/mK (measured at 30 °C for the nanocomposite with 0.06 wt% of rGO) represents an increase of ~40% compared to the unmodified epoxy. This value is one of the highest thermal conductivity values ever reported for rGO/epoxy nanocomposites having such relatively low contents of rGO. It has been attributed to the excellent dispersion of rGO and large lateral flake size particular to these nanocomposites. These results, taken together, show that it is now possible to tune the properties of an epoxy polymer with a simple and viable method of GO addition.Electron microscopy revealed a good dispersion of rGO in the nanocomposites, except for the highest content of 0.06 wt% rGO where agglomeration was observed. This was attributed to the tendency of GO to form liquid crystalline structures above certain critical contents. Although addition of rGO led to a decrease in the


Cancers are heterogeneous at the cellular level. Cancer stem cells/tumor initiating cells (CSC/TIC) both initiate tumorigenesis and are responsible for therapeutic resistance and disease relapse. Elimination of CSC/TIC should therefore be able to reverse therapy resistance. In principle, this could be accomplished by either targeting cancer stem cell surface markers or “stemness” pathways. Although the successful therapeutic elimination of “cancer stemness” is a critical goal, it is complex in that it should be achieved without depletion of or increases in somatic mutations in normal tissue stem cell populations. In this perspective, we will discuss the prospects for this goal via pharmacologically targeting differential Kat3 coactivator/Catenin usage, a fundamental transcriptional control mechanism in stem cell biology. There are minimally four broad resistance-inducing strategies that are employed by cancer cells including: (1) direct target reactivation; (2) activation of signals upstream or downstream of oncogenes; (3) engagement of parallel oncogenic pathways; and (4) adaptive survival mechanisms. Despite tremendous advances in targeted therapeutics and personalized medicine, which have significantly increased progression free survival, maximum clinical success as defined by overall survival or “cures”, remain limited due to therapeutic resistance. These resistance mechanisms can be attributed to a subpopulation of self-renewing, highly tumorigenic, drug-resistant cancer stem cell/tumor initiating cell (CSC/TIC), in which therapeutic pressure leads to the selection of therapy resistant clone-8.Cancer is a major contributor to worldwide mortality. The existence of CSC has now been demonstrated in many tumor types including leukemia, brain, breast, bladder, prostate, colon, etc., where their presence has been associated with disease recurrence, multidrug resistance and metastasis. Therefore a critical goal to change the course of cancer therapy is to develop strategies to safely eliminate CSC without deleterious effects to normal spermatogonial stem cell (SSC) populations.All stem cells by definition, have the capacity to both self-renew  as well as to differentiate into more mature, albeit less potent, specialized cells. The concept of CSC is not new. Cohnheim, more than 150 years ago, proposed that cancer might arise from rare cells with stem cell-like properties. Rather the term CSC refers to cells that have “stem-like” properties. CSC can originate from tissue stem cells, transiently amplifying cells or potentially even differentiated cells. SSC, due to their longevity and self-renewing properties, have a far greater propensity to accumulate carcinogenic mutations, which could markedly influence the behavior of those cells, e.g., accelerate self-renewal via a switch from asymmetric to symmetric division, which will be further discussed,14. It is also possible that the initial mutations occur in SSC, yet the final mutations that confer oncogenesis occur during neoplastic transformation in downstream progeny that have blocks in terminal differentiation. Further, interaction with the environment or signaling changes within a cell can lead to epigenetic or phenotypic state changes relevant to CSC generation. Regardless of the exact origin of CSC, therapeutic resistance in CSC has been associated with (1) quiescence, as most conventional cytotoxic agents target proliferating cell,18; (2) high expression of drug-efflux pumps, e.g., ATP binding cassette (ABC) family transporters; (3) increased DNA repair and detoxifying enzymes; (4) acquisition of an EMT-like phenotype; and (5) utilization of hypoxic niche microenvironments that provide survival fostering signals.Two mechanisms are proposed to account for the generation of CSC. In the stochastic model, cancer cell plasticity endows non-CSC with the ability to dedifferentiate into CSC. Alternatively, in the hierarchical model, CSC are able to self-renew thereby expanding the CSC pool from which escape mutants can be selected. CSC and SSC share multiple characteristics, including self-renewal and the potential to differentiate. As previously pointed out, the term “cancer stem cell” does not have to refer to the cell of origin. CSC express similar “stemness” markers and exhibit similar cellular behaviors to SSC as described above. SSC in tissues preferentially inhabit specialized hypoxic niches and are critical for both normal tissue homeostasis and regeneration after injury-26. Long-lived SSC are quiescent and rarely become activated under homeostatic conditions, however upon injury to repair damaged tissue, they enter the cell cycle. CSC occupy the same hypoxic niches, thereby competing with normal SSC for this limited environment. The same signal transduction pathways utilized in SSC maintenance, proliferation and differentiation  also regulate CSC-29. For both CSC and SSC, there are multiple points of intersection and crosstalk, including feedback and feed forward loops, connecting the various signaling cascades that modulate “stemness” allowing for escape from driver directed therapeutics. These targets and therapies blocking these pathways are summarized in recent reviews,30-32.Targeting CSC could in principle be accomplished via the targeting of CSC specific cell surface markers or through alternatively “stemness” pathways. Although the successful therapeutic elimination of “cancer stemness” offers enormous promise, it will require significant precision to avoid deleterious effects  in normal SSC populations. Unfortunately in this regard, the similarities between normal adult SSC and CSC far outweigh their differences or even less. Quiescent SSC, once they enter the cell cycle, can undergo mitosis to give rise to two daughter cells. Mitotic stem cells can divide either symmetrically or asymmetrically , which postulated that the stem cell desires to retain its original uncopied strands of DNA and to pass on the duplicated strands that contain multiple copy errors, inherent in the DNA replication process, to its differentiated daughter cell, thereby minimizing the total number of DNA mutations that accumulate in the long-lived SSC population. In order to make the decision to divide symmetrically versus asymmetrically, a stem cell undergoing mitosis must read an enormous array of information from its environment . How is all of this information integrated to decide a stem cell’s fate, i.e., to exit quiescence and subsequently divide either asymmetrically or symmetrically, be it a normal SSC or a CSC?Adult SSC are present in limited numbers. They are believed to be essentially immortal and remain with us for our entire lives. The “dark side” of the immortality of SSC is their capacity to be corrupted into CSC. Like their normal counterpart SSC, CSC exhibit self-renewal capacity and differentiation potential, albeit with aberrant and incomplete differentiation, thereby having the capacity to maintain or renew and propagate a tumor. Under normal homeostatic conditions, long-term SSC divide relatively infrequently, perhaps only once every few months. Loss of the tumor suppressor PTEN leads to premature exhaustion of the normal hematopoietic stem cell population, presumably via increased symmetric differentiative divisions and expansion of the leukemic stem cell population via increased non-differentiative symmetric divisions. Indirect perturbation of Notch signaling, via genetic activation of the Hedgehog pathway, also causes an increase in neural stem cell symmetric divisions. Symmetric differentiative divisions by “corrupted” SSC prior to the accumulation of additional deleterious mutations generates bona fide CSCand can stochastically eliminate this SSC population. This mechanism prevents non-differentiative symmetric divisions expanding the “pre-CSC” pool. An example of this expansion of the “pre-CSC” pool is represented by clonal hematopoiesis of indeterminate potential (CHIP). CHIP is defined by the presence of somatic hematologic-cancer-associated gene mutations and can be seen in the peripheral blood of at least 10% of people older than 60 years of age without any history of hematologic disorders. The presence of CHIP is associated with an increased risk of hematologic cancers and an increased overall mortality.Interestingly, a preference for symmetric over asymmetric divisions appears to be one of the fundamental differences between CSC and SSC. Breast cancer stem cells with p53 mutations preferentially undergo symmetric divisions that initiates a broad range of intracellular responses broadly classified as either canonical (involving nuclear β-catenin mediated transcription) or non-canonical ,43. Canonical Wnt signaling is generally associated with proliferation and lack of differentiation (for example in cancer), whereas the non-canonical pathway regulates cellular patterning and tissue organization. β-catenin is critical in both pathways via its roles either in the nucleus or cytoskeleton and cytoplasmic membrane, respectively. Although designating Wnt signaling as either canonical or non-canonical allows for simplified conceptual discourse, there is great crosstalk between the two responses, and Wnt crosstalk regulates complex nonlinear networks in development and homeostasis. Nuclear β-catenin, although additional catenins, including γ-catenin/plakoglobin, may additionally participate under particular circumstances, in transcription is controlled by the so-termed “canonical Wnt” or “Wnt/β-catenin” signaling cascade. Nuclear translocation of β-catenin and its subsequent transcriptional activity can also be induced by non-Wnt signaling. Epithelial to mesenchymal transition, leads to β-catenin nuclear translocation, perhaps through down-regulation of β-catenin’s cytoplasmic binding partner E-cadherin. Receptor tyrosine kinases and non-receptor tyrosine kinases including Src and Abl can enhance β-catenin-mediated transcription by disrupting the E-cadherin/β-catenin interaction. Prostaglandins, hypoxia,53, high glucose levels, and cholinergic innervation additionally may activate Wnt/β-catenin signaling. A wide range of inputs an influence β-catenin dynamics and β-catenin-dependent transcription-58. Balancing self-renewal versus differentiation in SSC, requires signaling from a number of other pathways  that must be integrated with nuclear β-catenin signaling . However, there is no consensus on whether Wnt signaling is important for either maintenance of potency,60 or the differentiation of stem cells. Wnt/catenin signaling clearly plays dichotomous roles in SSC biology.Wnt signaling is an ancient and highly evolutionarily conserved pathway that is important throughout embryonic development and the life of an organism. It is a very complex signaling cascade. Not surprisingly, aberrant regulation of Wnt signaling is a recurrent theme in cancer biology,65 and has been implicated in the tumorigenic potential of stem cells.Wnt signaling plays a critical role in SSC homeostasis. Wnt/β-catenin regulation of telomerase activity endows stem cells and cancer stem cells with unlimited self-renewal capacity. Slug, a strong inducer of EMT in tumors, is associated with nuclear accumulation of transcriptionally active β-catenin. Over-expression of either of the putative Wnt target gene EMT inducing factors twist and snail increases the expression of CSC markers. The connection between enhanced nuclear β-catenin signaling and EMT is strengthened further by the significant number of β-catenin target genes  associated with invasion, migration and metastases. Wnt signaling in CSC is associated with metastasis, and the regulation of organ specific tropism of CSC during metastasis, as well as in the formation of the pre-metastatic niche that nurtures metastasizing CSC. Cdx-1 and Id2, two transcription factors associated with the maintenance of a stem like” state, have been shown to be β-catenin regulated. Many cell surface markers in stem cell and cancer stem cells are direct Wnt targets, including LGR5/GPR49, CD44, CD24, CD133, ABC cassette genes,81 and EpCAM. The first identified CSC in solid tumors had a CD44high CD24low phenotype and comprised a population of breast cancer CSC possessing tumor-initiating capacity. These genes and related references are listed in ,85 and epigenetic silencing of negative regulators of the Wnt signaling cascade is frequently associated with leukemia. Moreover, aberrant activation of tumor associated Wnt/β-catenin signaling has been correlated with resistance to radiation, cytotoxic and targeted chemotherapy,88 and most recently checkpoint immunotherapy resistance in multiple tumor types including, melanoma, bladder and head and neck cancers. Tumor-intrinsic Wnt/β-catenin signaling mediates cancer immune evasion by preventing T-cell and/or dendritic cell infiltration, migration and function, and thereby resistance to immune checkpoint inhibitors,91 and has been shown to maintain T-cells in a differentiated exhausted dysfunctional state.Continued expression of BIRC5/Survivin, a Wnt target gene, in hES cells is essential for teratoma formation,41,93. It may seem obvious to target the Wnt signaling pathway in both SSC and CSC and indeed this has engendered substantial efforts to develop therapeutic agents. Despite these efforts, no therapeutic agents to date specifically targeting the Wnt pathway have been approved for use in patients. A number of factors have thwarted progress in this regard. First, the Wnt signaling cascade is highly complex,42. For example, in addition to classical canonical Wnt/β-catenin/TCF transcription, Wnt proteins elicit a variety of alternative non-canonical responses,95. Secondly, crosstalk from various non-Wnt factors can also modulate nuclear β-catenin accumulation as previously discussed. Overall, the ability to target Wnt signaling holds enormous potential; however, like the sword of Damocles, it brings substantial risks and concerns as it is also a crucial pathway in normal SSC maintenance and tissue homeostasis.Successful pharmacologic manipulation of aberrant catenin-regulated transcription of endogenous “stemness” in SSC and CSC holds enormous potential. However, significant concerns arise in regards to potential deleterious effects on normal SSC populations, including increasing DNA lesions or elimination of normal SSC while attempting to eliminate CSC or activate quiescent or senescent SSC. As stated previously, a preference for symmetric over asymmetric divisions appears to be one of the fundamental differences between CSC and SSC. The question then is: can we safely manipulate endogenous stem cell populations by taking advantage of their preferred modes of division to differentiate away the CSC population without eliminating normal SSCs?All-trans retinoic acid provided a breakthrough differentiation therapy for acute promyelocytic leukemia. However, broad scale success with differentiation therapy has not been achieved to date,97 to promoters and enhancers. Kat3 coactivators, by binding to hundreds of proteins, play critical roles as master regulators of transcription. Kat3 activation has been previously reviewed by our team,41, and is driven by multiple signals including Wnts, high glucose, hypoxia, and EMT inducers. Historically, CBP and p300 have been considered largely redundant due to their significant protein sequence identity and even higher similarity. However, CBP and p300 are clearly not redundant and carry out definitive and unique roles both in vitro and in vivo,98-101. From a library of 5000 secondary structure mimetics, we identified ICG-001 (IC50 = 3 μM) in a Wnt reporter screen in colon cancer cells. We subsequently identified and validated that the molecular target of ICG-001 was CBP and that ICG-001 binds specifically and with high affinity (~1 nM) to the N-terminus of CBP but not to p300,103. We subsequently found that selectively blocking the CBP/catenin interaction with ICG-001, with an increase in p300/catenin-mediated transcription leads to the initiation of differentiation in stem and progenitor cells including ES, iPS, SSC and CSC [Figure 3A]-108. These investigations allowed us to propose our model of differential coactivator usage. The critical non-redundant roles that CBP and p300 play in catenin-mediated transcription are highlighted in our model in vitro and in vivo,109-112In order to form a transcriptionally active complex, β-catenin must recruit one of the two Kat3 transcriptional coactivators, Kat3A, cAMP response element binding protein [CREB-binding protein (CBP)] or its closely related homolog Kat3B, p300 ,113-115. CBP/catenin antagonists can activate SSC and induce asymmetric differentiation thereby enhancing repair pathways in preclinical models of pulmonary and renal fibrosis,117, myocardial infarction and neurodegeneration,108,119. The differential effects of CBP/catenin antagonists on CSC versus SSC, specifically forced differentiation and elimination versus differentiation and enhanced repair without depletion, are cell intrinsic. CBP/catenin antagonists utilize the intrinsic propensity of CSC to preferentially divide symmetrically,37 thereby stochastically eliminating CSC via forced symmetric divisions [Figure 4A]. Asymmetric differentiation can be activated by CBP/catenin antagonists thereby enhancing repair without damaging the normal SSC population. Therefore, CSC when treated with CBP/catenin antagonists will stochastically be cleared from their niche via symmetric differentiative divisions We have extensively examined the therapeutic potential of selectively antagonizing the CBP/catenin interaction, and have demonstrated the ability to safely eliminate drug-resistant CSC, via forced differentiation, without deleterious effects on the normal endogenous stem cell populations. It is important to note that neither pre-clinical nor clinical studies have shown toxicity when utilizing specific small molecule CBP/catenin antagonists are safe. PRI-724 (IC50 ~150 nM), a second-generation clinical CBP/catenin antagonist demonstrated an excellent safety profile in preclinical IND enabling toxicology studies. The no-adverse-event-level for PRI-724 in dogs was 120 mg/kg/day administered for 28-day via continuous infusion. Clinically, PRI-724 had an excellent safety profile, demonstrating no dose limiting toxicities with escalation from 40 to 1280 mg/m2/day administered by continuous i.v. infusion. Down regulation of the biomarker survivin/BIRC5 with upregulation of the differentiation antigen CK20 in EpCAM selected circulating tumor cells strongly correlated with increasing plasma concentrations of drug. PRI-724 also demonstrated safety and efficacy with increased liver function in a trial conducted in patients with HCV-induced hepatic fibrosis.This degree of safety was initially surprising. We believe this is due to the high biochemical specificity of ICG-001/PRI-724 for binding to CBP, and its limited impact on only a fraction of all CBP interactions. The unique non-redundant roles that the N-termini of the two Kat3 coactivators CBP and p300 play in stem cell biology and the intrinsic preference for asymmetric division in normal SSC are critical to the safety of these agents.Significant concerns about specificity arise when targeting the coactivator protein CBP, as it has as many as 500 molecular partners, including a vast array of transcription factors“Nothing in Biology Makes Sense Except in the Light of Evolution” - Theodosius Dobzhansky.,123. SSC and CSC preferentially utilize glycolysis over oxidative phosphorylation despite the inefficiency in regards to ATP generation of glycolysis compared to oxidative phosphorylation. The activation of quiescent SSC and the initiation of differentiation involves a metabolic change from glycolysis and entry into the Krebs cycle. Reprogramming to pluripotency, on the other hand is associated with “anaerobicizing”. With the dawn of the evolution of vertebrates, roughly 450 million years ago, a new lifestyle having a relatively long-lived adult stage began. To accommodate this successfully a mechanism for long term homeostatic maintenance and tissue repair was essential. This was accomplished via quiescent “immortal” SSC maintaining an “anaerobic” metabolic state in specialized niches as opposed to their more proliferative aerobic-differentiated daughter cells. This mechanism evolved in order to protect genetic material integrity in long lived vertebrates. Maintaining the two different populations resulting from asymmetric division; one daughter being a long-lived quiescent SSC utilizing anaerobic metabolism and the other a rapidly expanding differentiating population utilizing aerobic metabolism, required tight regulation. The Kat3 coactivator family CBP and p300 diverged via gene duplication just prior to the vertebrate radiation over 450 million years ago. CBP and p300 are extremely large proteins encoded over 33 and 31 exons respectively. CBP and p300 retain extremely high identity, up to 93%, particularly over a large central core that includes the CH1, KIX, Bromodomain, and CH2 and CH3 regions ,129.Quiescence provides safeguards the functionality of SSC by restricting the damage caused by mitochondrial respiration and reactive oxygen species generated during oxidative phosphorylation. These safeguards limit DNA mutations and prevent uncontrolled cell cycle entry,111,130. This least conserved region between the two coactivators, which has only 66% identity, binds both β-catenin and through a highly conserved LXXLL sequence, nuclear receptor family members. The N-termini within each orthologous coactivator are extremely conserved with human and mouse CBP being 98% identical at the amino acid level within this region. The nuclear receptor family and Wnt signaling appeared significantly earlier in evolution approximately 600 million years ago in the first multicellular animals (metazoans),133 and are found in nematodes, flies, and vertebrates.The small molecules CBP/catenin antagonists, ICG-001/PRI-724, and p300/catenin antagonists, YH 249/250, bind the CBP and p300 N-termini, respectively. This co-evolution resulted in high fidelity control over the differential cell fates generated by asymmetric stem cell division, thereby enabling two inherently different cell populations and providing the expanding daughter cell population integrated pathways to generate divergent cell types. This joint genetic divergence and signaling integration additionally provided a mechanism to “read” multiple signals affecting SSC quiescence and DNA integrity. For example, a huge number of lesions in DNA can be induced by ultraviolet light.Therefore, circadian regulation of normal SSC activation and division is critical. Shift workers, with aberrant circadian regulation, have an increased risk for the development of cancer,137. Metabolic and circadian regulation control the timing and the mode of SSC division and metabolic pathways through nuclear receptors  play critical roles in circadian integration of metabolism energetics,140. Clock, part of the master circadian regulatory circuit mediated by the Clock/Bmal1 transcriptional complex, is recruited to p300 in vivo in a time-dependent manner. Evolutionarily, it would seem logical that mechanisms to enhance SSC asymmetric differentiation and symmetric differentiative divisions of CSC or pre-CSC would have evolved. In fact, numerous naturally occurring CBP/catenin antagonists have evolved. Returning to the concept of differentiation therapy, all-trans retinoic acid (ATRA), a vitamin A derivative, via its nuclear receptor complex (RAR/RXR) acting as a CBP/catenin antagonist is very effective in treating Acute Promyelocytic Leukemia. ATRA, similar to ICG-001 does not kill malignant cells but rather induces them to differentiate. Vitamin D plays an important role in cancer prevention through the (VDR/RXR) nuclear receptor complex and both ATRA and VitD have been shown to antagonize aberrant Wnt signaling in the context of malignancy. Nuclear receptor family members, via competition with β-catenin for binding to the N-terminus of CBP, phenocopy CBP/catenin antagonists. However, synergistic effects on the activation of gene expression by nuclear receptors and Wnt signaling have been demonstrated  and nuclear receptors also on their own control the expression of various transcriptional cassettes. Thus, nuclear receptor family members are not simply “pure antagonists” of CBP/catenin transcription and therefore have significant differences from small molecule direct CBP/catenin antagonists.Previously, we proposed that gene duplication generated the two Kat3 coactivators and a subsequent rapid divergence within their N-terminal regions occurred at the same time as the integration of Wnt and nuclear receptor family signaling,144. However, in contrast to modulation of nuclear receptors, which can cause developmental defects, selectively antagonizing the CBP/catenin interaction with ICG-001, even at very high levels, is extremely safe and has no deleterious effects on mouse embryonic development,145. Female mice treated topically or orally with high doses of ICG-001 throughout pregnancy have normal litters. The pups exhibited normal weight and size compared to their control littermates and can reproduce normally, demonstrating no deleterious effects to germ cell populations, which interestingly, also prefer asymmetric divisions,147. Interestingly, a 27 bp/9aa deletion in CBP between the β-catenin-binding region (DELI-sequence) and the nuclear receptor (LXXLL) binding sequence is a strongly evolutionarily conserved. Using CRISPR/Cas9 editing of p300, we recently demonstrated that this deletion in CBP provided a mechanism via steric inhibition, for nuclear receptors to antagonize CBP/catenin signaling, allowing for the maintenance of quiescence and initiation of asymmetric divisions in SSC. Whereas β-catenin and nuclear receptor signaling can synergize to effect a feed-forward mechanism to drive differentiation and lineage commitment utilizing p300, as steric constraints removed by the conserved 9 amino acid insertion is sufficient to allow for the simultaneous binding of nuclear receptors and β-catenin.The LXXLL sequence present in the amino termini of both CBP and p300 is highly conserved and can recruit both RAR/RXR and VDR/RXR complexes, and potentially all other nuclear receptor complexes including AR, PPAR, and others. Not surprisingly, multiple nuclear receptors can effect stem cell maintenance or initiate differentiation in a manner similar to small molecule p300/catenin or CBP/catenin antagonistsBIRC5/Survivin required for self-renewal of stem cells. In this regard, targeting CBP/catenin signaling appears to represent a common “Achilles’ Heel” in CSC in both solid and liquid tumors,113,148. Aberrant regulation of catenin/Kat3 coactivator usage enhances CBP/catenin activation at the expense of p300/catenin-mediated transcription. Preferential use of this coactivator can arise from a vast array of mutations, either inherited or acquired, and a wide variety of insults . Resistance to therapy, radiation, chemotherapy of immunotherapy is associated with selection of resistant clone(s) from a pre-existing CSC pool. CBP/catenin antagonists, by taking advantage of this fundamental difference between SSC and CSC can safely stochastically differentiate away symmetrically dividing CSC without depleting the SSC population that is dividing symmetrically. However, in cancer, the transient amplifying population is not sensitive to CBP/catenin antagonists and still must be targeted to eliminate the disease, as these populations rely on other pathways  to maintain their non-terminally differentiated proliferative status,105,114. The robust safety profile of CBP/catenin antagonists could eventually provide an opportunity to utilize them in a “vitamin-like” manner as a prophylaxis to the accumulation of pre-CSC or CSCs.SSC and CSC utilize Wnt/catenin signaling and differential Kat3 coactivator usage to regulate stem cell homeostasis and the balance between self-renewal and differentiation. The fundamental difference between SSC and CSC appears to be a preference for asymmetric over symmetric divisions respectively. Increased CBP/catenin transcription is associated with enhanced telomerase activity and the expression of


Hepatocellular carcinoma (HCC) is one of the most common cancers with an insidious onset, strong invasiveness, insensitivity to chemotherapy, and poor prognosis, thus makes clinical treatment challenging. The mechanisms require further elucidation for developing novel therapies and targeting drug resistance. Here, we observed high Shc3 expression in patients with chemoresistant and recurrent HCCs. Shc3 overexpression induced a significant increase in MDR1/P-glycoprotein expression, whereas Shc3 knockdown impaired this expression. Further, Shc3 inhibition significantly restored HCC cell sensitivity to doxorubicin and sorafenib. Mechanistically, Shc3 interacted with β-catenin, inhibited destruction complex stability, promoted β-catenin release, and dampened β-catenin ubiquitination. Shc3 bound β-catenin and facilitated its nuclear translocation, prompting the β-catenin/TCF pathway to elevate MDR1 transcription. β-catenin blockage abolished the discrepancy in drug resistance between Shc3-depleted HCC cells and control cells, which further validating that β-catenin is required for Shc3-mediated liver chemotherapy. We also determined the effect of Shc3 on the sensitivity of HCC to chemotherapy in vivo. Collectively, this study provides a potential strategy to target these pathways concurrently with systemic chemotherapy that can improve the clinical treatment of HCC. Currently, the most commonly used clinical treatment for HCC include percutaneous ablation, curative resection, liver transplantation, transarterial chemoembolization (TACE), and systemic targeted agent like sorafenib. However, most patients are diagnosed at an advanced stage and are not eligible for curative surgery. TACE is the standard of care for patients with unresectable HCC; however, standard chemotherapy has not been found to be beneficial in prolonging the overall survival of HCC patients as it is often associated with substantial toxicity and acquired multidrug resistance2. The cause of multidrug resistance (MDR) in cancer is multifaceted, with MDR1  overexpression being the key determinant of HCC drug resistance4. Inherent high resistance of HCC to chemotherapeutic drugs and early HCC recurrence are the main causes of HCC related death. Accumulating research findings has shown that recurrence and chemoresistance of HCC are closely related to HCC stemness, and that the epithelial-mesenchymal transition (EMT) program also appears to contribute to liver cancer stem cells (CSCs) phenotype through its effects on the intracellular signaling pathway5. For these reasons, identification of the key molecules involved in multidrug resistance of HCC is imperative for improving the clinical outcome.Hepatocellular carcinoma (HCC) is one of the most aggressive and common human malignancies globallySHC3  gene encodes for two isoforms, p52Shc3 and p64Shc3; all isoforms have conserved domains with an PTB-CH1-SH2 domain modular structure and p64Shc3 has an additional N-terminal CH2 domain6. Shc3 has been implicated in cell survival and differentiation; the downregulation of Shc3 could induce abnormal alterations in hypoxic signaling, apoptosis, and inflammatory response7. Recently, several studies on Shc3 in malignant tumors have been conducted. Shc3 is ectopically overexpressed in various cancers, such as high-grade astrocytomas, high-grade glioblastomas8, neuroblastomas9, thyroid carcinoma10, and hepatocellular carcinomas11. Shc3 interacts with Gab1 and recruits the p85 subunit of PI3K, which leads to downstream activation of the Akt pathway in papillary thyroid carcinoma. In neuroblastoma cells, the interplay between Shc3 and HIF-1α may protect of cancer cells against hypoxia. Also studies have reported that Shc3 is a new regulator of cancer stem cell migration, and Shc3 silencing in glioblastoma can reduce migration and invasion13. Our previous study reported that Shc3 forms a complex with MVP, MEK, and ERK to potentiate ERK activation independent of c-Raf. This interaction consequently induces EMT and promotes HCC cell metastasis, thus we speculate that Shc3 may plays an important role in sorafenib resistance in HCC11. Moreover, recent reports have indicated that EMT induction in tumor cells not only contributes to increased metastasis, but also leads to MDR, and we presume that Shc3 is associated with drug sensitivity. However, little is known about the molecular mechanism interplay Shc3 with MDR.Src homolog and collagen homolog 3 (Shc3) is an adaptor protein belonging to the Shc family. The 15. Ectopic activation of the β-catenin pathway has been found in a wide range of tumors of intestinal, liver and hematopoietic cell origin16. β-catenin is the key molecule in this pathway, and its protein levels and nuclear translocation are tightly controlled by the multiprotein β-catenin destruction complex. Cytoplasmic β-catenin is constitutively degradated by two scaffolding proteins, adenomatous polyposis coli (APC) and Axin, which requires scaffolding the Ser/Thr kinases glycogen synthase kinase 3 (GSK-3), casein kinase 1 (CK1) and β-catenin to facilitate the amino terminus phosphorylation of β-catenin. Phosphorylated β-catenin is recognized by ubiquitin ligase β-transducin repeat-containing protein (β-TrCP), and is subsequently targeted for ubiquitin-mediated proteasomal degradation15. Disassembly of the destruction complex can block β-catenin degradation that results in the translocation of the accumulated β-catenin into the nucleus, where β-catenin binds to the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of transcription factors and triggers the protooncogene-induced transcription of several target genes, such as c-myc, MDR1, OCT4, and cyclin D1 (refs. 19). Hyper-activation of the β-catenin/TCF signal is frequently detected in human HCCs20 and high nuclear expression of β-catenin correlated with reduced recurrence-free survival and vascular invasion, suggesting the β-catenin activation are involved in the promotion of HCC recurrence21. Hence, a better understanding of the mechanisms underlying the activation of β-catenin/TCF signaling would increase HCC therapeutic benefit.β-catenin/T-cell factor signaling plays a central role in carcinogenesis by regulating cell differentiation, proliferation, metastasis, drug resistance, and stemnessIn this study, we investigated the function of Shc3 in HCC recurrence and drug resistance. We firstly observed high expression of Shc3 in both MDR1 overexpression and recurrent HCCs from patients. Then we verified the effect of Shc3 expression on the stemness and drug resistance by cell function experiments. Mechanistically, Shc3 interacted with the β-catenin, promoted β-catenin release from the destruction complex and dampened the ubiquitination of β-catenin. Consistently, Shc3 facilitated the nuclear translocation of β-catenin and activated MDR1 expression in HCC cells via the β-catenin/TCF-dependent pathway. Our study has uncovered a new mechanism between Shc3 and MDR1 expression, which emphasizes the importance of the β-catenin/TCF pathway in the regulation of drug resistance of HCC.Our previous study has shown that aberrant expression of Shc3 may play an important role in sorafenib resistance in HCC, thus we are wondering that whether Shc3 is involved in HCC multidrug resistance. To determine the relationship between Shc3 and multidrug resistance, the clinical relevance of Shc3 and MDR1 messenger RNA (mRNA) expression level in 52 matched HCC and non-tumor liver tissues was examined by quantitative reverse-transcription PCR (qRT-PCR). We observed a dramatically higher expression of Shc3 in HCC tumor than in the para-tumors ; higher MDR1 transcript levels were observed in 80.76% (42 of 52) of HCC samples than its corresponding para-tumor tissues Fig. . The mRNχ2 = 7.127, P = 0.007). Furthermore, expression of Shc3 was associated with maximal tumor size , microvascular invasion , malignant differentiation , TNM stage . As MDR1 overexpression is the key determinant of HCC drug resistance, so we further detected the effects of Shc3 on the expression of MDR1. As shown in Fig. To further investigate the relationship between Shc3 and MDR1, we examined the expression levels of Shc3 and MDR1/P-gp in numerous HCC cells. As anticipated, the mRNA level of Shc3 significantly increased in malignant and P-gp high-expression MHCC97L, MHCC97H, and HCCLM3 cells Fig. , and wes22. Results of the spheroid formation assay shown that stable Shc3 overexpressed MHCC97L cells formed more spheroids than MHCC97L/Vector cells  assay. As shown in Fig. lls Fig. , right. lls Fig. , left. TWe next sought to understand the mechanisms by which Shc3 promotes cancer cell stemness and drug resistance in HCC. Immunoprecipitation and mass spectrometry were carried out to identify proteins that interact with Shc3. Mass spectrometry results showed that β-catenin associates with Shc3 Fig. . Then co24. Thus, we speculated that Shc3 might regulate β-catenin protein stability. To define the underlying molecular mechanism, we firstly tested the degradation of β-catenin in the presence of cycloheximide (CHX), a potent inhibitor of protein translation. As CHX (10 μg/mL) treatment time was prolonged, degradation of β-catenin increased, and the degradation was impaired when Shc3 was overexpressed, suggesting that Shc3 might be involved in β-catenin degradation  that binds to the MDR1 promoter region in HCC cells. In chromatin-immunoprecipitation (ChIP) assays, the MDR1 promoter was pulled down in anti-β-catenin IP assays in MHCC97L/Shc3 cells much more than in MHCC97L/Vector cells, suggesting that β-catenin binds to the MDR1 promoter under Shc3 regulation . qRT-PCR and western blot results revealed that downregulation of β-catenin blocked Shc3-mediated expression of MDR1 and stemness-associated transcription factors  Fig. . In addi6 cells per mouse). Ten days later, mice were treated with doxorubicin (20 μg/g) via intravenous injection every other day for four times consecutively, and the tumor volumes were monitored and recorded. Xenograft tumors grew much faster in the MHCC97L/Shc3 group when treated or not treated with doxorubicin compared with the MHCC97L/Vector group , vascular endothelial growth factor receptor 2 (VEGFR-2), c-Kit, and so on. We previously reported that ectopic expression of Shc3 promotes metastasis and proliferation via c-Raf–independent activation of the MEK-ERK pathway; thus, it can be speculated that this alternative pathway regulated by Shc3 may play an critical role in sorafenib resistance11. However, the potential molecular mechanism and clinical significance remains unknown and requires further research. In this study, we presented preliminary evidence for a novel mechanism in which Shc3 promotes HCC multidrug resistance.Various targeted and immune therapies have been tested on HCC patients and only sorafenib, a multi-kinase inhibitor, has demonstrated modest efficacy. Moreover, only approximate 20% of HCC patients respond positively to sorafenib treatment28. An important feature of CSCs is that they overexpress ABC transporter proteins, which pump many foreign substances including chemotherapeutic drugs out of the cells that may lead to chemotherapy failure. Considering the importance of cancer stem cells in chemoresistance and cancer recurrence, we examined the influence of Shc3 on HCC stemness. We detected more expanded stem cell population in MHCC97L/Shc3 cells compared with MHCC97L/Vector cells. In addition to MDR1, Shc3 modulated the expression of stemness-associated transcription factors, including OCT4, SOX2, and NANOG. These findings revealed that Shc3 is important for HCC stem cells expansion and that targeting Shc3 could be a promising strategy for HCC treatment.Our data suggested that Shc3 and P-gp were both upregulated in HCC samples and demonstrated a strong positive correlation. We also found that Shc3 was required for the activation of MDR1 in HCC cells. Moreover, univariate and multivariate analyses showed that elevated Shc3 expression in recurrent HCC. The existence of cancer stem cells is considered the origin of chemoresistance and HCC recurrence30, and MDR1 has been recognized as the driver of cancer multidrug resistance. Here, we revealed a novel finding that MDR1 expression is regulated by Shc3 via β-catenin/TCF pathway activation. Shc3 is significantly upregulated and its expression has a significantly positive correlation with MDR1 expression in HCC tumors. Our results further shown that Shc3 overexpression promotes MDR1 expression in HCC cells and decreases chemotherapeutic drug sensitivity. Additionally, inhibition of Shc3 expression could reduce MDR1 expression and increase drug sensitivity. This prominent correlation between the gene expression levels of Shc3 and MDR1 reveals that Shc3 may regulate MDR1 expression. However, the regulation of MDR1 expression remains poorly understood. Although recent reports have confirmed an upstream promoter region of MDR1 containing seven consensus binding sites of LEF/TCF in neuroblastoma, colorectal cancer, and breast cancer32, this regulation has not been investigated in HCC. As shown in Fig. Intrinsic or acquired MDR are major causes of chemoresistance, especially to doxorubicin. HCC chemoresistance is often attributed to overexpression of the MDR1 gene34. The “β-catenin destruction complex” and ubiquitin proteasome pathway regulated degradation of β-catenin are the central mechanism for the regulation of intracellular β-catenin levels35. Activation of the upstream signal transduction cascade leads to the disassociation of the destruction complex and suppresses phosphorylation and ubiquitin-proteasome-mediated degradation of β-catenin, which can enter the nucleus and then bind to the LEF/TCF transcription factors36. Aberrant β-catenin–LEF/TCF complex activation is frequently detected in human HCCs; hyper-activation of the β-catenin pathway mediates the expression of downstream target genes related to different cellular processes. Meanwhile, the elevated level of intracellular β-catenin expression correlated with shortened relapse-free survival, indicating the involvement of β-catenin activation in promoting recurrence of HCC37. Consequently, a better dissection of the mechanisms underlying the β-catenin/TCF pathway activation would improve HCC therapy effect. An important finding of this research was that Shc3 reduced the ubiquitination of β-catenin and activated β-catenin/TCF pathway in the absence of Wnt stimulation. First, we found that Shc3 interacted with the cytoplasmic components of β-catenin. Second, our results demonstrated that Shc3 is involved in the ubiquitin-proteasome-mediated degradation process of β-catenin and can function as a stabilizer. Knockdown of Shc3 promoted β-catenin ubiquitination, whereas overexpression of Shc3 significantly impaired the level of β-catenin ubiquitination. Shc3 is a member of the adaptor proteins that conserve domains with an identical PTB-CH1-SH2 domain. The PTB and SH2 domains are phospho-tyrosine-recognition modules that interact with tyrosine phosphorylated growth factor receptors, such as EGFR, RET, and TrkB (ref. 38). The CH1 and CH2 regions contain tyrosine-phosphorylation residues that recruit other adapter proteins, such as Grb2, MVP, and Grb2-associated binding protein 1 (Gab1)39. For the first time, our results reveal that Shc3 regulates β-catenin protein ubiquitination and degradation. We also found that β-catenin degradation by the destruction complex is destabilized by the binding between Shc3 and β-catenin, indicating that Shc3 mediated the β-catenin/TCF pathway in the cytoplasm, but not in the membrane where the Shc3 adaptor protein functions the best. These observations are consistent with previous studies that Shc3 was located in both the membrane and the cytoplasm. Therefore, this study verified the novel mechanism by which Shc3 independent Wnt activated β-catenin/TCF pathway. Thus, Shc3 promotes HCC recurrence via the potentiation of β-catenin signaling. This finding could partially explain the observation of high nuclear β-catenin in recurrent HCC compared to β-catenin.Recent research has implied that the β-catenin/TCF signaling plays a key role in carcinogenesis by mediating cell chemoresistance and stem cell maintenanceIn summary, we identified that Shc3 expression is significantly upregulated in HCC tumors and is strongly associated with MDR1/P-gp expression and HCC recurrence. We further reinforced the notion that Shc3 regulates MDR1 expression and reduces the sensitivity of HCC cells to sorafenib and doxorubicin treatment. First, Shc3 enhances stem cells maintenance by upregulating stemness-associated transcription factors, including SOX2, OCT4, and NANOG. Second, Shc3 interacts with β-catenin and inhibits destruction complex stability, promotes β-catenin release from the destruction complex, and dampens the β-catenin ubiquitination. Third, the Shc3-mediated β-catenin/TCF pathway can induce the activation of MDR1 and stemness-associated transcription factors, thus promoting HCC stemness and reducing sensitivity to sorafenib and doxorubicin. Moreover, we demonstrated that Shc3 can increase the drug resistance of HCC both in vitro and in vivo. The novel identified Shc3-β-catenin/TCF-MDR1 axis provides new insight into HCC MDR and represents a valuable target for HCC therapy.11. All experiments used early-passage cells and cells were tested for Mycoplasma.Huh7 was purchased from the Japanese Collection of Research Biosources. HEK293T, PLC and HepG2 cells were purchased from the American Type Culture Collection biobank. MHCC97H, MHCC97L, and HCCLM3 cells were cultured as describedThe xenograft mouse model was constructed by transplanting MHCC97L/Vector and MHCC97L/Shc3 cells subcutaneously to the nude mice. Animal care and handling procedures were performed in accordance with the guide for the care and use of laboratory animals, and the animal study protocol was approved by the institutional animal care and use committee of Tianjin Medical University.HCC tumor and adjacent para-tumor tissues from 52 patients were collected from September 2016 to December 2018 and the expression of mRNA level were detected by quantitative reverse-transcription PCR (qRT-PCR). Eight paired of HCC tissue samples were randomly selected from 52 HCC patients for western blot analysis Table . What’s Expression plasmids of p64Shc3 were constructed to pCDH-CMV-MCS lentivector . Recombinant genes coding short hairpin RNAs (shRNA) against Shc3 were constructed to pLKO.1-TRC cloning vector . The details of constructions of the expression plasmids or shRNA are described in Supplementary Materials and Methods .We observed the intracellular location of doxorubicin in MHCC97L cells by the confocal microscope. Cells were seeded on glass slides in 12-well culture plates at a density of 5 × 105 cells/well and allowed to attach for 24 h. After that, cells were incubated separately with doxorubicin concentration of 1 μg/mL. At 4 h after incubation, the cells were washed with phosphate-buffered saline (PBS) solution, digested with trypsin and resuspended in PBS solution. Finally, cell resuspensions were analyzed by a flow cytometer .To investigate the amounts of doxorubicin uptake by MHCC97L cells using flow cytometry, cells were seeded in 6-well plates at a concentration of 2 × 10Cells (5000 cells per 1 mL) were cultured in ultra-low attachment 6-well plates in serum-free Dulbecco’s modified Eagle’s medium/F12  supplemented with B-27 , 20 ng/mL epidermal growth factor , 20 ng/mL basic fibroblast growth factor  and 10 ng/ml hepatocyte growth factor . Cells were cultured for more than 7 days and the cells were fed every 3 days.40. The following antibodies were used in western blot analysis: P-gp , Shc3 , GAPDH , β-catenin , Axin , GSK-3β , H-3 , OCT4 , SOX2 .For coimmunoprecipitation, cell lysates were incubated on a rotator with antibody, at 4 °C overnight. The protein antibody protein A/G-dynabead complexes were prepared by adding 30 µL of protein A/G dynabeads , a normal IgG control was assayed simultaneously. After centrifugation to pelletize the dynabeads, the supernatants were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and IB. The immunocomplexes were analyzed by using liquid chromatography-tandem mass spectrometry (LC/MS-MS) or western blot analysis and these are performed as described previouslyg for 10 min. The supernatants were incubated overnight with 30 μL Dynabeads Protein A  precoated with anti-β-catenin , or anti-ubiquitin  antibodies. The immunocomplexes were analyzed by western blots. A normal IgG  control was assayed simultaneously.Cell lysates were prepared by incubating cells in 1% Tris-Triton cell lysis buffer  containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and the protease inhibitor cocktail on ice for 30 min, following which, lysates were centrifuged at 12,000 × Promoter reporters and dual-luciferase assay. MHCC97L cells were transfected with the TOP-FLASH and FOP-FLASH reporter plasmids  together with pRL-TK. Luciferase activity was measured in a 1.5 mL Eppendorf tube with the Promega Dual-Luciferases Reporter Assay kit  according to manufacturer’s protocols after transfection. Relative renilla luciferase activity was normalized to firefly luciferase activity.6 cells for each mouse, n = 3 mice per group) with different treatment. Two weeks later, the mice were treated with doxorubicin (20 μg/g) via intravenous injection every other day for consecutive four times, and the tumor volumes were monitored and recorded every three days. The investigator who monitored the tumor volumes was blinded to group allocation. The tumor volumes were estimated using the following formula: 0.5 × length × width2.Animal care and handling procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, and the animal study protocol was approved by the Institutional Animal Care and Use Committee of Tianjin Medical University. The animals used to test the treatment were 5-week-old female BALB/c athymic nude mice. Briefly, 12 nude mice were randomly allocated to four groups and subcutaneously inoculated with the above MHCC97L/Vector and MHCC97L/Shc3 cells , P-gp  and β-catenin  were used for routine IHC stains on Dako EnVision two-step method in accordance with the manufacturer’s instructions.χ2 test. The two-way ANOVA or Student t-test were used for comparison differences among three groups or between two groups, respectively. All statistical tests were two-sided, and differences were considered statistically significant for P-values less than 0.05. SPSS 22.0 software was used for all data analyzes.Clinicopathologic correlations were analyzed by Pearson’s Supplementary FiguresSupplementary Figure legendsSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4