To understand the differential role of NRP1 in tumorigenesis process, we utilized cells from two different cancer types, pancreatic and lung, each containing either wild type KRAS (KRASwt) or mutant KRAS (KRASmt). Inhibition of NRP1 expression by shRNA in both pancreatic and lung cancer cells containing dominant active KRASmt caused increased cell viability and tumor growth. On the contrary, inhibition of NRP1, in the tumor cells containing KRASwt showed decreased tumor growth. Importantly, concurrent inhibition of KRASmt and NRP1 in the tumor cells reverses the increased viability and leads to tumor inhibition. We found that NRP1 shRNA expressing KRASmt tumor cells caused increased cell viability by decreasing SMAD2 phosphorylation. Our findings demonstrate that the effects of NRP1 knockdown in cancer cells are dependent on the genetic status of KRAS.Neuropilin-1 (NRP1), a non–tyrosine kinase receptor, is overexpressed in many cancers including pancreatic and lung cancers. Inhibition of NRP1 expression, however, has differing pro-tumor  Recent studies have shown that NRP1 is multifunctional and capable of binding multiple growth factors and mediators, including integrins, fibroblast growth factors, transforming growth factor β-1 (TGFβ1) and its receptors, hepatocyte growth factor and its receptor c-Met, and platelet-derived growth factor and its receptors4. These interactions link NRP1 to multiple pathways including tumorigenesis and angiogenesis. A growing collection of literature have shown that increased expression of NRP1 correlates with tumor progression and poor prognosis in hematologic malignancies, such as acute leukemia7 and various solid tumors, including pancreatic cancer8 and lung cancer10. In several cancers like lung cancer11 and renal cancer12 blocking of NRP1 expression has been shown to suppress tumor growth. Conversely, NRP1 overexpression has been shown to promote tumor progression in certain cancers, glioma13, prostate cancer15, and colon cancer16. Thereby, it was assumed that NRP1 might constitute a promising target for cancer therapy.Neuropilin-1 (NRP1) is a non–tyrosine kinase receptor involved in neuronal guidance and angiogenesiset al.17 showed that overexpression of NRP1 in pancreatic cancer could decrease tumor incidence and inhibit tumor growth, whereas reduced expression of NRP1 by small interfering RNA enhanced tumor growth. Therefore, the experimental data on the role of NRP1 in cancer are conflicting, and the molecular mechanisms of differential role of NRP1 in tumorigenesis remain unknown.Interestingly, differing from the established notion that NRP1 promotes tumor growth, Gray KRAS in tumorigenesis. Our results showed that NRP1 has different effects in pancreatic and lung cancers depending on their KRAS genotype. We further established the role of the TGFβ/SMAD pathway in NRP1/KRAS-interdependent signaling. In cancer cells harboring KRASmt, NRP1 down regulation promotes tumorigenesis through the suppression of SMAD2 pathway. These novel findings provide an opportunity to tailor precise, individualized therapies for effective cancer treatment.Here, we would like to elucidate the mechanism through which NRP1 plays opposing roles in oncogenesis and tumor progression. We demonstrated a novel link between NRP1 and the genetic status of KRASmt, PANC-1 and A549 respectively and was decreased in cells with KRASwt, BxPC-3 and H226 respectively -mediated NRP1 knockdown in human pancreatic cancer (PDAC) and non–small cell lung cancer (NSCLC) cell lines. NRP1 knockdown was confirmed by Western blot analysis Fig. . We examely Fig. . A similely Fig. . We alsoin vivo studies, we utilized orthotopic human PDAC and NSCLC tumor xenografts in immunocompromised SCID mice. Tumor volume was monitored by bioluminescence imaging and quantified with imaging software. NRP1 downregulation in the PDAC cell line PANC-1 and NSCLC cell line A549 (KRASmt) resulted in increased tumor growth, whereas NRP1-deficient PDAC BxPC-3 cells and NSCLC H226 cells (KRASwt) exhibited decreased tumor growth as compared to that of parental cells  promotes tumorigenesis, whereas NRP1 down regulation in KRASwt cells  inhibits tumorigenesis  signaling upon TGFβ stimulation to modulate myofibroblast phenotype26. Based on this finding, we investigated whether TGFβ affects NRP1 expression or function by stimulating KRASmt  and KRASwt  cells with TGFβ for 24 hours. The protein expression level of NRP1 decreased after TGFβ treatment in KRASmt cells   thereby contributing to tumor growth in our model.Next, we studied the biological significance of our findings. For our 49) Fig. . We next49) Fig. . The decFurther, as we had earlier seen that in PANC-1 cells with reduced NRP1 expression there is increased phosphorylation of ERK (p44/42) we examined the effect of TGFβ treatment on the phosphorylation of ERK (p44/42). We observed that there was increased ERK (p44/42) phosphorylation in PANC-1 cells with reduced NRP1expression on TGFβ treatment  receptors. Semaphorin (SEMA) 3 F and VEGF165 have been shown to compete for NRP receptors on the cell surface27. By associating with plexins, NRP1 transduces SEMA signaling, whereas it transduces VEGF signals when associated with VEGF receptors. SEMA3B and SEMA3F have been implicated in mediating tumor-suppressing effects, whereas VEGFA has been shown to promote tumor cell proliferation and survival by binding to NRP133. Similarly, NRP1 can associate with GIPC1 and promote tumor progression through the KRAS/ERK signaling pathway34.NRP1 was originally defined as a co-receptor for many growth factors and has been associated with tumors growth and angiogenesis. The role of NRP1 in tumorigenesis is complex and context dependent. Published data indicate that upregulation of NRPs correlates with tumor progression, advanced disease stage, and poor prognosis in various cancers. However, the experimental data investigating the functional role of NRPs in cancer are inconsistent22. We have also previously shown that NRP1 promoted an undifferentiated phenotype in cancer cells12. Later, Gray and colleagues17 demonstrated that NRP1 could suppress tumor growth in the pancreatic adenocarcinoma cell line PANC-1, thereby opposing the existing notion that NRP1 promotes tumors. The relevant literature suggests that these discrepancies may be attributed to tissue and cell type differences or in vitro versus in vivo differences37. We carefully scrutinized these differences and found that the mutation status of KRAS in tumors most likely affects the role of NRP1 in tumorigenicity. In the current study, we demonstrated that the knockdown of NRP1 promotes tumor formation in KRASmt cells  but suppresses tumor growth in KRASwt tumor cells . The knockdown of both KRAS and NRP1 in PANC-1 cell lines (KRASmt) inhibited tumor formation. These data suggest that the KRAS genotype is involved in NRP1modulated tumorigenesis. Of importance, KRAS is mutated in more than 90% of pancreatic cancers and 35% of lung adenocarcinomas38.All of the initially published data examining the role of NRP1 in cancers showed that NRP1 stimulated tumor growth25. The TGFβ pathway is known to induce epithelial-mesenchymal transition, which results in an increased ability to invade and metastasize39. We have previously demonstrated that NRP1 regulates the myofibroblast phenotype through divergent RSMAD signaling26. Supporting our finding, Glinka et al. published NRP1 acts as a TGFβ co-receptor via SMAD2/3 signaling40. Furthermore, other studies have reported the interaction of RAS signaling with the TGFβ family, in which the antitumor effect of TGFβ is altered to pro-tumor signaling via SMAD proteins41. On the basis of these previous findings, we analyzed whether TGFβ and the canonical TGFβ pathway could be involved in KRASmt/NRP1-mediated tumorigenesis. In cells with KRASmt (PANC-1 and A549), NRP1 expression is decreased at the protein level 24 hours after stimulation with TGFβ. The mechanism regulating NRP1 downregulation in different cell types is different. In PANC-1 cells, NRP1 is downregulated at the mRNA level, whereas in A549 cells protein stability may be affected in addition to the decrease in mRNA level.To understand the biological relevance of our studies, we investigated how NRP1 might be downregulated in actual cellular settings. Cancer cells and stellate cells are known to secrete various cytokines including TGFβKRASmt cells decreased the phosphorylation of SMAD2, but there was no change in SMAD2 phosphorylation in KRASwt cancer cells upon NRP1 knockdown. Using PANC-1 cells with confirmed SMAD2 knockdown in orthotopic mice models; we observed an increase in tumors compared with controls. This is in agreement with our in vitro findings that SMAD2 has an antitumor role in our model. In KRASmt cells, NRP1 knockdown results in decreased SMAD2 phosphorylation and enhanced tumor growth. SMAD2 knockdown via shRNA in KRASmt cells also led to increased tumor growth. KRASwt BxPC-3 cells did not survive after shRNA-mediated SMAD2 knockdown.To delineate the pathway involved in NRP1 signaling, we analyzed phosphorylation levels of SMAD2. Down regulation of NRP1 in et al.42 reported that in PDAC cells, oncogenic KRAS/ERK helps in TGFβ-induced downregulation of PTEN at the mRNA level, which in turn could help promote tumor growth. ERK1/2 has been reported to promote pancreatic  cancer43, ovarian cancer44, and prostate cancer45. We found that phosphorylated ERK1/2 was upregulated in PANC-1 cells with reduced NRP1 24 hours after TGFβ stimulation. This result agrees with the literature suggesting that ERK1/2 promotes tumor growth and likely facilitates it in our model, as well.Regarding potential downstream targets, a publication by Chow 47. It has been published that SerpinB1 can promote pancreatic β cell proliferation48. Our results show that Serpine is upregulated at the mRNA levels in PANC-1 cells post 24hrs of TGFβ induction. This data suggests that Serpine might likely help promote tumor growth in KRASmt cells. However, more detailed studies are needed to fully understand the role of Serpine in this cross -talk.Serpine is a SMAD2/TGFβ targetKRAS influences the role of NRP1 in tumorigenesis. We found that in KRASmt cells, NRP1 acts as a tumor suppressor. NRP1 knockdown enhanced cell viability and tumor growth and resulted in decreased SMAD2 phosphorylation. However, in KRASwt cells, NRP1 knockdown suppressed cell viability and tumor growth. Future studies are needed to fully understand the regulatory circuitry involved.In summary, our study provides some insight regarding the pro-tumor and anti-tumor functioning of NRP1 in different cancer types. Our results demonstrate that the genetic status of 49.The human pancreatic ductal adenocarcinoma cell line PANC-1 was maintained in Dulbecco’s Modified Eagle Medium plus 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic , and 0.02% plasmocin . Human pancreatic adenocarcinoma cell lines AsPC-1 and BxPC-3 were maintained in RPMI 1640 medium plus 10% FBS, 1% anti-anti, and 0.02% plasmocin. Lung cancer cell lines A549 and H226 were maintained in RPMI 1640 medium plus 10% FBS, 1% anti-anti, and 0.02% plasmocin. PANC-1 KRAS-inducible cell lines were maintained in 10% tetracycline-free FBS plus 1% anti-anti and 0.02% plasmocin. All cell lines were purchased from American Type Culture Collection. Cells were serum starved for 16 h before TGFβ  treatment. For 3D cell culture, cells were grown in medium containing carboxymethylcellulose at a ratio of 8:2 prepared as previously described50  generously provided us with the shRNA plasmids for KRAS (sh236) and control (shNT). Lentiviruses for NRP1 shRNA and control shRNA were prepared and infected into the target cells as described51. After infection, 2 μg/mL of puromycin was added to the medium for antibiotic selection. For the Tet-On–inducible KRAS shRNA, 0.1 μg/mL of doxycycline was used to induce shRNA expression in the stably infected cells. The sequences of the shRNAs used are listed in Supplementary Table The plasmids for NRP1 shRNA and control shRNA were purchased from Open Biosystems, US. Dr. Francesco Hofmann KRAS sh26 and conCells  were seeded into 96-well plates and cultured for 72 hours in complete medium. After 72 hours, the MTS cell proliferation assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) Promega, Madison WI) was performed for 2D cell culture; the CellTiter-Glo Luminescent Cell Viability Assay  was used for cells grown in 3D cell culture.Western blot antibodies for β-actin, KRAS and horseradish peroxidase–conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Antibodies to NRP1, SMAD2 and p-SMAD2 were purchased from Cell Signaling Technology Inc, Danvers, MA. Ki-67 antibody used for immunohistochemistry was purchased from Santa Cruz, US.Cells were washed 3 times with ice-cold phosphate-buffered saline  and lysed with ice-cold NP-40 lysis buffer  with 1% proteinase inhibitor cocktail  and 1% Halt phosphatase inhibitor cocktail . Cells were incubated on ice for 30 minutes and centrifuged at 14,000 rpm at 4 °C for 10 minutes. The supernatant was collected, and protein concentration was measured by the bicinchoninic acid assay (BCA assay).Proteins were denatured by adding 6× Laemmli SDS sample buffer and heating for 5 minutes. SDS gel electrophoresis was performed with equivalent protein in each lane, followed by wet transfer of the protein to PVDF membrane. The membrane was blocked in TBS-T buffer  containing 5% nonfat milk or BSA. The membrane was incubated overnight at 4 °C with primary antibody diluted in TBS-T containing 5% nonfat milk or BSA, followed by incubation for 1 hour at room temperature with the horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology) diluted in TBS-T. The SuperSignal West Pico Chemiluminescent Substrate  was used for immunodetection.6 luciferase-labeled cells—control shRNA or NRP1 shRNA PANC-1 or BxPC-3 cells, resuspended in 100 μL of PBS—were injected into the pancreas of each mouse. For the lung cancer orthotopic model, the model was established in SCID mice as described before52. Briefly, animals were anesthetized using ketamine and placed in the left lateral decubitus position; 3 × 106 luciferase-labeled cells—control shRNA or NRP1 shRNA A549 or H226 cells in PBS and 10 µL of Matrigel —were injected percutaneously into the right lung of the animals using 1-mL tuberculin syringes. The mice were returned to their cages after confirming they were fully recovered.All animal work was conducted under protocols approved by the Mayo Clinic Institutional Animal Care and Use Committee. All procedures were performed according to the approved guidelines. For the pancreatic cancer orthotopic model, 8-week-old male SCID mice were obtained from the NCI Animal Production Program. Mice were anesthetized, and 1 × 102/steradian and was quantified using the Living Image software  using the IVIS system 200 series (Xenogen Corp). At the end of the treatment, mice were sacrificed, and tumors were harvested for morphologic analysis and immunostaining.Bioluminescent imaging of luciferase-expressing cells was detected in the orthotopic tumor models. Beetle Luciferin (Promega) (substrate for the luciferase-expressing tumor cells) was injected intraperitoneally at 150 mg/kg in PBS 15 minutes before imaging. Mice were anesthetized with 2% isoflurane and imaged once a week with a cooled CCD camera . The exposure time was 1 second to 1 minute. Signal was displayed as photons/second/cmTumors were removed and fixed in neutral buffered 10% formalin at room temperature for 24 hours before being embedded in paraffin and sectioned. Sections were deparaffinized, subjected to immunochemical staining, and imaged with an Olympus BX51 microscope.t test with GraphPad software. Statistical significance was defined as P < 0.05, and a high level of statistical significance were defined as P < 0.01.Statistical analyses were performed with the SPSS 11.0 statistical software . Data are presented as mean (SD). Statistical analysis was performed using 1-way analysis of variance, followed by SNK tests as post hoc testing. The Kruskal-Wallis test was used to evaluate for differences in categorical values, followed by Mann-Whitney U tests as post hoc testing. Analysis of NRP1 mRNA expression was performed using an unpaired Supplementary File


In contrast, β2-AR expression was unaltered among the groups while Gαi expression was significantly higher in the LV post-burn (p = 0.023). B-arrestin-1 and G-protein coupled receptor kinase-2 mRNA expression were significantly increased in the left ventricle post-burn . cAMP production and PKA activity were significantly lower in the LV post-burn . These data indicate that burn injury affects the β-AR signaling pathway in the RV independently of the LV. Additionally, non-canonical β-AR signaling may be activated in the RV as cAMP production and PKA activity were unchanged despite changes in β1-AR protein expression.Burn injury detrimentally affects the myocardium, primarily due to over-activation of β-adrenergic receptors (β-AR). Autopsy reports from our institution reveal that patients often suffer from right ventricle (RV) failure. Since burn injury affects β-AR signaling in the left ventricle (LV), we proposed that β-AR signaling may also be altered in the RV. A rodent model with a scald burn of 60% of the total body surface area was used to test this hypothesis. Ventricles were isolated 7 days post-burn. We examined the expression of β-ARs via Western blotting and the mRNA expression of downstream signaling proteins via qRT-PCR. Cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) activity were measured in membrane and cytosolic fractions, respectively, using enzyme immunoassay kits. β Cardiovascular dysfunction after severe burn injury has been demonstrated clinically in patients as well as in rodent and mammalian burn models. , 2 Howev1- and β2-adrenergic receptor  signaling has been well studied in various non-burned states. Overstimulation of β-ARs by catecholamines results in dysregulation of the associated signaling pathways. Among other things, there can be desensitization of the receptors, altered protein phosphorylation, and activation of alternate signaling pathways. [Circulating catecholamines activate myocardial adrenergic receptors to modulate cardiac function.  Increaseathways. Both right and left ventricles predominately express β-ARs, but α-adrenergic receptors also modulate cardiac function. It has also been hypothesized that the right ventricle is more dependent upon α-adrenergic receptor activity than the left ventricle. The right and left ventricles respond to catecholamine administration in vastly different manners. Irlbeck et al., reported that norepinephrine infusion resulted in altered right ventricular hemodynamics but left ventricular hypertrophy.  The two Guide for the Care and use of Laboratory Animals approved this study’s protocol (Protocol#:0506032). A full-thickness scald was induced in male Sprague-Dawley rats as previously described. [The Institutional Animal Care and Use Committee of University of Texas Medical Branch-Galveston in accordance with the National Institutes of Health scribed. , 11 BrieThe groups for this experiment were control and burn. Control animals received analgesia, anesthesia, and were shaved but did not undergo the burn procedure or resuscitation. We chose 7 days post-burn as the study period because of previously published data showing that there were alterations in β-ARs, cyclic adenosine monophosphate (cAMP) production, and protein kinase A (PKA) activity 1 week after burn injury. 1-AR, β2-AR, β3-AR , Gαs, Gαi, and GAPDH  protein expression was determined via Western Blot analysis. Thirty micrograms of left and right ventricular homogenates were resolved on SDS-PAGE gradient gels  and transferred to polyvinylidine difluoride membranes. Primary antibody incubation occurred at 4°C for a minimum of 16 hours. Blots were then washed with 0.1% TBST, followed by incubation with anti-HRP secondary antibodies  for a minimum of 1 hour at room temperature. Band intensities were visualized using chemiluminescence  and quantified using NIH ImageJ Data Acquisition Software .βcAMP production and PKA activity were measured using commerically available kits from Enzo LifeSciences, Ann Arbor, MI. Aliquots of 30ug of ventricular membrane fraction was used to determine cAMP production as previously described.  ExperimeT method. [Left and right ventricular tissue was homogenized and RNA extracted using Ambion TRIzol Reagent  and the RNeasy Mini Kit . cDNA was generated using 250ng of purified RNA and the iScript cDNA Synthesis Kit . cDNA products were amplified in triplicate using 10ul iTaq Universal SYBR Green Supermix and 400nm primer mix to a total volume of 20ul . Samples method.  Two valipost hoc test (GraphPad Prism 5.0). Data are expressed as means ± standard error of the mean. A p-value <0.05 was considered significant.Data were analyzed with one-way ANOVA followed by Tukey-Kramer’s 1-AR protein expression was significantly higher than expression in the right ventricle (1-AR protein expression was significantly increased (168%) in the right ventricle after burn injury but not in the left ventricle (p = 0.002). While there was a trend for stimulatory G-protein (Gαs) expression to be increased in the right ventricle after burn, this was not significant  was expressed higher in the left ventricles of burned animals  and β-arrestin-1 are two proteins involved in β-AR desensitization to terminate signaling through the receptors in response to increased and/or prolonged stimulation by circulating catecholamines or administration of β-AR agonists. , 16 Simiαi) or stimulated (Gαs). Adenylyl cyclase is the primary producer of cytosolic cAMP, the activator of PKA, a kinase that phosphorylates proteins that regulate cardiac contractility, relaxation, and function. [1-AR and Gαs protein expression in the right ventricle, cAMP production and PKA activity remained unchanged. In contrast, as would be expected with increased expression of Gαi, burn injury significantly reduced left ventricular cAMP production and PKA activity  mRNA and insulin-like growth factor 1 (IGF-1) mRNA expression was significantly decreased.  Addition2-AR mRNA in the left ventricle [In disease conditions, sympathetic nervous system (SNS) tone is increased in response to either reduced cardiac output or contractility. The enhanced SNS tone causes an increased secretion of neurohormones such as norepinephrine and epinephrine. β-AR stimulation initially enhances cardiac contractility but prolonged catecholamine release and subsequent β-AR stimulation triggers a series of adaptive signaling cascades that will eventually produce cardiac dysfunction and, in some cases, heart failure. To date, there are very few mechanistic reports concerning the effect of prolonged catecholamine release in the right ventricle although there have been reports of interventricular differences in the response to β-AR stimulation. Molina et al. have shown that the response to short-term β-AR stimulation in the canine heart is significantly more robust in the right ventricle compared to the left ventricle . Environentricle . However1- and β2-AR) were increased in the right ventricle of patients suffering from tetralogy of Fallot. This was accompanied by increased adenylyl cyclase activity which the authors attributed to β-ARs coupling with Gαs [In heart failure, a disease state characterized by β-AR overstimulation, changes in β-AR density, signaling partners, and the termination of β-AR signaling have been well described , 24–27. with Gαs . However2-ARs can couple with both inhibitory and stimulatory G-proteins and, in the presence of chronic stimulation, it has been shown that β2-ARs begin to preferentially couple with Gαi [αi expression after prolonged catecholamine release [αi inhibits adenylyl cyclase to reduce cAMP production and thus PKA activity. Similar to what has been shown in other disease conditions characterized by chronic stimulation of β-ARs, we observed both increased Gαi expression and reduced cAMP production and PKA activity in the left ventricle after burn injury [3-ARs is not as clearly defined as the other β-ARs. Some have reported increased β3-AR expression after chronic stimulation whereas others and the present study were unable to determine any differences [βwith Gαi , 24, 29. release . Gαi inhn injury , 31. Theferences , 32.Increased GRK2 and β-arrestin gene expression and/or protein expression has been associated with β-AR desensitization and downregulation after chronic β-AR stimulation , 24, 25.A previous study also reported decreased cAMP content as well as PKA activity for up to two weeks after burn injury . These cVEGF mRNA expression can be increased in a β-AR/cAMP/PKA dependent mechanism . Both VEDrake et al. showed that IGF-1 gene expression was significantly increased in right ventricular hypertrophy but not in right ventricular failure . As its Changes in β-AR density, cAMP production, and GRK2 expression occurs in both right and left heart failure. However, the extent of signaling changes in the right ventricle appears to depend upon the cause of failure . TherefoWhile our study did not observe any clear indications of right ventricular or left ventricular failure after burn injury, our data does indicate that burn injury affects the β-AR signaling pathway in the RV independently of the LV. While the LV displayed characteristic perturbations of canonical β-AR signaling expected with enhanced catecholamine release after burn injury, these data indicated non-canonical β-AR signaling may be occurring in the RV as cAMP production and PKA activity were unchanged. Our future directions are to investigate these biventricular differences over a time-course ranging from 24 hours post-injury up to 28 days post-injury.


Exosomes are small vesicles that cells secrete into the extracellular space, and there is increasing evidence that they have pivotal roles in cell-to-cell communication in malignancy. It is reported also that EBV-associated malignant cells, including those derived from nasopharyngeal carcinoma (NPC) and B-cell lymphoma, secrete exosomes. These EBV-related exosomes may contain viral products such as latent membrane protein 1 (LMP1) and may contribute to cancer progression. The aim of this study was to investigate the mechanism by which those viral products are loaded in exosomes. In this study, we show for the first time that ubiquitin C-terminal hydrolase-L1 (UCH-L1) and its C-terminal farnesylation, a posttranslational lipid modification, contribute to this mechanism. Our results also suggest that inhibition of UCH-L1 farnesylation is a potential therapeutic target against cancer metastasis and invasion. Increasing evidence shows that exosomes are key regulators in cancer cell-to-cell communication. Several reports on Epstein-Barr virus (EBV)-related malignancies demonstrate that latent membrane protein 1 (LMP1) secreted by exosomes derived from EBV- or LMP1-positive cells can promote cancer progression and metastasis. However, the mechanism by which LMP1 is loaded into exosomes is still poorly understood. Here, we examined whether the process of LMP1 loading into exosomes is linked to the multifunctional molecule of the ubiquitin system—ubiquitin C-terminal hydrolase-L1 (UCH-L1). For the first time, we demonstrate that LMP1 is physically associated with UCH-L1 and that directing of LMP1 to exosomes is mediated by C-terminal farnesylation of UCH-L1. Additionally, we found that the FTI-277 farnesyltransferase inhibitor reduces motility- and anchorage-independent growth of EBV-positive cells in functional assays. On the basis of our results, we conclude that C-terminal farnesylation of UCH-L1 is one of the key mechanisms by which LMP1 is sorted to exosomes. We hypothesize that inhibition of farnesylation with specific small-molecule inhibitors blocks exosome-mediated transfer of prometastatic molecules such as LMP1 during cancer cell-to-cell communications and thereby impedes the process of cancer invasion.IMPORTANCE Exosomes are small vesicles that cells secrete into the extracellular space, and there is increasing evidence that they have pivotal roles in cell-to-cell communication in malignancy. It is reported also that EBV-associated malignant cells, including those derived from nasopharyngeal carcinoma (NPC) and B-cell lymphoma, secrete exosomes. These EBV-related exosomes may contain viral products such as latent membrane protein 1 (LMP1) and may contribute to cancer progression. The aim of this study was to investigate the mechanism by which those viral products are loaded in exosomes. In this study, we show for the first time that ubiquitin C-terminal hydrolase-L1 (UCH-L1) and its C-terminal farnesylation, a posttranslational lipid modification, contribute to this mechanism. Our results also suggest that inhibition of UCH-L1 farnesylation is a potential therapeutic target against cancer metastasis and invasion. Nasopharyngeal carcinoma (NPC) is a highly metastatic malignancy compared with other head-and-neck carcinomas. In fact, the most common clinical symptom of NPC is a neck mass that results from cervical lymph node metastasis , 2. Worl1–Among them, LMP1 is considered the primary EBV oncogene participating in normal cell transformation as well as in cancer metastatic progression , 4. We h6–Exosomes, which represent the best-explored set of extracellular vesicles derived from multivesicular bodies (MVBs), are membrane vesicles 30 to 180 nm in diameter that participate in the transfer of active transduction molecules  –22 to ne–Recent studies revealed that exosomes are key regulators of cell-to-cell communications in cancer –28. In t–29–However, the mechanism by which cargo molecules are loaded into exosomes is still poorly understood , 37–40. 37–de novo expression has been reported in numerous cancers such as lung cancer  is mainly known as a deubiquitinating enzyme (DUB), although its other activities have also been reported previously –44. Expr–g cancer , 46, colg cancer , bladderg cancer , and breg cancer . Tumor vormation –54. A surmation –, 55–60. Endogenous UCH-L1 can be found in virtually any cell part and organelle not only inside but also outside both normal and transformed cells, including extracellular membrane vesicles. The latest experimental data reported by a number of groups indicate that, as a multifunctional oncogenic molecule of the ubiquitin system, UCH-L1 is involved in the regulation of cellular processes responsible for transport under normal  and pathological (cancer development and progression) conditions. UCH-L1 regulates secretory trafficking pathways in neurons, including those involved in synaptic structures  and neurFarnesylation represents a lipid posttranslational modification that is catalyzed by farnesyltransferase (FTase), which attaches farnesyl to the thiol group of cysteine of the CAAX motif  in the carboxyl terminus of a protein , 67–69. 67–Farnesyl transferase inhibitors (FTIs) belong to a class of experimental cancer drugs that target protein farnesyltransferases . Origina71–Farnesylation is also implicated in UCH-L1 function: membrane-associated UCH-L1 is farnesylated at C220 in the C terminus, and the farnesylated form of UCH-L1 has been shown to promote α-synuclein neurotoxicity . InsteadIn this study, we demonstrated for the first time that C-terminal farnesylation of UCH-L1 is critical for its physical association with the EBV primary oncogene LMP1. Moreover, LMP1 sorting to exosomes depends on C-terminal farnesylation of UCH-L1: point mutation abolishing C-terminal farnesylation of the protein dramatically reduces its binding to LMP1 and transfer of the LMP1/UCH-L1 complex to the exosomes. Additionally, we demonstrate that blocking of farnesylation with FTI-277 inhibitor significantly reduces cell motility and anchorage-independent growth of EBV-positive epithelial cells in functional assays. We hypothesize that the mechanism of LMP1 sorting to exosomes includes UCH-L1 C-terminal farnesylation. Considering the recently established functional role of extracellular vesicles (ECV) in cancer progression, we suggest that specific inhibition of C-terminal farnesylation of UCH-L1 may reduce invasion and metastasis of EBV-associated LMP1-positive metastatic carcinomas.We previously reported that UCH-L1 expression is upregulated during EBV-mediated transformation of B-lymphocytes  and by eNext, we examined the molecular requirements for association between LMP1 and UCH-L1. Since both LMP1  and UCH-Next, we investigated whether specific UCH-L1 C-terminal farnesylation at cysteine 220 affects LMP1/UCH-L1 association. We compared the ability of wild-type UCH-L1, a DUB-dead UCH-L1 mutant (C90S mutant) , and a ng) from conditioned media as described previously  did not result in significant changes of LMP1 levels in exosomal fractions. In contrast, in the protein fractions collected from C220S UCH-L1 mutant-expressing exosomes, LMP1 levels were visibly reduced compared to those from cells transfected with LMP1 alone  cells (293 cells) and EBV-expressing HEK-293 cells (293EBV cells) were used for these experiments. 293EBV cells were a gift from Wolfgang Hammerschmidt  . All celAntibodies were purchased as follows: UCH-L1 (381000) from Thermo Fisher Scientific ; myc (sc-40) and HSC-70 (sc-7298) from Santa Cruz Biotechnology ; Flotillin-2 (610383) from BD Biosciences ; Flag (F3165) and β-actin (A1978) from Sigma-Aldrich ; and GAPDH  from Abnova . Anti-mouse (NA931V) and anti-rabbit (NA934V) secondary antibodies for Western blotting were purchased from GE Healthcare .Chemical agents were purchased as follows: LDN-57444 (L4170) and FTI-277 (F9803) from Sigma-Aldrich .pcDNA3-based LMP1 has been previously described . Wild-tyCells were lysed with buffer containing 1% sodium dodecyl sulfate (SDS) , 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and cOmplete EDTA-free protease inhibitor cocktail . Total cell lysates were denatured in 6× Laemmli’s buffer and boiled for 5 min. Samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes . Membranes were blocked with a mixture of 5% milk with Tris-buffered saline–Tween 20 (TBST) and incubated overnight at 4°C with primary antibodies. Membranes were then washed with TBST and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Membranes were washed again with TBST, and bands were visualized with enhanced chemiluminescence reagent .293 cells were grown in 100-mm-diameter plates and transfected with 3 μg of plasmids with the use of polyethylenimine . Empty vector was used to equalize total amounts of DNA in the transfections.Cells were lysed with buffer containing 1% Triton, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and cOmplete EDTA-free protease inhibitor cocktail . Cell lysates were incubated with EZview Red anti-Flag M2 Affinity Gel  according to the manufacturer’s instructions, and then protein was eluted from anti-Flag beads with 2× Laemmli’s buffer.g for 5 min. To remove large cellular debris, the supernatant fluids were then spun at 2,000 × g for 10 min. Next, larger extracellular vesicles (ectosomes) were collected by centrifugation at 20,000 × g for 60 min. Finally, the exosome fraction was collected by centrifugation at 100,000 × g for 60 min. Exosomes and ectosomes were washed in phosphate-buffered saline (PBS) and pelleted again by centrifugation at the same speed.Exosomes were purified by sequential centrifugation as previously described . In briehttp://rsbweb.nih.gov/ij/), and the healing rate of the wounds was calculated by the following formula: (width after 24 h or 48 h/width at the beginning) × 100%.Confluent cell monolayers incubated with either FTI-277 or control DMSO were scratched with a micropipette tip, and spontaneous cell migration was monitored for 24 h and 48 h. The widths of the “wounds” (scratched areas) were measured by the use of ImageJ . A soft-agar assay was performed by seeding 24,000 cells per well in a layer of 0.4% agar–DMEM–FBS over a layer of 1% agar–DMEM–FBS on 6-well plates. Cultures were maintained at 37°C for 10 days. Colonies were stained by the use of an iodonitrotetrazolium chloride  solution, and then the colonies which had been stained by the agent and which were larger than 200 μm in diameter were counted using light microscopy.t test. All statistical analysis was performed using EZR Software . Statistical significance was analyzed using Student’s Software . A P val


Moreover, AR was borderline significantly associated with poor progress free survival (PFS) after adjustment with other variables . Knockdown of ERα inhibited the proliferation, migration and invasion of GC cells possibly via modulating the expression of p53, p21, p27, cyclin D1 and E-cadherin. Downregulation of AR suppressed the migration and invasion of GC cells and inhibited the epithelial-mesenchymal transition (EMT) associated pathways.Despite the mounting studies exploring the role of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ) and androgen receptor (AR) in gastric cancer (GC), there remain controversies in those findings. The present study investigated the expression of ERα, ERβ and AR in Chinese gastric cancer by immunohistochemistry, analyzed their clinical relevance in gastric cancer, and examined the potential mechanisms by which ERα and AR modulated GC progression. The positive rate of ERα, ERβ and AR in GC tissues was 6% (9/150), 93.5% (143/153), and 42.4% (59/139), respectively. The expression of ERα was an independent unfavorable risk factor for overall survival (OS) (hazard ratio [HR] = 3.639, 95% confidence interval [CI] = 1.432-9.246, The present study showed that positive ERα was associated with poor prognosis of Chinese GC patients. ERα might modulate the proliferation, migration and invasion via regulating the expression of p53, p21, p27, cyclin D1 and E-cadherin. ERα could be a valuable prognostic biomarker and promising therapeutic target for Chinese GC patients. Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer-related death in the world . A largeIn the past decades, hormonal therapy has been well established in the treatment of hormone-dependent tumors such as prostate cancer and breast cancer , 4. HoweThe relationship between hormone receptors and GC was first reported by Tokunaga et al . Since tThe positive rate of ERα, ERβ and AR in GC tissues was 6.0% 9/150), 93.5% (143/153), and 42.4% (59/139), respectively . Moreover, higher AR expression was observed in patients with better differentiated tumors (p=0.009).Clinicopathological characteristics including patient sex, age, whether received adjuvant chemotherapy, tumor grade, vascular and nerve invasion, TNM classification and stage of the patients enrolled in this study were listed in Table p=0.032; Figure p=0.103; Figure p=0.167; Figure p=0.462; Figure p=0.05; Figure p=0.025; Figure The median follow-up time for the patients was 48.5 months (range 24.7-70.1 months), and nine patients who failed to contact through telephone or email were lost to follow up. Kaplan-Meier survival curves for ERα, ERβ and AR expression were shown in Figure Moreover, univariate analysis of OS demonstrated in Table We next investigated the effects of ERα on the malignant behavior of GC cells. We examined the expression of ERα in GC cell lines, and found that ERα was expressed in low levels in GC cells compared with the normal gastric epithelial cell line (GES-1), which was in accordance with the low positive rate of ERα in GC tissues examined by immunohistochemistry. MKN45 and SNU601 cells with ERα silenced were established by lentivirus infection, as the two cell lines showed relative high basal levels of ERα expression Figure . DownregTo further explore the mechanisms in the decreased proliferation, migration and invasion by ERα knockdown, proliferation associated molecules  as well as the important molecular in adherens junctions of epithelial cells (E-cadherin) were examined by western blot Figure . The expAlthough the present study found that AR was only borderline significantly associated with poor PFS, considering studies on the expression and function of AR in GC was insufficient, we also investigated the effects of AR on the malignant behavior of GC cells. AR was silenced in MGC803 and SGC7901 cells which showed relative high basal levels of AR expression by lentivirus infection . No signin vitro via regulating the expression of p53, p21, p27, cyclin D1 and E-cadherin. And AR suppressed the migration and invasion of GC cells in vitro possibly through inhibiting EMT process.The present study demonstrated 6% (9/150) of GC tissues expressed ERα, 93.5% (143/153) expressed ERβ, and 42.4% (59/139) were AR positive. The correlation coefficients among ERα, ERβ and AR were too small (r < 0.4), indicating that the correlation was too weak to have any clinical significance in GC. The positive expression of ERα was an independent prognostic factor for OS, and AR was borderline associated with PFS for GC patients after adjustment with other variables. In addition, downregulation of ERα might suppress the proliferation, migration and invasion ERα expression patterns varied in different GC patients, for example, the data from TCGA database  showed that among 205 stomach adenocarcinoma samples, 5 samples were detected with ERα amplification, 1 with deep ERα deletion, and 6 with ERα mRNA upregulation. In light of this, high-quality clinical trials that examine the effect of anti-ER therapy on the treatment of ER positive GC patients are needed, and anti-ER therapy might be only effective on ER positive individuals.Treatment of ER positive breast cancer patients with ER antagonist has achieved great success . Howeverp21 [Limited researches have explored the effect of ERα and the underlying mechanisms in GC. Several previous experimental studies has demonstrated that ERα could promote the proliferation of GC cells possibly by interacting with hedgehog pathway , c-Src pp21 . P21 andp21 . E-cadhep21 . Taken tin vitro experiment, and revealed that downregulation of AR suppressed the migratory and invasive ability of GC cells, possibly via inhibition of EMT associated molecules including α-SMA, β-catenin, snail, and slug. In fact, Kominea et al firstly reported that AR was an unfavorable prognostic factor for the OS of GC patients [Since the prognostic role of AR in GC and the potential mechanisms are insufficient, even though the association of AR with PFS only reached borderline significance in the present study, we performed patients . Later Zpatients . Taken tIn summary, the present study showed that positive expression of ERα was significantly associated and positive expression of AR had a tendency to associate with poor prognosis of Chinese GC patients. In addition, knockdown of ERα suppressed the proliferation, migration and invasion of GC cells probably via modulating the expression of p53, p21, p27, cyclin D1 and E-cadherin. And downregulation of AR suppressed the migration and invasion of GC cells possibly via inhibiting EMT process. The results suggested that ERα and AR might serve as prognostic biomarkers and therapeutic targets to improve the survival of Chinese GC patients. In the next period of time, precise approaches are needed to identify the expression patterns of ERα and AR in GC patients, and clinical trials involving highly selected ERα and AR positive patients are required to illustrate the potential of anti-ERα and anti-AR therapy in GC.Formalin-fixed and paraffin-embedded tumor tissues were collected from 155 patients with gastric carcinoma who underwent surgical resection at the Department of Gastric Cancer and Soft Tissue Sarcomas, Shanghai Cancer Center of Fudan University, Shanghai, China, from Oct 2007 to Jan 2010. The samples were used for tissue microarray construction and immunohistochemistry. The patients were followed up every 4 months until death or the end of the study , except those lost to follow up. Overall survival (OS) was defined as the interval between the date of surgery and the date of death or last follow-up visit. Progress free survival (PFS) was defined as the time from the date of surgery to the time of disease progression, death, or last follow-up visit if the disease did still not progress.Informed consents were obtained from all patients, and the research was approved by the Clinical Research Ethics Committee of Fudan University Shanghai Cancer Center, and complied with the principles of the Helsinki Accord.ERα, ERβ and AR expression was detected by immunohistochemistry using UltraSensitive™ SP kit  according to the manufacturer's instructions. In brief, the sections were deparaffinized, rehydrated and subjected to antigen retrieval . The sections were then incubated overnight at 4°C with the primary mouse monoclonal antibodies to ERα , ERβ  and AR , respectively. The sections were subsequently washed and incubated with a secondary antibody. Reaction products were visualized with 3, 3’diaminobenzidine tetrahydrochloride and counterstained with hematoxylin and eosin.Cases displaying brown cytoplasmic and/or nuclear stainings were regarded as positive. The staining intensity was graded by the Allred score system . A scoreNine human GC cell lines , one normal gastric epithelial cell line (GES-1) and HEK293T cell line were obtained from the Cell Resource Center, Shanghai Institute of Biochemistry and Cell Bank at the Chinese Academy of Sciences. All cell lines were routinely authenticated by DNA-fingerprinting and isoenzyme analysis and free of contamination by mycoplasma. GC cell lines were cultured in RPMI 1640, MEM, or DMEM containing 10% fetal bovine serum at 37 °C under humidified atmosphere with 5% CO2.® HD Transfection Reagent (Promega) to generate lentivirus. MKN45, SNU601, MGC803 and SGC7901 cells were infected with the recombinant lentivirus with 10μg/mL Polybrene® .Lentiviral vector expressing shRNA targeting ERα was purchased from Obio Tech , and lentiviral vector expressing shRNA targeting AR was purchased from Genechem . Those shRNAs were transfected into HEK293T cells using FuGeneMKN45 and SNU601 cells were plated at a density of 2000 cells per well in 96-well plates. MGC803 and SGC7901 cells were plated at a density of 1000 cells per well in 96-well plates. Cell proliferation was determined with Cell Counting Kit 8  for 5 days.in vitro were carried out in chambers of 8-μm transwell inserts  in the presence or absence of Matrigel . Eighty thousand MKN45 and SNU601 cells, 40,000 MGC803 cells and 60,000 SGC7901 cells were seeded in the top chamber in serum-free medium, while medium containing 20% serum was in the lower chamber as the attractant. After 24-48 hours, migrated cells were fixed with 4% paraformaldehydeand then stained with 0.1% crystal violet. The number of migrated cells was counted using an IX71 inverted microscope .Migration and invasion assays Cells were lysed and the protein concentration was measured using the bicinchoninic acid protein assay kit . The lysates were subjected to SDS-PAGE gel and transferred to a PVDF membrane . The membrane were blocked with 5% non-fat milk and incubated with indicated primary antibodies, and then probed with the horseradish peroxidase–conjugated secondary antibody. The primary antibodies against ERα was purchased from Abcam. The primary antibodies against p53 and p27 were purchased from Santa Cruz Biotechnology. The primary antibodies against p21 were purchased from Abways Technology. The primary antibodies against AR, snail, slug, α-SMA, and β-catenin were purchased from Cell Signaling Technology. The antibody against GAPDH was purchased from Proteintech Group. The blots were visualized with Pierce™ enhanced chemiluminescence reagents (Life Technologies).p<0.05) variables remained in multivariate survival analysis. The data of functional experiments were expressed as the mean ± standard deviation from three independent experiments, and analyzed using Student's t-test. All statistical analyses were performed by the SPSS 19.0 for windows . A two-tailed p< 0.05 was considered statistically significant.The associations of ERα, ERβ and AR expression with clinicopathological characteristics were evaluated by Chi-square test or Fisher's exact test. Survival data were analyzed using Kaplan-Meier method (log-rank test). Cox proportional hazards was used for univariate and multivariate survival analysis, clinical factors considered relevant for the prognosis including age, adjuvant therapy, TNM classification and stage according to the REMARK criteria , as well


Androgen receptor (AR) is widely expressed in breast cancer (BC) but its role in estrogen receptor (ER)-positive tumors is still controversial. The AR/ER ratio has been reported to impact prognosis and response to antiestrogen endocrine therapy (ET).We assessed whether AR in primary tumors and/or matched metastases is a predictor of efficacy of first-line ET in advanced BC. Patients who had received first-line ET (2002–2011) were recruited, while those given concomitant chemotherapy or trastuzumab or pretreated with > 2 lines of chemotherapy were excluded. ER, progesterone receptor (PgR), Ki67 and AR expression were assessed by immunohistochemistry, and HER2 mainly by fluorescent in-situ hybridization. Cut-offs of 1 and 10% immunostained cells were used to categorize AR expression.p = 0.028). AR status in primary tumors or metastases was not associated with progressive disease (PD) as best response. In contrast, Ki67 ≥ 20% and PgR < 10% showed a statistically significant association with PD as best response.Among 102 evaluable patients, biomarkers were assessed in primary tumors in 70 cases and in metastases in 49, with 17 patients having both determinations. The overall concordance rate between primary tumors and metastases was 64.7% (95% CI 42%-87.4%) for AR status. AR status did not affect TTP significantly, whereas PgR and Ki67 status did. AR/PgR ≥0.96 was associated with a significantly shorter TTP (HR = 1.65, 95% CI 1.05-2.61, AR expression does not appear to be useful to predict the efficacy of ET in advanced BC, whereas Ki67 and PgR exert a greater impact on its efficacy. Androgen receptor (AR) is emerging as an important mediator in the biology and therapy of breast cancer (BC). It has a role in breast carcinogenesis, varying on the basis of the expression of other biomarkers, such as estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2/neu), and a prognostic effect has also been observed .AR is a nuclear transcription factor, which is activated by the binding of steroid hormone androgens, similarly to other members of this family of receptors including ER and PgR .AR protein is expressed in about 70–90% of BC. Its levels may however vary depending on the location considered (cytoplasmic and/or nuclear), the cutoff of expression , and the antibody used for immunohistochemistry (IHC) , 4.AR expression in different BC subtypes is also variable. It is found in about 75% of ER-positive, 50–60% of HER2-enriched, and 20-40% of triple negative BC, respectively [Depending on the expression of other hormone receptor proteins and their ligands, the AR pathway may promote or inhibit cell proliferation. Moreover, this interplay between AR, ER and their ligands is complicated by the possible conversion of androgens to estrogens. A cross talk between AR pathway and HER2/neu pathway is also known , 8.AR and ER can compete for the binding to estrogen response elements (EREs) on specific genes . So the Moreover a high AR expression can activate the epidermal growth factor receptor (EGFR), promoting an agonist effect of tamoxifen on ER pathway, and this aberrant mechanism could be blocked by enzalutamide +/− gefitinib .Currently AR is under investigation in the clinical setting as a therapeutic target in BC. Enzalutamide and other anti-androgens appear suitable treatment options for AR-expressing BC , 14.A favorable prognostic role of AR in BC was identified in various studies. This effect was confirmed, in terms of both disease-free survival (DFS) and overall survival (OS), in a large meta-analysis including almost 8000 patients with stage I-III BC. In other studies AR expression independently predicted longer BC specific survival and lower risk of recurrence , 15, 16.We previously showed the value of PgR and Ki67 to predict benefit from first-line endocrine therapy in a case series of patients with advanced luminal breast cancer . In thisA retrospective analysis was performed for this study. Patients with locally advanced or metastatic BC histologically confirmed treated with first-line ET from 2002 to 2011 at Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, IRST , were included. Patients who received concomitant chemotherapy or trastuzumab or pretreated with more than 2 lines of chemotherapy were excluded. All patients were aged ≥18 years and were followed up for a period ranging from 5 to 13 years. We collected data from medical records of these patients. Subsequently we gathered tumor tissue samples of these patients for AR assessment.The original hematoxylin-eosin stained sections were reviewed by two pathologists responsible for selecting pathological inclusions representative of tumor tissue and for the analysis of clinical pathological features, such as histologic grade.The predictive value of AR expression, alone or in relation to the other conventional biomarkers, to select the patients responsive to first-line endocrine therapy was the primary endpoint.IRST and AVR (Area Vasta Romagna) Ethics Committee , reviewed and approved the study and informed consent was obtained from all patients.Data were collected from electronic databases. The following characteristics were evaluated for each single patient.Patient’s data: age at diagnosis. Tumor’s data: 1) histotype ; 2) tumor classification at diagnosis  according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual 7th Edition; 3) nodal classification ; 4) presence of metastases at diagnosis (M0-1); 5) ER, PgR, Ki67, HER2 status in primary tumor and metastasis. Treatments and outcomes: 1) adjuvant chemotherapy; 2) adjuvant ET; 3) type of first-line ET; 4) best objective response (complete response (CR), partial response (PR), stable disease (SD), PD, according to RECIST criteria); 5) time-to-progression (TTP), meant as the time in months from the beginning of first-line ET until progression or last tumor response evaluation available.All datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Hormone receptors, Ki67, HER2 and AR status of the primary tumor, metastasis or both were assessed for all patients enrolled. The major part (79%) of the biomarkers detection was performed in the Pathology Unit of Morgagni Pierantoni Hospital in Forlì .Tumor samples obtained during surgery were fixed in neutral buffered formalin and embedded in paraffin. Four-micron sections were mounted on positive-charged slides for each patient . REporting recommendations for tumour MARKer prognostic studies (REMARK) and the European Quality Assurance guidelines were used. Immunostaining for conventional biomarkers and AR expression was performed using the Ventana Benchmark XT staining system  with the Optiview DAB Detection Kit . ER, PgR, Ki67 , HER2  and AR  antibodies were used. For ER, PgR, Ki67 and HER2 detection, tissue sections were incubated for 60 min with antibodies diluted 1:80, 1:40, 1:100 and 1:350, respectively, in antibody diluent . The AR antibody was pre-diluted by the supplier. Sections were incubated for 16 min and automatically counterstained with hematoxylin II . Biomarker positivity was detected and semiquantitatively quantified as the percentage between immunopositive tumor cells and the total number of tumor cells. Two independent observers evaluated all the samples and any disagreement was resolved by consensus after joint review using a multihead microscope. Hormone receptors, Ki67 and HER2 biomarkers were classified on the basis of the St. Gallen and ASCO-CAP guidelines –20. In pPatients and tumor features were described according to descriptive statistics.Positive or negative AR status in both tumor and metastasis was used to define the concordance between tumor and metastasis, while discordance was defined as positivity at one site and negativity at the other one. The concordance rate was calculated as the proportion of concordant cases with respect to the total number of patients. The two-sided exact binomial 95% confidence interval (95% CI) was estimated.AR expression was analyzed in relation to the other conventional biomarkers , best response to therapy , and time to progression (TTP) (months). Kaplan-Meier method was used to estimate TTP and compared with the logrank test. Hazard ratios (HR) and their 95% CI were calculated using the Cox regression model.The Chi Square test was used to evaluate the association between categorical variables and best response and the non-parametric ranking test (Wilcoxon test) was used to evaluate the relationship between median value of AR expression and Ki67 classes .To determine the optimal cutoff values of AR/ER and AR/PgR ratios at a median TTP time Receiver operating characteristic (ROC) curves were used.p values < 0.05.Analyses were performed using SAS Statistical software . Statistical tests were significant for One hundred and two patients were evaluable according to the selection criteria and the availability of tissue samples. Median age was 60 years (range 33-85). Seventy eight percent of them were diagnosed as ductal and 14% as lobular histotype. Metastatic disease at diagnosis was found in 26 out of 102 (25.5%). Ninety two percent were treated with an aromatase inhibitor as first-line endocrine therapy, and letrozole was the most frequent aromatase inhibitor administered (45%). For detailed description of patients’ characteristics see Table Biomarker’s expression  was assessed in primary tumors in 70 cases and in metastases in 49, with 17 patients having both assessments Fig.  (Table 2ER was negative (< 1%) in 3 samples (4.3%) of primary tumors and in none of the metastatic samples. PgR was negative (< 1%) in 10 (14.3%) primary tumors and in 12 (24.5%) metastases. Ki67 was low (< 20%) in 59.1% of primary tumors and in 61.2% of metastases. HER2 status was positive in about 15% of cases both in primary tumors and metastases. In Fig. p = 0.017).AR status Fig. , consideAR status in primary tumors or metastases was not associated with PD as best response Table .Table 3Rp = 0.62); clinical benefit was 90.7% and PD 9.3% for AR-positive tumors defined by a cutoff ≥10%, versus 79% and 21%, respectively, for AR-negative tumors (p = 0.18). Conversely Ki67 showed a significant association with PD as best response, with 26.9% PD with high Ki67 versus 4.3% PD with low Ki67 (p = 0.009). PgR-negative status defined by a cutoff < 1% showed just a trend in terms of association with PD as best response (p = 0.079), while significant results (p = 0.031) were obtained by using ≤10% PgR as cut off value.Clinical benefit rate, defined as CR or PR or SD, was 88.5% and PD 11.5% for AR-positive tumors defined by a cutoff ≥1%, versus 83.3% and 16.7%, respectively, for AR-negative tumors (p = 0.884); similarly for AR ≥10% median TTP was 16 months (95% CI 13.0–19.0) vs 13.8 months (95% CI 11.0–42.1) for AR < 10% (p = 0.935). AR/PgR ≥0.96 was associated with a significantly shorter TTP  . Differences in TTP according to AR status were not statistically significant (Table  95% CI 1.0–19.0 vMost of ER-positive BCs are AR-positive (about 80-90% of them) and the coexpression of AR, ER and PgR is associated with a better prognosis and well-differentiated phenotype –23.Some studies suggest that the levels of expression of AR and its relation to ER expression levels in primary tumors predict benefit from adjuvant endocrine therapy with tamoxifen .This study was not powered to determine whether AR expression could predict response to tamoxifen or fulvestrant, as the majority of patients received aromatase inhibitors and very few patients received those therapies.Nevertheless our findings suggest that AR expression does not predict the efficacy of first-line endocrine treatment (mainly based on aromatase inhibitors in our study) in ER- or PgR-positive advanced BC, both in term of TTP and PD as best response.These results might be influenced by the limited amount of AR negative cases in this subtype of BC. Moreover, the stronger predictive effect of PgR and Ki67 could make the role of AR expression less evident, as suggested by the predictive significance of the AR/PgR ratio.Cochrane et al. . evaluatMoreover we evaluated AR/PgR ratio and we found a potential predictive value of this parameter at the cut-off of 0.96. This finding could be explained by the stronger predictive value of PgR alone in comparison with AR alone, as PgR < 10% and Ki67 ≥ 20% showed a significant association with PD as best response, and PgR ≤1% and Ki67 ≥ 20% were significantly associated with shorter TTP.PgR and Ki67 seem to be useful to select patients with a higher probability of being responsive to first-line endocrine therapy for metastatic BC, whereas AR expression does not appear to be useful. The AR expression could acquire more relevance when anti-androgen therapy will be available for BC patients.


Compensatory mechanisms leading to increased androgen receptor expression and activity after androgen ablation or anti-androgen treatment have been identified in prostate cancer. After hydroxyflutamide and bicalutamide were used in therapy of prostate cancer over many years, novel anti-androgen enzalutamide showed improved clinical activity. However, enzalutamide resistance develops over a certain time period, and molecular mechanisms responsible for this process are heterogeneous.As with other anti-androgens, these mechanisms include alterations of AR but also may be associated with overexpression of oncogenes which should be targeted by novel therapies. Androgen receptor splice variants have been frequently described in patients who developed enzalutamide resistance. Mutant AR F876L has been detected in patients who are resistant to enzalutamide. Glucocorticoid receptor overexpression has been observed in patient tissues and in pre-clinical models of enzalutamide resistance.There is a heterogeneous picture of enzalutamide resistance in prostate cancer and, therefore, the development of appropriate post-enzalutamide treatment remains a challenge. The role of androgen receptor (AR) in castration therapy-resistant prostate cancer is a subject of intensive investigation in urological oncology. It is well-known that the expression of AR may critically influence cancer progression. Androgen ablation therapy and blockade of AR are considered palliative treatments for non-organ confined prostate cancer. Anti-androgens hydroxyflutamide and bicalutamide have been used over many years in prostate cancer treatment, and the studies on resistance mechanisms were particularly focused on AR mutations which emerge during endocrine therapy with these compounds. Alterations in expression and function of several AR coactivators are also relevant for acquisition of resistance to anti-androgens in prostate cancer. The development of novel anti-androgenic compounds resulted in a worldwide use of the non-steroidal anti-androgen enzalutamide, a drug that binds to the ligand-binding domain of the AR thus preventing nuclear transactivation and expression of target genes. Several clinical studies with enzalutamide revealed that the use of this anti-androgen leads to improved survival of prostate cancer patients . HoweverConstitutively active ARs are of special interest in castration therapy-resistant prostate cancer. These receptors lack ligand-binding domain and exhibit transcriptional activity in the absence of androgenic hormones. Li and associates • demonstInhibition of variant AR V7 could be achieved with niclosamide •. NiclosIn case of hydroxyflutamide and bicalutamide treatment, several mutations have been discovered during chronic treatment with the drugs , 11. In Importantly, glucocorticoid receptor expression is increased in cells which become resistant to enzalutamide therapy . AdaptivThere are several other interactions between molecules involved in regulation of chemosensitivity and androgen signaling pathway in enzalutamide resistance. Twist is a transcription factor that is implicated in regulation of epithelial to mesenchymal transition and docetaxel resistance. Twist itself is up-regulated during androgen ablation therapy. Inhibition of Twist signaling was found to be associated with enhanced efficacy of enzalutamide therapy . This coExpression of several cytokines in normal and malignant tissue is under control of nuclear factor kappa B. It is particularly important that interleukins (IL) are involved in regulation of many functions in prostate cancer, and IL-6 is highly expressed in castration-resistant tumors. IL-6 increases transcriptional activity of the AR in a ligand-independent and synergistic manner with low concentration of androgens. Experimental therapies against IL-6 have been established; however, clinical trials were performed in patients with late stage prostate cancer and were not successful , 24. NucThere may be different approaches on how to develop post-enzalutamide therapies. An important issue that should be critically discussed is administration of novel anti-androgens. Although important, the duration of post-enzalutamide response in such a case will be rather limited. The AR itself may be a primary target of therapy of enzalutamide-resistant cancers. The use of novel AR antisense oligonucleotides targeting exon 1 and exon 8 has revealed that there is a therapeutic potential to target resistant tumors . This exThus, compound 30, developed by optimizing receptor-binding efficiency of aryloxy tertramethylcyclobutyl lead compounds identified in a high throughput cellular screen, was found to suppress growth of castration (LNCaP C4-2)- and enzalutamide-resistant cells in vitro and in vivo . The mecAnother approach to reverse enzalutamide responsiveness is targeting autophagy. Nguyen and associates showed that treatment of cells with enzalutamide induced autophagy in prostate cellular models . AutophaA common feature of resistance to several types of androgen deprivation is up-regulation of activity of Akt. Enzalutamide treatment may be therefore enhanced if an inhibitor of the phosphotidylinositol 3 kinase-Akt pathway AZD5363 is combined with enzalutamide . This coFurther studies may be focused on the role of a potential target in immunotherapy, PD-L1 in enzalutamide resistance. PD-L1 is expressed at a high level in enzalutamide-resistant prostate tumors , 42. XenBromodomain (BET) inhibitors target bromodomain-containing proteins BRD/2/3/4 and BRDT. They enhance efficacy of therapy and disrupt resistance to enzalutamide in prostate cancer treatment as evidenced in the LNCaP and VCaP models . MultivaReplacement of a single anti-androgen with another one is a task which could in most clinical cases lead to a temporary improvement of clinical symptoms and biochemical status. This approach could be in part useful in patients who failed enzalutamide treatment. As elaborated in this review, several mechanisms related to changes in AR itself have to be considered in patients with enzalutamide resistance. Most importantly, AR mutation(s) certainly contribute to drug resistance. However, identification of novel targets will likely lead to therapies which are not solely limited to AR inhibition. Similarly as with other mechanisms related to anti-androgen resistance, development of enzalutamide resistance seems to be very heterogeneous. This factor strongly limits durable success in therapy for castration therapy-resistant prostate carcinoma.