New therapeutic targets for oral squamous cell carcinoma (OSCC) are urgently needed. We conducted genome-wide CRISPR-Cas9 screens in 21 OSCC cell lines, primarily derived from Asians, to identify genetic vulnerabilities that can be explored as therapeutic targets. We identify known and novel fitness genes and demonstrate that many previously identified OSCC-related cancer genes are non-essential and could have limited therapeutic value, while other fitness genes warrant further investigation for their potential as therapeutic targets. We validate a distinctive dependency on YAP1 and WWTR1 of the Hippo pathway, where the lost-of-fitness effect of one paralog can be compensated only in a subset of lines. We also discover that OSCCs with WWTR1 dependency signature are significantly associated with biomarkers of favorable response toward immunotherapy. In summary, we have delineated the genetic vulnerabilities of OSCC, enabling the prioritization of therapeutic targets for further exploration, including the targeting of YAP1 and WWTR1. Many types of cancer now have 'targeted treatments', which specifically home in on genes cancer cells rely on for survival. But there are very few of these treatments available for the most common type of mouth cancer, oral squamous cell carcinoma, which around 350,000 people are diagnosed with each year.Designing targeted treatments relies on detailed knowledge of the genetic makeup of the cancer cells. But, little is known about which genes drive oral squamous cell carcinoma, especially among patients living in Asia, which is where over half of yearly cases are diagnosed. One way to resolve this is to use gene editing technology to find the genes that the cancer cells need to survive.Now, Chai et al. have used a gene editing tool known as CRISPR to examine 21 cell lines from patients diagnosed with oral squamous cell carcinoma. Most of these lines were from Asian patients, some of whom had a history of chewing betel quid which increases the risk of mouth cancer. By individually inactivating genes in these cell lines one by one, Chai et al. were able to identify 918 genes linked to the survival of the cancer cells. Some of these genes have already been associated with the spread of other types of cancer, whereas others are completely unique to oral squamous cell carcinoma. The screen also discovered that some cell lines could not survive without genes involved in a signalling pathway called Hippo, which is known to contribute to the progression of many other types of cancer.Uncovering the genes associated with oral squamous cell carcinoma opens the way for the development of new targeted treatments. Targeted therapies already exist for some of the genes identified in this study, and it may be possible to repurpose them as a treatment for this widespread mouth cancer. But, given that different cell lines relied on different genes to survive, the next step will be to identify which genes to inactivate in each patient. Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous tumor arising from the mucosal surfaces lining the upper aerodigestive tract. The commonest subtype, oral squamous cell carcinoma (OSCC) is especially prevalent among Asian countries . OSCC haGenomic sequencing technology has enabled the delineation of the comprehensive mutational and transcriptomic landscape of cancers, including OSCC . HoweverThe Cancer Dependency Map project (a consortium effort by the Wellcome Sanger Institute and the Broad Institute) have conducted CRISPR-Cas9 screen on a large number of cell lines including some OSCC models . HoweverYAP1) and WW domain-containing transcription regulator protein 1 (WWTR1) and revealed mutually exclusive dependency and compensable functions of these paralogs in different subsets of OSCC models. We identified OSCC tumors with a gene expression signature similar to cell lines with validated dependencies. Among which, OSCC resembling the WWTR1-dependent cell lines showed significant enrichment of immune-related pathways and are associated with biomarkers of response towards checkpoint inhibitors. In summary, our study demonstrated the robustness of genome-wide CRISPR-Cas9 screen in identifying genetic vulnerabilities in diverse OSCC models, offering new molecular insights into this disease.To identify genetic vulnerabilities in OSCC, we performed genome-wide CRISPR-Cas9 screens on 21 highly annotated OSCC cell lines, most of which are unique models derived from Asian patients . Our stuIn order to identify genetic vulnerabilities of OSCC, particularly those that are more relevant to tumors of Asian origin, we conducted genome-wide CRISPR-Cas9 knockout screens . We screFitness genes were identified after an unsupervised computational correction with CRISPRcleanR , followeAs we aimed to identify genetic vulnerabilities of OSCC that can be safely targeted therapeutically, we filtered the significantly depleted genes to exclude previously defined core fitness genes , that wePathway enrichment analyses on the 918 context-specific fitness genes was conducted on each individual cell line . ConsistPIK3CA and CDK6 are already in clinical trials for HNSCC treatment . We also examined the dependency profile of 44 cancer genes with driver mutations known to be associated with HNSCC (PIK3CA were observed in four OSCC cell lines with a hotspot mutation in E545K (BICR10), Q546R (ORL-150) and H1047R (HSC-2), and to a lesser extent, in ORL-115 with H1047L mutation. Intriguingly, HSC-4, which harbors the same E545K mutation as BICR10, did not show any dependency on the mutated PIK3CA, this is consistent with findings from Project Score  a negative regulator of NFE2L2. The R320Q mutation has been reported to stabilize NRF2 (encoded by NFE2L2) and enhances cell fitness as reported previously in lung cancer (HRAS (G12C). Another gene, encoding ZFP36L1 with truncating mutation at S324 in ORL-48 also showed preferential dependency, suggesting that the effect of this mutation should be studied further.Next, we sought to determine which components of the common oncogenic pathways altered in HNSCC  were reqth HNSCC  and founth HNSCC . Based oth HNSCC . Dependect Score . A splict cancer . Dependeferation . On the g cancer . FinallyNFKB2, TNFAIP3, CSNK2A1, and TRIM25. When cross checking with the DepMap and Project Score data, three out of four (75%) of these genes  were not found among the screened OSCC models, which were mostly derived from Caucasians, or from Asians not known to chew betel quid. Interestingly, our findings are coherent with previous studies from Taiwan and India, where betel quid chewing is common, which have demonstrated that extract from the areca nut of betel quid can directly activate the NF-kB signaling pathway, favoring OSCC cells survival . The only transcription factor that falls within tractability group one is the ESR2, with several antagonists available owing to its well-studied ligands and structure (KLF4 and KLF5) and zinc finger proteins (ZNF148 and ZNF236)  .YAP1 or WWTR1 amplifications occur in approximately 19% of HNSCC , which puts it among the top five cancers with the highest amplification of these genes amongst 33 cancer types  are transcription co-activators, which are the major effectors of the Hippo pathway . YAP1 anYAP1 or WWTR1 (WWTR1 gene locus (3q25) is at close proximity to the locus of PIK3CA, SOX2 and TP63 at 3q26-28, whereby their focal amplification is frequently reported in HNSCC  among cell lines that are neither dependent on YAP1 or WWTR1, whereby five out of six such lines have PIK3CA hotspot mutations   .Intriguingly, mutually exclusive copy number gains of chromosome 3q and 11q22 (where YAP1 is mapped to) have been reported in squamous cell carcinoma  and consYAP1 and WWTR1 is associated with the dependency, we examined baseline mRNA and protein expression of YAP1 and WWTR1 in representative OSCC lines -tagged YAP1 sgRNA and mCherry-tagged WWTR1 sgRNA . The co-PIK3CA mutation, and that alterations in the PI3K signaling pathway have been linked to multiple metabolic dysregulations in cancer  analysis based on the OSCC cell lines with validated dependency . Using tn cancer .H17 cell differentiation , we examined the changes in gene expression level of PD-L1 upon depletion of WWTR1 and other co-amplified genes. qPCR results revealed strongest suppression of PD-L1 gene expression upon WWTR1 knockout, but remain largely unchanged when the other genes were knockout   . We alsond PD-L1 . TogethePIK3CA and HRAS and even in these, mutations are only found in a small subset of patients. Therefore, determining the oncogenic pathways and specific therapeutic targets have not been straight forward for this disease. We performed genome-wide CRISPR-Cas9 screens in a unique collection of OSCC cell lines derived from patients with diverse risk habits to identify genetic vulnerabilities that can serve as a basis for further therapeutic development for OSCC. Adding to existing genomics datasets in OSCC, this approach identifies fitness genes required for the survival of cancer cells where targeting these will result in the killing of these cells. The majority of the cell lines used in this study were from Asian patients where OSCC is more prevalent, and where causative factors include betel quid chewing in addition to smoking that is more often found in Caucasian patients . Notably, we showed that about 5% (45/918) of these genes are highly tractable with approved drugs, or have drugs that are in late-stage of clinical testing, demonstrating that these screens could help to prioritize drugs that could be repurposed for OSCC treatment. We compared our screens with previous genome-wide RNAi screens and found that candidate genes such as those relating to the cell cycle  were also identified in our dataset  did not seem to be fitness genes in the other cell lines not associated with betel quid chewing, nor those from the Project Score or DepMap , most other driver mutations did not confer preferential gene dependency and their value as a drug target remains unclear.Driver mutations are often expected to be robust biomarkers in precision medicine. However, upon analysing the dependency profiles on cancer genes that are commonly mutated in HNSCC and those with driver mutations among our 21 OSCC cell lines, we show that with the exception of some genes with driver mutations leading to oncogene addiction  are common events reported in OSCC, often in mutual exclusive manner  were derived spontaneously from surgically resected OSCC tissue specimens in Cancer Research Malaysia. Briefly, tissues were collected in α-MEM containing 20% (v/v) FBS, 200 iu/l penicillin, 200 μg/ml streptomycin and 0.1 μg/ml of fungizone. Subsequently, tissues were washed in absolute ethanol for 20–30 s and then washed twice with phosphate-buffered saline (PBS) under sterile conditions. Tissues were minced, washed twice in culture media and re-suspended in α-MEM containing 20% (v/v) FBS, 200 iu/l penicillin, 200 μg/ml streptomycin, 0.4 ng/ml EGF, 2 μg/ml hydrocortisone and 2 mM L-glutamine, and seeded into tissue culture dishes. Cultures were continuously maintained for more than 100 population doublings . HSC-2, 2, transfection complex medium was replaced with fresh DMEM high glucose (Gibco) complete medium. Medium containing virus was collected at 48- and 72 hr post-transfection and filtered using PVDF 0.45 μm syringe filter.HEK293 cells were transfected using jetPRIME transfection reagent (Polyplus Transfection) according to the manufacturer’s instructions. Briefly, transfection complex consisting of jetPRIME buffer, jetPRIME reagent, vector of interest, pMD2.G and psPAX2 were prepared and mixed with Opti-MEM (Gibco). Next, the transfection complex medium was added to HEK293 cells with 90% confluency. After overnight incubation at 37°C, 5% COTo perform virus transduction, selected cell lines were transduced with lentivirus containing the vector of interest, in the presence of 8 μg/ml polybrene. After overnight incubation, medium containing lentivirus was replaced with fresh DMEM/F12 complete medium. Cells were incubated for 48 hr and harvested to evaluate the transduction efficiency via flow cytometry analysis with BD LSR Fortessa X-20 cell analyser (BD Biosciences). Gating strategy for flow cytometry analysis of transduced cells carrying fluorescence marker is exemplified in Selected cell lines were transduced with lentivirus containing the pKLV2-EF1α-Cas9Bsd-W . Cells stably expressing Cas9 enzyme was established via blasticidin selection 3 days post-transduction. Cas9 enzyme cutting efficiency was routinely checked via lentivirus transduction of the reporter plasmid pKLV2-U6gRNA5(gGFP)-PGKmCherry2AGFP-W . The efficiency of Cas9 cutting activity was accessed using a reporter plasmid and analyzed using flow cytometry analysis. The screening will only be conducted on those cell lines with >80% Cas9 cutting activity, as indicated by the efficiency of GFP knockout in cell lines transduced with the reporter plasmid.The Human Improved Genome-wide Knockout CRISPR Library v1  containing 90,709 gRNAs targeting a total number of 18,010 protein-coding genes was used in the genome-wide CRISPR-Cas9 screening . For eacGenomic DNA was extracted from 60 million post-CRISPR screened cells using QIAGEN blood and cell culture DNA Maxi kit (Qiagen), according to manufacturer’s instruction. Extracted DNA was quantified using Qubit 2.0 fluorometer (Thermo Fisher Scientific).4. For the CRISPR library v1 plasmid, 10 independent PCR reactions were set up using 2.0 μg of the plasmid. While for the CRISPR-screen cell lines, 2.0 μg of genomic DNA harvested from day-18 post-transduction was used in each of the 36 independent PCR reactions. The PCR conditions were as follows: 98°C for 30 s, 26–28 cycles of 98°C for 10 s, 61°C for 15 s and 72°C for 20 s, and the final extension, 72°C for 2 min. The PCR products were analyzed on 2% agarose gel and additional PCR cycles were added if necessary.To prepare and generate Illumina libraries for deep sequencing, amplification of sgRNA was performed using Q5 Hot Start High-Fidelity 2 × Master Mix and forward/reverse primers pair (gLibrary-HiSeq_50bp-SE-U1 F and R) in 50 μl reaction, as previously describedAbout 5 μl of PCR products were pooled from all 36 reactions and QIAquick PCR purification kit (QIAGEN) was used to purify the amplified gRNA. Concentration of purified PCR products were quantified with Qubit dsDNA broad-range (BR) assay kit (Thermo Fisher Scientific), using the Qubit 2.0 fluorometer (Thermo Fisher Scientific). PCR enrichment was then carried out using 200 pg of purified PCR products with 2x KAPA HotStart ReadyMix. 1 μl of forward P5 fusion primer (PE 1.0 p5 Top_PE_C) and 1 μl of different reverse primers (indexed iPCRTags) were used. The PCR conditions were as follows: 98°C for 30 s, 10–12 cycles of 98°C for 10 s, 66°C for 15 s and 72°C for 20 s, and the final extension, 72°C for 5 min. Finally, SPRISelect beads (Beckman Coulter) were used to purify the PCR products at a PCR-product-to-bead ratio of 1:0.8. Purified libraries were dissolved in 30 μl nuclease-free water and quantified using Agilent High Sensitivity DNA kit (Agilent Technologies) on Agilent 2100 Bioanalyzer (Agilent Technologies).Purified libraries of the triplicate screens tagged with different iPCR tags were pooled and sequenced at about 300x coverage on Illumina HiSeq 2500 with 19 bp single-end (SE) deep sequencing at the Wellcome Sanger Institute (WSI). About 30–40 million reads were obtained for each of the three replicates. Sequences of the Read one sequencing primer (U6-Illumina-seq2) and index sequencing primer can be found in https://github.com/francescojm/CRISPRcleanR) and used for pre-processing of the sgRNA raw counts  <0.5).Next, in order to identify and prioritize genes that can be safely targeted for the treatment of OSCC, we curated a list of core fitness essential genes from four different sources and used it to further filter the list of MAGeCK hits from each cell line. The first two sources were the core essential genes (CEG) list, published by All 21 OSCC cell lines were subjected to WES at the WSI using HiSeq2500. WES data were processed using an established pipeline as previously described to identify driver mutations . A totalhttp://ConsensusPathDB.org)  . A thresOSCC models with known betel quid chewing as the only risk habit were included in this analysis – , to compare with the other OSCCs not associated with betel quid chewing . Venn diagram was used to depict the number of unique and overlapping fitness genes that are found between those OSCCs with or without association with betel quid chewing.http://www.pantherdb.org/)  .https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). List of sgRNA used and their sequences can be found in BbsI enzyme (NEB R0539S) and the concentration was adjusted to 20 ng/μL. Target oligos were phosphorylated and annealed using T4 PNK (NEB M0201). The thermocycler condition used are as follows: 37°C for 30 min, 95°C for 5 min, followed by a ramp down to 25°C at 0.1°C /s. The annealed oligos were next diluted twice for prior to ligation: 1st dilution = 139 μL EB buffer + 2 μL of 10 μM double-stranded oligos; 2nd dilution = 57 μL EB buffer + 3 μL of 1st dilution. Following that, overnight ligation at 4°C was carried out using T4 ligase (NEB M0202S) and 10X ligase buffer (NEB M0202S). The ligation products were then transformed into DH5α chemically- generated competent cells and plated onto Luria Broth (LB) agar plates containing 100 μg/ml Ampicillin. The plasmids were then extracted using QIAprep Spin Miniprep Kit (QIAGEN) and the sgRNA sequences were verified by Sanger sequencing prior to use.To validate the results obtained from the screen, individual targeted genetic knockouts were generated using CRISPR/Cas9 and plasmid expressing sgRNAs targeting the gene of interest. Two sgRNA sequences were used for each target gene, one was selected from the CRISPR library v1 while another sequence was designed using the Genetic Perturbation Platform (GPP) web portal (YAP1 and WWTR1), two different sgRNA were used, one from the Kosuke Yusa’s CRISPR Library v1  and another independently designed sgRNA using Broad’s sgRNA-designer tool . sgRNA targeting a core fitness gene, Polo-like kinase 1 (PLK1) was included as a positive control, choline acetyltransferase (CHAT)-targeting sgRNA was used as a non-fitness gene negative control and a non-targeting (NT) sgRNA w also included.The relative growth rate of sgRNA-transduced and non-transduced cells was compared using co-competiton assay, as described previously . BrieflyTo achieve double gene knockout, sgRNAs targeting two different genes were cloned into either one of the plasmids tagged with BFP or mCherry (pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W or pKLV2-U6gRNA5(BbsI)-PGKpuro2AmCherry-W). Thereafter, the changes in the BFP- and mCherry double-positive cell population was measured as mentioned above.3 dish and cultured until they reached 70–80% confluency. To assess the differential expression of the protein of interest after gene knockdown, Cas9-expressing cells were transduced with the target sgRNA at above 90% transduction efficiency. Next day, transduced cells were selected using 2 μg/ml of Puromycin. Day four post-transduction, percentage of BFP-expressing cells were determined using flow cytometry and total cell lysates (TCL) were extracted with RIPA buffer  NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 150 mM NaCl) supplemented with Halt Protease and Phosphatase Inhibitor (PI) Cocktail (Pierce Biotechnology) on ice. TCL was collected by centrifugation and quantified using the BCA method (Thermo Fisher Scientific). About 20 µg of the TCL was resolved on SDS-PAGE gel and proteins were transferred onto PVDF membranes (Millipore). Membranes were blocked with 5% (w/v) milk in TBST (0.1% (v/v) Tween 20) and probed with primary antibodies (1:1000 dilution in 1% (w/v) BSA) overnight at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies  were probed for one hour at room temperature. For signal development, WesternBright Quantum HRP substrate (Advansta Inc) was used and visualized using the FluorChem HD2 imaging systems (Alpha Innotech). To normalize for loading, the blots were re-probed with an anti-tubulin monoclonal antibody (1:1000 dilution in 1% BSA) and processed as described above. List of primary and secondary antibodies used is found in To determine the baseline protein expression level, OSCC parental cell lines were seeded in 100 mmCas9-expressing cells were transduced with selected sgRNA to achieve single gene or double gene knockout, as described above. On day 4 post-transduction, total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific). Total RNA (1 μg) was used for reverse transcription to complementary DNA (cDNA) using high-capacity cDNA reverse transcription kit (Applied Biosystems). Real-time quantitative PCR was performed using 1 μl of 5x diluted cDNA with PowerUp SYBR Green Master Mix and corresponding primers in 7500 Real-Time PCR System (Applied Biosystem). All reactions were performed in technical triplicates and repeated twice. Cycling conditions used are as follows: 50°C for 2 min, 95°C for 2 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. A default melt curve stage was included to allow inspection of primer specificity. Ribosomal protein L13 (RPL13) was used as an endogenous reference control for normalization. Sequences of all primers used can be found in On 4- or 6 days post-transduction of sgRNA-containing lentiviruses, 2000 cells were seeded into six-well plate. After a week, cells were fixed using ice-cold methanol followed by staining with crystal violet solution.The 3-−2,5-diphenyltetrazolium bromide (MTT) assay was used to access the effect of target gene knockdown/knockout on cell viability. Briefly, 4- or 6 days post-transduction with sgRNA, 2000 cells were seeded in triplicates in 96-well plate. 72 hr later, 50 μl MTT was added to each well and incubated for 4 hr at 37°C. After removing the media, 150 μl dimethyl sulfoxide was added to dissolve the formazan crystal and optical density was measured using Synergy H1M microplate reader  at 570 nm.On 4- or 6 days post-transduction of sgRNA-containing lentiviruses, 30,000 cells were seeded into 24-well plate and harvested after 72 hr. Cells were pelleted down and washed with PBS. For detection of apoptotic cells, the cell pellet was resuspended in 1x Annexin V buffer containing 2.5 μl of Annexin V solution (BD Biosciences) and 2.5 μl of propidium iodide and incubated for 15 min in the dark. The proportion of apoptotic cells were analyzed using the LSR Fortessa X-20 cell analyser (BD Biosciences) and FlowJo , considering all single- and double-stained cells as apoptotic cells. Gating strategy for detection of apoptotic cells using flow cytometry can be found in https://www.cbioportal.org/)  on the iRAP-processed, ComBat corrected FPKM matrix for these nine cell lines. Non-overlapping DEGs with significant p-value threshold <0.01 and log fold change >2 were retained. The final list of DEGs is found in al.org/) . Gene exFor all DEGs, Z-score was computed and a ‘dependency signature score’/ ‘compensable signature score’ was generated for each cell line/tumor sample, taking the difference between the average of all Z-score of upregulated DEGs and that of downregulated DEGs. For example, ‘YAP1-dependency signature score’ =  - .https://software.broadinstitute.org/morpheus) using the ‘YAP1 dependency signature score’, ‘WWTR1 dependency signature score’ and ‘Compensable signature score’.Subsequently, cell lines and OSCC tumors were analyzed using hierarchical clustering and visualize with heatmap    with thern.org/) .cor.test function in Rstudio  was used.All statistical significance analyses were performed using unpaired parametric two-tailed t-test in GraphPad Prism  unless otherwise stated. Unpaired t-test with Welch’s correction (Welch’s t-test) was used for all analyses in No unreported or custom code was used in this study. Open source softwares were used for data analysis and codes are available upon request. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This work defines genes required for survival and robust growth by oral squamous cell carcinoma (OSCC) cells in Western and Asian populations. Following a genome-wide CRISPR screen, the authors identified components of the Hippo/Yap pathway as important for growth of multiple OSCC cell lines. In dissecting the pathway, the authors determined that some OSCC cell lines depend more on YAP1, some more on WWTR1/TAZ, while some depend on both, suggesting more heterogeneity in response than was previously appreciated. Importantly, the work also suggests some specific features of transcriptional pathways, including activation of the NF-κB pathway, in OSCC derived from Asians with a habit of betel quid chewing. These different dependencies may have clinical consequences, as the data suggests that OSCC cells specifically dependent on WWTR1 are associated with a distinct transcriptome and immune signature associated with response to immunotherapy.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Päivi Ojala as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Marius Sudol (Reviewer #2).Thank you for submitting your article "Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:This manuscript by Annie Chai and colleagues from the laboratory of Sok Ching Cheong reports in the CRISPR/Cas9-based screen of cells derived from oral squamous cell carcinoma (OSCC), which were predominantly of Asian, Malaysian origin. The screen was aimed at the identification of "fitness genes". Several of these genes, some known and some new, were given non-essential status and a new set of essential genes was identified as worthwhile targets of a potential therapy. The authors identified components of the Hippo/Yap pathway as important for growth of multiple OSCC cell lines. For more than a decade, the two main pro-oncogene effectors of the Hippo pathway, namely YAP and TAZ, have been considered acting in unison in cancers. The current report indicates that YAP and TAZ may play specific roles in cancer. In dissecting the pathway, the authors determined that some OSCC cell lines depend more on YAP1, some more on WWTR1/TAZ, and in some either protein can complement the function of the other, suggesting more heterogeneity in response than was previously appreciated. The copy number amplification of WWTR1/TAZ was shown as likely to constitute a functional oncogenic role of this gene and its protein in OSCC. However, non-genomic mechanisms (with some exceptions) could likely activate YAP. These different dependencies may have clinical consequences, as the data suggests that OSCC cells specifically dependent on WWTR1 appear to be associated with a distinct transcriptome and immune signature associated with response to immunotherapy. Given the considerable interest in therapeutically targeting immune pathways, these findings could provide a valuable response biomarker, as indicating that two Hippo pathway endpoints that are often grouped together have quite distinct roles. The results also could guide the use of therapies under development for targeting YAP1 or WWTR1/TAZ. For example, another of the important conclusions of this study is that TAZ-dependent cell lines may represent fiduciaries of their original tumors, which could be primary candidates for checkpoint inhibitors treatment via modern immunotherapy.There are some points that should be addressed by the authors to solidify the conclusions and improve the study.Essential revisions:(1) The authors emphasize that a rationale for their study is the use of cell materials developed from Asian populations with different risk factors, such as use of betel. They hypothesize that these features of the OSCC cell set may result in different dependency profiles than those already published for OSCC from the DepMap project . However, they do not explicitly compare results with those from the DepMap project. This analysis should be added, to determine if the hypothesized difference exists.(2) A lot of the analysis pathway appears to be derivative of approaches from Behan et al., 2019. This includes data exclusion for core fitness genes, assignment of tractability groups, and other points. The authors need to make clear the novelty of the current work.(3) From data shown in Figure 1C, most of the cell lines have a limited number of non-core fitness genes (40 or less), whereas 4 lines have a very large number of dependencies. What genomic features characterize those with large numbers of dependencies? If these are excluded, how does this affect the statistical analysis present in Figure 2? Similarly, 10 of the 21 cell lines appeared to have <10 core dependencies. Do these models specifically have unique genomic features, and YAP1 or WWTR1 dependency?(4) In Figure 3—figure supplement 3, the authors note correlation between WWTR1 gene essentiality and expression across 273 cell models. What are the cancer types that are most dependent on WWTR1 ?(5) In Figure 6, the authors analyze the YAP1 and WWTR1 gene signatures across a large group of OSCCs in the TCGA. Earlier in the study, the authors note that WWTR1 is often amplified as part of an amplicon including PIK3CA, TP63, and SOX2. To what extent is the transcriptional signature associated with amplification of these other genes, and dependent on their transcriptional activity, rather than that of WWTR1? Using their cell line models, they should complement TCGA analysis by using shRNA or drug inhibition to deplete WWTR1 versus other key genes in the amplicon, to determine which causes a specific loss of the immune-associated transcriptional signature. This is a really important data point for interpretation of the results.+ T cell inflamed phenotype, Martin et al., 2018, dissecting the Hippo pathway and demonstrating the targetability of YAP1 in head and neck cancer in a detailed Nat Comm paper, and an extensive review of the pathway at the end of 2019 by Santos-de-Frutos and colleagues. This work should be cited and discussed by the authors. It is important that the Discussion addresses more about how the generated data relate to previously published reports. For example, the fact that YAP, TAZ, PI3K, TP63, and SOX2 are amplified in OSCC is curious as all these genes are part of the extended signaling network of the Hippo pathway. Please see the examples of relevant references and consider a short paragraph discussing this point.  Also when the role of TA in eliciting the immune response is mentioned, it would be important to reference one of the first publications (as far as the reviewer is aware of) that indicated the role of TAZ  via LATS1 KO in mice. The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity. Moroishi T, (many authors) Guan KL. Cell. 2016 Dec 1;167(6):1525-1539.(6) Besides papers cited by the authors, work relevant to the topic of this study includes Omori et al., 2020, for YAP1 as a driver, Saloura et al., 2019, showing YAP1 mutation is associated with a low CD8(7) The authors should specifically state how many of their cell lines were highly dependent on WWTR1, how many on YAP1, and how many on both, out of the group of 21.(8) The cell lines used in the screen are critical for the study. Therefore, it is suggested to provide in the Materials and methods and/or in the Results more information about how these lines were derived. It would help the readers a lot without referring to a previous publication  and various commercial data for cell repositories. Please address if these cell lines were derived spontaneously  or were engineered by transfections of immortalizing genes . If the latter was the case, please comment if the process of immortalization did not affect the actual transcriptional profile of the lines.(9) As stated above, one of the critical conclusions this study is that TAZ-dependent OSCC lines may represent fiduciary (direct references) of their original tumors, and therefore patients with tumors driven by TAZ could be primary candidates for checkpoint inhibitors treatment via immunotherapy. Please address directly in the Discussion the fact that most of the tumors are quite heterogeneous in terms of oncogenic drivers and either the tumor biopsy or the cell lines derived from the tumor might not represent the "main" oncogenic drivers of the entire tumor. Essential revisions:(1) The authors emphasize that a rationale for their study is the use of cell materials developed from Asian populations with different risk factors, such as use of betel. They hypothesize that these features of the OSCC cell set may result in different dependency profiles than those already published for OSCC from the DepMap project . However, they do not explicitly compare results with those from the DepMap project. This analysis should be added, to determine if the hypothesized difference exists.We thank the reviewers for the suggestion. We have added the analysis of enriched dependencies seen among betel quid-associated OSCC in the revised manuscript and discussed the results accordingly. In particular, by comparing the gene dependencies identified from betel quid-associated OSCC lines with those non-betel quid associated OSCCs, we found that the NF-κB pathway is significantly enriched in the betel-quid associated lines. When cross checking the fitness genes in this pathway with those from the DepMap project, where models not known to be associated with betel quid were used, all except one were not identified as fitness genes . Notably, the literature that reported on betel quid as a risk factor in OSCC have demonstrated that aberrant NF-κB pathway activation is common in these cancers. Furthermore, extracts from betel quid have been shown to directly activate NF-κB signaling in OSCC as reported in previous studies. By contrast, this is rarely reported among the OSCC studies from the Western countries, where betel quid is uncommon.We have added the results of this analysis to Figure 2 , and explained this in the Results section (subsection “Identification of unique dependencies in betel-quid associated OSCC”). We also discussed our findings in the Discussion section (third paragraph). The description of the analysis has been added to the Materials and methods section of the revised manuscript . Figure legends and figure source data have also been included accordingly.2) A lot of the analysis pathway appears to be derivative of approaches from Behan et al., 2019. This includes data exclusion for core fitness genes, assignment of tractability groups, and other points. The authors need to make clear the novelty of the current work.The publication from Behan et al. is one of the most comprehensive essential screens that was conducted across many types of cancers (pan-cancer) and in their project, they have developed comprehensive and robust methods in filtering and analyzing data from such screens particularly in identifying tractable genes for further therapeutic development. As one of our aims in conducting the essential screens for OSCC was to identify gene targets that could be translated to clinical benefit through the development or identification of new therapies, we applied similar filtering criteria to our data including removing the core-fitness genes :1515-26. doi: 10.1016/j.cell.2015.11.015), and assigning tractability groups as described by Behan et al. Whilst we may have adopted a similar approach, our work is novel in that (i) our study focuses on OSCC and it would be the first to describe non-core fitness genes for OSCC and it is also the largest dataset of dependencies in OSCC models; (ii) we included models derived from Asians (particularly those with betel-quid chewing habit) where the disease is most prevalent and describe possible distinctiveness in the non-core fitness genes in models that are representative of OSCC in Asians; (iii) we looked specifically at known oncogenes and recurrently amplified regions to interrogate the function of OSCC cancer genes, and (iv) we report distinct dependencies on YAP1 and WWTR1 among the different subsets of OSCC where these dependencies could have implications in cancer therapies including immunotherapy.3) From data shown in Figure 1C, most of the cell lines have a limited number of non-core fitness genes (40 or less), whereas 4 lines have a very large number of dependencies. What genomic features characterize those with large numbers of dependencies? If these are excluded, how does this affect the statistical analysis present in Figure 2? Similarly, 10 of the 21 cell lines appeared to have <10 core dependencies. Do these models specifically have unique genomic features, and YAP1 or WWTR1 dependency?NELFCD) is a fitness gene for all the 21 cell lines (top most bar). This demonstrates the heterogeneity of the essentiality across the cell lines. To make this clearer, we have changed the y-axis label to “Number of dependent cell lines” and added more description in the figure legend.We would like to clarify that the y-axis on Figure 1C refers to the number of dependent cell lines; while x-axis refers to the number of non-core fitness genes. For example, there are 366 genes that are essential in one out of 21 cell line (bottom most bar) – indicating fitness genes unique to a single cell line. In other words, we found 366 unique genes that is essential in only one cell line. On the other hand, only one gene (4) In Figure 3—figure supplement 3, the authors note correlation between WWTR1 gene essentiality and expression across 273 cell models. What are the cancer types that are most dependent on WWTR1 ?We observed the correlation of WWTR1 gene dependency with its gene expression in several cancer types including squamous cell lung carcinoma, breast carcinoma, glioblastoma, ovarian carcinoma and low-grade glioma in addition to OSCC. Therefore, this observation is not entirely unique to OSCC or squamous cell carcinoma. We have added this information on other cancers in Figure 3—figure supplement 3D and included the results in the fourth paragraph of the subsection “Differential dependency pattern on YAP1 and WWTR1”.5) In Figure 6, the authors analyze the YAP1 and WWTR1 gene signatures across a large group of OSCCs in the TCGA. Earlier in the study, the authors note that WWTR1 is often amplified as part of an amplicon including PIK3CA, TP63, and SOX2. To what extent is the transcriptional signature associated with amplification of these other genes, and dependent on their transcriptional activity, rather than that of WWTR1? Using their cell line models, they should complement TCGA analysis by using shRNA or drug inhibition to deplete WWTR1 versus other key genes in the amplicon, to determine which causes a specific loss of the immune-associated transcriptional signature. This is a really important data point for interpretation of the results.We thank the reviewer for the suggestion. We agree that it would be important to show that the correlation between WWTR1-dependency gene signatures with the immune signatures are indeed specific to WWTR1, and not due to other co-amplified genes such as PIK3CA, TP63 and SOX2. We did not previously include this analysis as using cell line model, most of the immune-associated transcriptional signature might not be directly measurable. Nevertheless, we have now taken two approaches to address this question as best as we could. First, we used a WWTR1-dependent model, ORL-214 to knockout WWTR1, PIK3CA, TP63 and SOX2 using two individual sgRNAs for each gene, and examined the down-regulation effect on PD-L1 gene expression. As we anticipated, only WWTR1-knock-out (KO) cells showed significant down-regulation of PD-L1 expression, but not in PIK3CA-, TP63-, nor SOX2-KO cells. These results are now reported in the subsection “ OSCC with WWTR1 dependency signature and immune biomarkers” and Figure 6—figure supplement 3A.In addition, we also examined the correlation between the expression of PIK3CA, TP63 and SOX2 with PD-L1 expression, using an OSCC microarray dataset from Hiemer et al., 2015. Consistently, only WWTR1 gene expression showed significant correlation with PD-L1 gene expression. This correlation was not observed between PIK3CA, TP63, or SOX2 with PD-L1. These results are now reported in the aforementioned subsection and Figure 6—figure supplement 3B.+ T cell inflamed phenotype, Martin et al., 2018, dissecting the Hippo pathway and demonstrating the targetability of YAP1 in head and neck cancer in a detailed Nat Comm paper, and an extensive review of the pathway at the end of 2019 by Santos-de-Frutos and colleagues. This work should be cited and discussed by the authors. It is important that the Discussion addresses more about how the generated data relate to previously published reports. For example, the fact that YAP, TAZ, PI3K, TP63, and SOX2 are amplified in OSCC is curious as all these genes are part of the extended signaling network of the Hippo pathway. Please see the examples of relevant references and consider a short paragraph discussing this point.  Also when the role of TA in eliciting the immune response is mentioned, it would be important to reference one of the first publications (as far as the reviewer is aware of) that indicated the role of TAZ  via LATS1 KO in mice. The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity. Moroishi T, (many authors) Guan KL. Cell. 2016 Dec 1;167(6):1525-1539.6) Besides papers cited by the authors, work relevant to the topic of this study includes Omori et al., 2020, for YAP1 as a driver, Saloura et al., 2019, showing YAP1 mutation is associated with a low CD8We thank the reviewer for the suggestion to discuss our findings in the context of the highlighted literature. We have now elaborated on the relatedness of our findings with these literature and proposed opportunities for further studies that will address current knowledge gaps. These are now included in the Discussion of the revised manuscript. In addition to the suggested literature, we have also discussed and cited two additional papers that are relevant including Saladi et al., 2017, and Garcia-Escudero et al., 2018.7) The authors should specifically state how many of their cell lines were highly dependent on WWTR1, how many on YAP1, and how many on both, out of the group of 21.We thank the reviewer for this suggestion. Seven of the lines are highly dependent on YAP1, four are highly dependent on WWTR1, two are dependent on both YAP1 and WWTR1, while the rest did not show dependency on either YAP1 or WWTR1. This information is depicted in the bottom color-coded bar of the Figure 3A, where the dependency of the cell line on YAP1, WWTR1 and PIK3CA were determined from the extent of the genes depletion in the CRISPR screen, at a false-discovery rate of 5%, as defined using MAGECK analysis pipeline. We have now added this information in the second paragraph of the subsection “Differential dependency pattern on YAP1 and WWTR1”. We have also revised the figure legend to add clarity.8) The cell lines used in the screen are critical for the study. Therefore, it is suggested to provide in the Materials and methods and/or in the Results more information about how these lines were derived. It would help the readers a lot without referring to a previous publication  and various commercial data for cell repositories. Please address if these cell lines were derived spontaneously  or were engineered by transfections of immortalizing genes . If the latter was the case, please comment if the process of immortalization did not affect the actual transcriptional profile of the lines.As requested by the reviewer, we have now included information on how the lines were derived under the heading “Cell lines” in the Materials and methods section.9) As stated above, one of the critical conclusions this study is that TAZ-dependent OSCC lines may represent fiduciary (direct references) of their original tumors, and therefore patients with tumors driven by TAZ could be primary candidates for checkpoint inhibitors treatment via immunotherapy. Please address directly in the Discussion the fact that most of the tumors are quite heterogeneous in terms of oncogenic drivers and either the tumor biopsy or the cell lines derived from the tumor might not represent the "main" oncogenic drivers of the entire tumor.We agree with the reviewer and understand the importance of highlighting the assumption and limitation in our claims. We have now addressed in the Discussion section (seventh paragraph).


Hepatoma cell lines are widely used to model the hepatocyte for insulin signaling and fatty liver disease. However, a direct comparison of insulin action in primary hepatocytes and in hepatoma cell lines is needed to validate this model and to better understand liver cancer. Here we have investigated insulin signaling, gluconeogenic gene expression, glucose production, and fatty acid synthase abundance in primary hepatocytes and in HepG2, Hepa 1–6, and McARH7777 hepatoma cell lines. Differences in the electrophoretic profiles of protein extracts from human and mouse primary hepatocytes and the hepatoma cells lines are shown. Compared to primary hepatocytes, hepatoma cells showed high basal phosphorylation of AKT at Thr 308 and constitutively activated RAS-MAPK signaling, which were resistant to the dominant negative Ras mutant H-Ras17N. Hepatoma cell lines also showed defective expression of gluconeogenic enzymes, insulin unresponsive GSK phosphorylation, and marginal glucose production. Hepatoma cells also showed lower protein levels of fatty acid synthase and a largely distinct protein electrophoresis profile from hepatocytes but similar between different hepatoma lines. We conclude that hepatoma cell lines do not accurately model the hepatocyte for insulin action but may be valuable tools to investigate the proteomic changes conferring to hepatocellular carcinoma its peculiar metabolisms. Furthermore, fatty liver disease is a condition closely associated with obesity and insulin resistance, which at advanced stage progresses to cirrhosis and hepatocellular carcinoma, and it was predicted that fatty liver disease will be the first cause for liver transplantation in the near future2. The liver is a major insulin target organ, is the main source of endogenous glucose production, plays a chief role in the control of systemic lipid metabolism, and is central to the link between obesity and type-2 diabetes5. Hence, identifying the molecular mechanisms linking obesity to the pathogenesis of hepatic insulin resistance and the progression of fatty liver disease is a major challenge of modern biomedical research.Obesity and type-2 diabetes have reached unprecedented proportions and may be the largest pandemic in the history of humanity6. However, recent studies indicate that direct insulin action on the hepatocyte plays a dominant role in the control of glucose metabolism8. A better understanding of the direct insulin action on hepatocyte metabolism in physiological conditions and in obesity is therefore necessary to unravel the link between obesity, insulin resistance, and fatty liver disease. This research field to progress needs a solid cell culture model to investigate and define the molecular mechanisms of insulin action in the hepatocyte and its role in metabolic homeostasis and disease progression. Cultures of hepatoma-derived cell lines display typical morphological features of hepatocytes, express specific hepatocyte markers and therefore can be seen as a practical and ethical alternative to primary hepatocyte cell cultures. Indeed, preparation of primary hepatocytes requires animals and is more technically demanding and labor intensive than immortalized cell lines. Furthermore, the availability of hepatoma cell lines of human origin, such as HepG2, could also be considered a significant advantage, as human primary hepatocytes have a limited availability at a prohibitive cost. It is therefore not surprising that thousands of studies used hepatoma cell lines, most commonly HepG2 cells, to model hepatocytes in insulin signaling or in metabolism. However, whereas HepG2 proteome was shown to be qualitatively similar to the one of human primary hepatocytes9, principal component analysis of these proteomes could distinguish between these cell-types, indicating significant quantitative differences10. Most importantly, insulin action in hepatoma cell lines remains largely uncharacterized. To our knowledge, only one recent study has directly compared insulin action in HepG2 and other immortalized hepatocyte cell lines, to the one in primary mouse hepatocytes, and several metabolic differences between these cell types were found11. However, the authors could not measure insulin-induced phosphorylation of insulin-receptor and AKT in HepG2 cells, probably because of technical difficulties, and have not evaluated MAPK signaling, which complicates the interpretation of this study. Furthermore, another recent study compared HepG2 cells with two lines of immortalized hepatocytes for insulin action and hepatokine gene expression12. This study concluded that HepG2 cells are a valid model to investigate insulin signaling, and that HepG2 show a gluconeogenic and hepatokine gene-expression pattern similar to the one observed in in-vivo settings12. Yet, this study has not directly compared HepG2 and immortalized hepatocytes to primary cell cultures, or to the liver in in-vivo settings.The metabolic function of the liver is highly integrated with other organs and insulin action on the hepatocyte implicates indirect mechanisms involving signals from adipocytes and the brainOverall, a direct comparison of insulin signaling in primary hepatocytes and in hepatoma cell lines is necessary to validate hepatoma cell lines as model to study insulin signaling and insulin action on glucose metabolism, and to better understand the biology of hepatocellular carcinoma (HCC).Here we have investigated insulin-driven PI3K and RAS-MAPK signaling, gluconeogenic gene expression, glucose production, and fatty acid synthase (FAS) protein levels in primary mouse hepatocytes and in three hepatoma cell lines from three different species: human HepG2; mouse Hepa 1–6 cells; and rat McARH7777. Finally, a Coomassie gel of protein extracts from primary human and mouse hepatocytes and from the three hepatoma cell lines above is shown to estimate the relative differences in protein expression profiles between species and between primary hepatocytes and hepatoma cells.7. To evaluate insulin signaling in HepG2 hepatoma cells we have measured insulin-induced receptor beta chain (IRβ) autophosphorylation, AKT phosphorylation, and ERK phosphorylation in HepG2 cells in parallel to primary mouse hepatocytes infected either with a control virus expressing the GFP or with a virus expressing the dominant negative mutant H-RAS17N  and phosphoenolpyruvate carboxykinase (PEPCK), which was dampened to basal levels by insulin Fig. a. HoweveWe next evaluated the effects of insulin on glycogen synthase kinase phosphorylation (GSK), a major molecular effector of the insulin-PI3K-AKT signaling cascade mediating insulin action on glucose mobilization from glycogen. The results indicate that despite the fact that insulin induced AKT Ser 473 phosphorylation to a similar extent in primary hepatocytes and hepatoma cells GSK phosphorylation was significantly increased by insulin only in primary hepatocytes Figs. a, b.Finally, to investigate the physiological relevance of these derangements we measured glucose production in primary hepatocytes and in the three hepatoma cell lines exposed to dbcAMP, insulin, or dbcAMP in presence of insulin.Primary hepatocytes displayed a substantial basal glucose production, which was significantly increased by dbcAMP, but not by dbcAMP in presence of insulin Fig. h. HoweveOverall, HepG2, Hepa1-6, and McARH7777 hepatoma cells display similar derangements of the glucose production molecular machinery, consisting in very low glucose production rates and aberrant expression of gluconeogenic genes which were unresponsive to either dbcAMP or insulin. Finally, the hepatoma cell lines largely dissociated insulin-induced AKT Ser 473 from GSK phosphorylation.Our data from above show that HepG2, Hepa1-6, and McARH7777 hepatoma cells display similarly aberrant insulin signaling and glucose production machinery. We have therefore compared the protein levels of the lipogenic enzyme fatty acid synthase (FAS), in primary hepatocytes with the one of the three hepatoma cell types, in three independent experiments each one with three replicates. The results show that FAS expression was significantly reduced in all the hepatoma cells analyzed Fig. c, d.FiguOverall, the expression of all the metabolic enzymes we have measured was quantitatively different between primary hepatocytes and hepatoma cells but similar between the different hepatoma cell lines that we have tested Figs. a, b. ThiAltogether, our data indicate that quantitative differences in protein expression profile between hepatoma cells and primary hepatocytes appear to be larger than the one between mouse and human hepatocytes, and that the three hepatoma cell lines derived from mouse, rat, and human display a similar protein expression profile.We have found that, compared to primary hepatocytes, hepatoma cell lines display aberrant insulin—receptor tyrosine kinase signaling consisting in elevated basal phosphorylation of AKT specifically at Thr 308 and constitutively activated RAS-MAPK signaling, which were resistant to the effects of the dominant negative Ras mutant H-RAS17N. Compared to primary hepatocytes, these hepatoma cell lines also showed defective expression of the key gluconeogenic genes G6P and PEPCK, insulin unresponsive GSK, and dampened glucose production which was unresponsive to either dbcAMP or insulin. Hepatoma cell lines also showed lower protein levels of FAS compared to primary hepatocytes, and showed a protein electrophoresis profile which was remarkably distinct from the one of hepatocytes but similar between them despite being derived from different animal species.13. Furthermore, it was reported that mice injected with H22 carcinoma cells develop tumors in their liver that showed largely reduced PEPCK and G6P expression compared to normal liver tissue14. The latter is consistent with our results and indicate that the blunted glucose production we observed in cultured hepatoma cell lines is also observed in-vivo.These results are relevant to the interpretation of a large bulk of scientific literature where these hepatoma cells have been used to model the hepatocyte to investigate insulin signaling, insulin resistance, glucose and lipid metabolism for the study of diabetes and fatty liver disease. These data are also relevant to the field of hepatocellular carcinoma, as we have found that different hepatoma cell lines derived from different tumor clones from three different animal species and that have been cultured for years, appear to converge toward similar derangements of PI3K-AKT and RAS-MAPK signaling, glucose metabolism, and protein expression profile. It is thus reasonable that this aberrant phenotype could be a general hallmark of HCC, and the full mechanistic elucidation of such phenotype may lead to new pharmacological targets for the treatment of HCC. Indeed, it was shown that the gene expression profile of several hepatoma cell lines is closely correlated with the one of primary tumors, with the HepG2 cell line showing the stronger correlation15. This apparent discrepancy can be explained by the fact that, in the normal hepatocyte, DNL is a highly regulated process which is minimal in animals fed with diets abundant in lipids, but that it can be dramatically induced during refeeding on a diet rich in carbohydrates after a period of starvation16. Because in our study cells are cultured in medium containing only glucose as energy source (but not lipids), it is expected that DNL is activated in the cultured primary hepatocytes.Whereas we have found that FAS levels were significantly reduced in cultured HCC cells compared to primary hepatocytes, it was shown that the expression of FAS and other DNL markers is elevated in primary HCC compared to normal liver of patients17.Our immunoblots from primary hepatocytes and from hepatoma cell lines were performed with antibodies from the same tube, which in principle should exclude the possibility that the elevated signals we measured for AKT Thr 308 and ERK phosphorylation in hepatoma cells but not in hepatocytes, are caused by an artefactual interaction with non-specific bands. However, it is likely that others before us have observed a similar pattern of AKT Thr 308 and ERK phosphorylation in hepatoma cells and ascribe it to suboptimal antibody quality or immunoblot conditions. Indeed, out of 100 randomly selected manuscripts measuring insulin-induced AKT phosphorylation in the hepatoma cell lines that we have read, only seven studies specifically showed AKT phosphorylation at Thr 308, whereas the other 93 either showed only AKT Ser 473 phosphorylation or a non-specified AKT phosphorylation site. Mechanistically, the constitutively activated ERK signaling and elevated AKT Thr 308 basal phosphorylation observed in the hepatoma cells are expected to be caused by genetic mutations. Thereby, we have examined the information available on the HepG2 cell line at Broad Institute Cancer Cell Line encyclopedia (CCLE), where we have found 386 entries for mutations in the HepG2 genome. These included the well-established NRAS activating mutation Q61L, which fully explains the constitutively elevated ERK phosphorylation and the insensitivity to the effects of the dominant negative H-Ras17N mutant that we observed in HepG2 hepatoma cells. Furthermore, according to data from CCLE, HepG2 also bear a S265R missense mutation on MAPK4, a kinase which was shown to directly phosphorylate AKT specifically on Thr 308Other mutations reported at CCLE for the HepG2 genome, which are expected to affect PI3K or MAPK signaling are a G573S substitution on IGF2R, and a nonsense mutation on the PI3K regulatory subunit PIK3R2, whereas a D69N mutation for FOXO1 is expected to affect gluconeogenic gene-expression. Although these mutations may contribute to the blunted glucose production observed in hepatoma cells, this dampened glucose production may be more generally linked to the ensemble of genetic aberrations driving altered glucose metabolism in HCC. Furthermore, the genetic and epigenetic alterations driving aberrant metabolism in HCC most likely involve indirect and complex interactions, which are not readily deducible from genetic and epigenetic signatures. However, our data indicate that hepatoma cell lines bear a conserved proteomic signature whose quantitative comparison to the one of primary hepatocytes may reveal the detailed mechanism causing the “malignant” metabolic transformation of hepatocellular carcinoma.Altogether, our data indicate that hepatoma cell lines from different species largely deviate from the original hepatocyte phenotype for insulin signaling, insulin action on glucose production, and protein expression signature.11. However, it is worth of attention that three different hepatoma cell lines from three different species, which have been cultured for years, show remarkable similarities in their aberrant phenotype for insulin signaling, glucose production, and protein electrophoretic profile. Thereby we conclude that hepatoma cell lines are most likely valuable tools to investigate the proteomic changes conferring to HCC its peculiar metabolisms.This observation implies that the large bulk of literature using hepatoma cell lines, such as HepG2, to model the hepatocyte for insulin action on glucose and lipid metabolism, should be carefully interpreted in light of the data in this manuscript and from emerging evidence from other studiesMice used for this study were wild type C57BL/6J. Mice were maintained at the EBM specific pathogen free facility of the University of Gothenburg at room temperature of 22 °C under 12 h of light and 12 h of dark cycles. All the protocols were carried out according to the relevant guidelines and regulations and were authorized by the Ethics Committee on Animal Care Use in Gothenburg, Sweden.g) and washed three times with 20–25 ml of culturing medium: 50% Dulbecco’s Modified Eagle’s Medium (DMEM) high-glucose, 50% HAM’S F-12, 10% FBS, 1% Penicillin Streptomycin and 100 µM dexamethasone. Alive cells were counted by trypan blue exclusion and plated 400,000 cells/ml in 12 well plates pre-coated one day earlier with type I collagen dissolved in 0.02 N acetic acid let dry overnight. From a 12–18 weeks old mouse we typically obtained 30–50 million hepatocytes with 80–98% viability.Murine primary hepatocytes were prepared from 12–18 weeks old mice. Mice were anesthetized with isofluorane (Baxter KDG9623) and sacrificed by bleeding. We perfused the liver with 60 ml of washing solution 1 × Hank`s buffered salt sodium [GIBCO 14170-112] in absence of Magnesium and Calcium with addition of 0.5 mM EGTA. After washing, the liver was perfused with a digestion medium: DMEM-low glucose (HyClone) with 1% Penicillin Streptomycin and 15 mM HEPES and 0.8 mg/ml of collagenase type IV (SIGMA). The digestion process was performed at 37 °C for 8–10 min, when liver structure is disrupted, Glisson’s capsule was opened releasing the hepatocytes in a 10 cm Petri dish filled with 10 ml digestion medium. Cells are filtered on a 70 µM cell strainer and collected by centrifugation  with 10% FBS, 1% Penicillin Streptomycin. Hepa 1–6 cells (from mouse hepatoma) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose with 10% FBS and 1% Penicillin Streptomycin. McARH7777 cells (from rat hepatoma) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose with 10% FBS and 1% Penicillin Streptomycin.The hepatoma cell lines were kindly provided by Prof. Stefano Romeo  and by Prof. Fredrik Bäckhed (an independent stock of Hepa 1–6), which were purchased from ATCC and all experiments were performed between passage 4 and 6. HepG2 cells (from a human hepatoma) were cultured at 37 °C, 5% COHuman hepatocyte lysate was purchased from Sciencell.7. Brifely, the empty VmAdcDNA3 virusmid used to generate the recombinant adenoviral vectors and the control adenovirus expressing the green fluorescent protein were kindly gifted by Luciano Pirola at INSERM 1,060 CarMeN Lyon. The recombinant adenovirus expressing the H-RAS17N dominant negative mutant was generated by homologous recombination of the VmAdcDNA3 virusmid and a pcDNA3.1 plasmid in which was cloned the H-RAS17N cDNA fused with three HA tags at the N terminus. The pcDNA3.1 H-RAS17N 3XHA plasmid was bought from the UMR cDNA Resource Center, University of Missouri-Rolla (GB Acc. NM_005343). For immunoblot analysis cells were serum starved for 12 h and stimulated with 10 nM of insulin or with the indicated dose  for 8 min. When indicated cells were infected 24 h before insulin treatment with either 100 MOI of either a GFP-expressing control adenovirus, or an adenovirus expressing the dominant-negative mutant H-RAS17N.The procedures to generate, amplify, and titer the viral vectors used in this manuscript were previously describedTo estimate the infection efficiency, cells were infected with 25, 50 and 100 MOI of the GFP adenovirus and 24 h post-infection the number of GFP positive cells was evaluated. Primary hepatocytes and hepatoma cell lines were also infected with 100 MOI of H-RAS17N and relative protein levels were compared by immunoblot. For qPCR analysis of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphate (G6P) gene expression, and measurement of glucose production, cells were serum starved for 12 h and then treated either with 10 nM of insulin, 100 μM of dbcAMP, or 10 nM insulin and 100 μM dbcAMP for 6 h.The RNA was extracted from primary hepatocytes and cell lines with guanidinium-thiocyanate method. The complementary DNA was prepared using reverse transcription kit (Promega A3802). qPCR was performed using SYBR green mix (Bio-Rad 172-5274) and the following primers: mouse G6P fwd 5′-CGACTCGCTATCTCCAAGTGA-3′, rev 5′GTTGAACCAGTCTCCGACCA3′; mouse PEPCK fwd 5′-AGCATTCAACGCCAGGTTC-3′, PEPCK rev 5′-CGAGTCTGTCAGTTCAATACCAA-3′; rat G6P fwd 5′-GGCTCACTTTCCCCA-3′, rev 5′-ATCCAAGTGCGAAACCAAACA-3′; rat PEPCK fwd 5′-GGTTGCAGGCCCAGTTGTTG-3′, rev 5′-GAC AGACTCGCCCTATGTGGTG-3′; human G6P fwd 5′-GCTTCGCCATCGGATTTTAT-3′, rev 5′-CACCACCTCTGGGCTTTCT-3′; human PEPCK fwd 5′-GCAAGATTATCGTCACCC-3′ and rev 5′-GGCATTGAACGCTTTCTCAAAAT-3’.For immunoblot, proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. Membranes were incubated for 1 h in blocking solution 3% bovine serum albumin (BSA) 0.3% tween in phosphate saline buffer; and then incubated with the primary antibody overnight at 4 °C in PBS 3% BSA 0.3% tween. Membranes were washed 3 times in PBS with 0.3% tween and incubated with horseradish peroxidase conjugated secondary antibody  in PBS 0.3% tween 3% BSA for one hour at room temperature. After three washes in PBS 0.3% tween, membranes were incubated with detection reagent and the signal was analyzed through BioRad ChemiDoc apparatus and BioRad Image Lab software.All primary antibodies were from Cell Signaling except for anti-phospho tyrosine 4G10 (Millipore), anti-FAS (abcam), and anti-H-RAS (Santa Cruz).https://www.bio-rad.com/en-se/product/image-lab-software?ID=KRE6P5E8Z).For coomassie staining, the acrylamide gel after the electrophoresis was covered with a warm fixation solution  for about one minute, and then incubated in 50 ml of 5% EtOH, 7.5% acetic acid to which were freshly added 0.5 ml of Coomassie stock solution  and warmed to about 50–60 °C in a microwave for about 1 min. The gel was then incubated for 10–20 min at room temperature on a shaker, and then destained with destained solution  for 3 h at room temperature on a shaker. Image acquisition was performed with BioRad ChemiDoc apparatus and BioRad Image Lab software Version 6.0.0 build 26 2017, Bio-Rad Laboratories . After 12 h starvation cells were treated with 10 nM of insulin, or with 100 µM dbcAMP, or with both 10 nM insulin and 100 μM dbcAMP added in the cell medium for 6 h. 50 μl of medium was collected and mixed with an equal volume of reaction mix. A standard curve was prepared by serial dilutions of a 1.25 mM NADH stock in assay buffer in as indicated by the vendor, and 450 nm absorbance was measured 30 min and 60 min after plating.Data are displayed as means and the error bands represent standard errors. For “biological replicate” is intended a primary hepatocyte preparation derived from one mouse. Values are expressed as relative to controls. P-values for immunoblot signals on the same gel, and for glucose production analysis were performed by two-tailed t-test, while Mann–Whitney test was applied to qPCR analysis of gene expression and for analysis of immunoblots data from different membranes (in the three independent FAS measurements). All the statistical analysis was performed with the GraphPad Prism software.Unprocessed blot images are provided in the supplemental material of this manuscript, and datasets from this study are available from the corresponding author on reasonable request.Supplementary figures S1–S4.Supplementary information.


There was no similar pattern of phenotypic alterations among eleven resistant cell lines, including expression/activity of crucial regulators, such as MITF, AXL, SOX, and NGFR, which suggests that patient-to-patient variability is richer and more nuanced than previously described. This diversity should be considered during the development of new strategies to circumvent the acquired resistance to targeted therapies.The clinical benefit of MAPK pathway inhibition in BRAF-mutant melanoma patients is limited by the development of acquired resistance. Using drug-naïve cell lines derived from tumor specimens, we established a preclinical model of melanoma resistance to vemurafenib or trametinib to provide insight into resistance mechanisms. Dissecting the mechanisms accompanying the development of resistance, we have shown that (i) most of genetic and non-genetic alterations are triggered in a cell line- and/or drug-specific manner; (ii) several changes previously assigned to the development of resistance are induced as the immediate response to the extent measurable at the bulk levels; (iii) reprogramming observed in cross-resistance experiments and growth factor-dependence restricted by the drug presence indicate that phenotypic plasticity of melanoma cells largely contributes to the sustained resistance. Whole-exome sequencing revealed novel genetic alterations, including a frameshift variant of RBMX found exclusively in phospho-AKT BRAF and alternative splicing of BRAF transcript, loss of cyclin-dependent kinase inhibitor 2A (CDKN2A) and alterations of genes encoding the components of the phosphoinositide-3-kinase–protein kinase B/AKT (PI3K/AKT) signaling pathway [A constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway due to genetic alterations is detected in the majority of melanomas . Addicti pathway ,12,13,14 pathway . Cell pl pathway . Diverse pathway ,18,19,20V600E) or trametinib . The original drug-naïve melanoma cell lines were previously characterized at the level of cell morphology, proliferation rate, activity of multiple signaling pathways and response to changes in the microenvironment [To address the challenges of extensive variability of acquired resistance mechanisms, we have taken the advantage of melanoma cell lines derived from tumor specimens to obtain their counterparts resistant to either vemurafenib , ERK1/2, phospho-MEK1/2 (Ser217/Ser221), p-AKT (Ser473), AKT, AXL, SOX2, EGFR , BRAF, DUSP6, PARP, SOX10, GAPDH , active β-catenin (dephosphorylated on Ser37 and Thr41) from Millipore or β-actin  were used followed by binding of the secondary HRP-conjugated anti-mouse or anti-rabbit antibodies . The membranes were incubated with Pierce® ECL Western Blotting Substrate , and the proteins were visualized by using ChemiDoc Imaging System , or on a medical X-ray film .Preparation of cell lysates, electrophoresis, protein transfer, and immunodetection were performed according to the procedures described previously ,25. BrieAXL: 5’-GGTGGCTGTGAAGATGA-3′ and 5′-CTC AGA TAC TCC ATG CCA CT-3′; IL-8: 5′-AGG TGC AGT TTT GCC AAG GA-3′ and 5′-TTT CTG TGT TGG CGC AGT GT-3′; MMP-2: 5′-AGC GAG TGG ATG CCG CCT TTA A-3′ and 5′-CAT TCC AGG CAT CTG CGA TGA G-3′; SOX2: 5′-GCT AGT CTC CAA GCG ACG AA-3′ and 5′-GCA AGA AGC CTC TCC TTG AA-3′; SOX9: 5′-GAC CCG CAC TTG CAC AAC-3′ and 5′-TCC GCT CTC GTT CAG AAG TC-3′. Relative gene expression was calculated versus a reference gene RPS17, and efficiency correction for real-time PCR was included.Total RNA isolation, cDNA synthesis, quantitative real-time polymerase chain reaction  procedures and data analysis were described elsewhere . In brieAnalysis of 43 phospho-proteins by using Human Phospho-Kinase Array  was performed according to the manufacturer’s protocol. In brief, drug-resistant cell lines and matched drug-naïve cell lines cultured in the same conditions for 19 h were used to prepare cell lysates. Antibody-coated membranes were incubated with Array Buffer followed by overnight incubation with cell lysates. After three washes, the membranes were incubated with biotinylated secondary antibodies and streptavidin conjugated with HRP. Chemiluminescence signal was developed on X-ray film or by using ChemiDoc Imaging System . Pixel densities of dots were analyzed using a transmission-mode scanner and ImageJ free software. Mean pixel densities of the pair of duplicate spots representing each protein of interest and HSP60 protein (a loading control) were calculated. Relative level of each protein was calculated by using the formula: (mean pixel density of the protein)/(mean pixel density of HSP60). Obtained values were used to prepare color coded matrix.LIVE/DEAD Fixable Violet Dead Cell Stain Kit  was used to exclude dead cells from the analysis. Antibodies against NGFR (PE-conjugated) were from BD Pharmingen . Appropriate isotype controls were included in each experiment. Flow cytometric data were acquired with FACSVerse  and analyzed using BD FACSuite.ELISA kit Quantikine High Sensitivity Human CXCL8/IL-8  was used to determine IL-8 secretion to the culture medium by melanoma cells. Briefly, culture media were diluted 40x, added to each well of the pre-coated plate and incubated for 2 h at room temperature. After 6 washes, the plate was incubated with IL-8 HS Conjugate for 1 h, and washing steps were repeated. This was followed by an incubation with the Substrate Solution, Amplifier Solution and Stop Solution. The optical density of each well was determined using a microplate reader Infinite M200Pro . Concentrations of IL-8 in the medium samples were obtained by applying a four parameter logistic (4-PL) curve fit, and normalized to cell number.http://www.ncb.nlm.nih.gov), and gene expression values were log2-transformed. The AXL expression profiles were developed from paired tumor specimens collected from patients before treatment and post-relapse with acquired resistance either to vemurafenib or combined treatment, dabrafenib and trametinib.The publicly available microarray data sets  were downloaded from the GEO database  derived from six different tumor specimens were used to develop resistance to vemurafenib (PLX), whereas to obtain melanoma cell lines resistant to trametinib (TRA), in addition to BRAFV600E melanoma cell lines, one Harvey rat sarcoma viral oncogene homolog (HRASQ61R) cell line (DMBC17) was employed. After 4–5 months of continuous exposure to increasing concentrations of PLX and TRA finally reaching 10 µM and 50 nM, respectively, six PLX-  and five TRA-resistant cell lines  were obtained. None of two attempts to obtain 12_TRAR and 33_TRAR was successful. 33_PLXR cell line derived from DMBC33, which originally exerted very high doubling time (107.7 h) [BRAF107.7 h) , althougCCND1 expression. Proliferation rates of resistant cells were high even if all PLXR cell lines had significantly lower transcript levels of cyclin D1 than their drug-naïve counterparts, whereas in TRAR cell lines, CCND1 expression was enhanced (11_TRAR), diminished (29_TRAR and 17_TRAR) or remained at similar level (21_TRAR and 28_TRAR) compared with parental cell lines   was even substantially reduced ), which revealed that the development of resistance to vemurafenib or dabrafenib combined with trametinib was accompanied with increased expression of AXL only in selected cases, and the AXL level either remained unchanged or was decreased in the majority of relapse samples  D,E. At t DMBC11) D. The pr reduced E. Althou samples F.SOX2, SOX9, and SOX10 at the transcript levels was compared between drug-naïve cell lines. SOX10 was quite uniformly expressed in all drug-naïve cell lines relative to the median value , and several substitutions in RAS guanyl-releasing protein 4 (RASGRP4) and son of sevenless homolog 1 (SOS1) were found in 21_PLXR and 28_PLXR cells  or was substantially diminished (11_TRAR and 12_PLXR cells) during the development of resistance  and hepatocyte growth factor (HGF), and lack of them in the culture medium do not affect cell morphology and activity of several signaling pathways in patient-derived melanoma cells . In thisAR cells B, which AR cells C. TRAR cAR cells C and 17_AR cells C. In conAR cells C.low resistant cell lines, 21_PLXR and 28_PLXR . 21_PLXR, 21_TRAR, and 28_PLXR cells were used for comparison. The activity of EGFR downstream signaling pathways remained unaffected in EGFR 28_PLXR D. In con 28_PLXR D. Notabl 28_PLXR D. Furthe 28_PLXR D. Result 28_PLXR E.217/221 (BRAFwt/HRASQ61R). Lack of cleaved PARP indicates that apoptosis was not induced in any of cell lines  and drug-naïve cells after short treatment (immediate response) were performed. Gene expression at the transcript and protein levels as well as activity of corresponding proteins were (re)-analyzed based on results of the present report and previously published data ,27 and sS473 also diverse in drug-naïve cells , remained unchanged (29_TRAR) or was significantly enhanced , whereas during immediate response the percentages of NGFR-positive cells were increased in all melanoma cell lines although to different extent , AXLhigh (invasive), and NGFRhigh . In acquired resistance phase, about 70% of melanomas present the AXLhigh phenotype and only about 23% are enriched with the MITFhigh phenotype. Our present study as well as previously published [high/AXLlow (five cell lines) or MITFlow/AXLhigh (two cell lines). Among eleven resistant cell lines, six vemurafenib-resistant and five trametinib-resistant, none became MITFlow/AXLhigh , MITFlow/AXLlow/NGFRlow , MITFlow/AXLlow/NGFRhigh (11_PLXR), MITFmedium/AXLlow/NGFRhigh , patient-to-patient variability might be richer and more nuanced than previously suggested. Especially interesting is MITFlow/AXLlow/NGFRlow phenotype of 28_PLXR cell line. This is vemurafenib-resistant cell line, in which dedifferentiation, shown as inhibition of expression of MITF and MITF-dependent pigmentation-related genes, is irreversible [FGFR, FMS-like tyrosine kinase 3 (FLT-3) and ERBB4, which is exclusively observed in this cell line. Moreover, enhanced activity of STAT3 followed by increased expression of SOX2, which were observed only in 28_PLXR cells could be also considered as the mechanism supporting cell survival. It has been demonstrated that SOX2 as a part of STAT3-SOX2-CD24 axis rescued melanoma cells against acute exposure to vemurafenib [BRAF or NRAS) was present [SOX2 expression in 29_TRAR and 17_TRAR cells were less pronounced probably because of an elevated MITF level and high frequency of differentiated cells [The plasticity of melanoma as enabler of drug resistance was a subject of most recent reviews ,37,53. Aublished , which btudy and ). Thus, tudy and ,34,35. Ttudy and ,34,35, ahibition ,60, but hibition . Our stuhibition ,63, it chibition . Therefoversible . In the versible ,66,67, dNGFRhigh _PLXR, MIversible . In 28_Purafenib . SOX2 wa present . Recipro present , which wed cells . SOX2 leed cells , vemurafed cells ,75,76,77CCND1 expression, including upregulation, was found in resistant cells. The stop-gained variants of CDKN2A were acquired by two vemurafenib-resistant cell lines, 21_PLXR and 28_PLXR, and a possibly damaging D148N variant of E2F transcription factor 3 (E2F3) was additionally found in 21_PLXR cells  [Proliferation curves of resistant cells and their drug-naïve counterparts were similar. While immediate response to PLX and TRA was associated with reduced cyclin D1 transcript levels in all drug-naïve cell lines , variablXR cells , but the of BRAF  and CCNDF [CCND1 , respect4 (CDK4) . Interes4 (CDK4) . In the 4 (CDK4) , IL-8 thF57C substitution exerted phospho-MEK2low/phospho-ERK1/2high signature [P124L variant [P124S or MEK1P124L variants faced rapid progression upon administration of BRAF inhibitor [Reactivation of the MAPK (ERK1/2) pathway ,81,82 orignature , (ii) re variant , and  TRAR cells neither expressed SOX2 protein at the detectable level nor activated JNK as shown by the phospho-protein array. In addition, mutually exclusive expression of MITF and EGFR was reported [low [EGFR expression in both 21_TRAR and 28_TRAR cells. Moreover, as deprivation of exogenous EGF was enough to trigger inhibition of EGFR signaling, production of EGFR-activating ligands in resistant melanoma cells was insufficient. Addiction to EGFR activation was, however, advantageous to melanoma cells only in the presence of drug since the cessation of trametinib treatment led to a growth factor-independent maintenance of TRA-resistant EGFR-expressing cells. This was an immediate effect, which was presumably due to a high plasticity of melanoma cells, and not as a result of depletion of EGFRhigh subpopulation as reported in BRAF inhibitor-resistant cells [EGFR is expressed in epithelial cells and its overexpression is found in cancers of epithelial origin [EGFR [EGFR expression is more frequent in metastatic melanoma [A high plasticity of resistant melanoma cells was also detected as the rapid regrowth and reprogramming of EGF-dependent EGFRurafenib ,104,105,of SOX10 ,103 follof SOX10 , and SOXof SOX10 . It is ureported ,102, andted [low , howeverted [low . In addited [low . Thus, al origin , whereasin [EGFR . Positivin [EGFR . EGFR exmelanoma  and has melanoma . In addimelanoma . Short tmelanoma ,115. OurThis in vitro study was performed to characterize melanoma cells that developed resistance to vemurafenib or trametinib after 4–5 months of treatment. A high variability of molecular and cellular changes was observed, as there was no similar pattern of genetic/non-genetic alterations among eleven investigated resistant cell lines, including phenotypic changes in expression/activity of crucial melanoma regulators e.g., MITF, AXL, NGFR, AKT, ERK1/2, and SOX2 . Moreove


Muir-Torre syndrome (MTS) a genetic disorder characterized by predisposition to cutaneous neoplasms. Lipedematous scalp is characterized by the presentation of a thick, sponge-like scalp due to the altered and increased deposition of adipose tissue. We present a case of Muir-Torre-associated sebaceous carcinoma of the scalp consistent with a lipedematous scalp. Sebaceous carcinomas are rare among the general population and are known to be characteristic of Muir Torre Syndrome (MTS). MTS is a genetic disorder that is characterized by a predisposition to sebaceous skin cancers, keratoacanthomas, as well as visceral cancers, most commonly gastrointestinal and genitourinary cancers; it is considered a variant of hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. Muir originally described MTS in 1967 with Torre reporting a patient in 1968 ,3. The sOriginally described by Combleet in 1935, the term ‘lipedematous scalp’ was not introduced until Lee described it in 1994 in an attempt to separate it from lipedematous alopecia . When a A healthy 37 year old African American female with no significant past medical history presented to the plastic surgery service for treatment of a recently diagnosed sebaceous carcinoma of the scalp. She denied any personal or family history of previous skin cancers or other malignancies. On first exam, the thickness of her scalp was not initially appreciated. However, during the surgical procedure, her scalp was noted to have a ‘sponge-like’ consistency and was noted to be thicker than normal tissue. An increased egress of fluid or increased bleeding was not noted during the operation. Due to the concern for possible satellite metastasis, a biopsy of her scalp was taken from the area inferior to the excision site  that code for proteins that function to correct errors that occur during DNA replication [MYH gene which encodes base excision repair [MTS is a rare genetic condition that predisposes to sebaceous skin conditions and visceral cancers, most frequently large bowel, endometrium, genitourinary tract, breast, and ovarian. The condition most commonly arises in an autosomal dominant inheritance pattern with a mutation in a DNA mismatch repair (MMR) gene – principally n repair .th and 7th decades of life, and women are more commonly affected than men, comprising about 70% of patients diagnosed with sebaceous carcinomas [The most common sebaceous neoplasms in MTS are sebaceous adenomas, sebaceous epitheliomas, and sebaceous carcinomas with varying reports of frequency. Sebaceous adenomas as the initial presentation for MTS have reported associations from 25% to 60%. Sebaceous carcinomas in patients with MTS have reported frequencies of 36–100%, while sebaceous epithelioma associations range from 31% to 86%. Sebaceous neoplasms are rare among the general population and are the most defining characteristic of MTS. Specifically, sebaceous carcinoma represents less than 1% of all cutaneous malignancies and 17% of all sebaceous neoplasms in the general population ,11. Althrcinomas . Twenty-rcinomas . Extraocrcinomas ,5,12.Clinical presentations of sebaceous carcinomas are varied, and it is known for masquerading as common benign conditions. In a study of 31 patients with extraocular sebaceous carcinoma, the most common presentations were blepharoconjunctivitis (32%), eyelid masses (29%), or eyelid thickening (26%), with regional lymphadenopathy accounting for 3% of initial presentations . ExtraocSebaceous carcinomas have the ability to invade and produce metastases; therefore, it is often recommended to excise with 5–6 mm margins. Lymph node investigation is sometimes recommended for larger and more invasive tumors due to the 3–28% risk for local metastasis . ColonosAnother condition seen in our patient was the presence of a lipedematous scalp (LS). LS as well as lipedematous alopecia (LA) are rare conditions that are, as stated previously, most often seen in African-American women. Due to the increased proportion of women affected, hormonal factors have been hypothesized to have some level of influence . TheorieLipedematous scalp is characterized by a thickened subcutaneous and adipose layer in the scalp that presents clinically as a soft, almost sponge-like consistency to the scalp with thickened areas mainly in the scalp vertex and occiput. The lipedematous scalp is measurably thicker ∼10–16mm) than the normal scalp (∼5–6mm) as can be seen in our biopsy specimens above in mm than tSebaceous carcinomas are rare and, when present, should encourage the investigation into possible connections with syndromes such as Muir-Torre Syndrome. MTS is associated with an increased risk for other malignancies, especially GI/GU, and warrants a thorough investigation to rule out the presence of any other tumors. A lipedematous scalp is a very rare finding that has only recently been reported and does not appear to have any connection with sebaceous carcinoma or MTS. We therefore present this unique case that has two rare skin related conditions, sebaceous carcinoma associated with MTS and a lipedematous scalp.Sebaceous carcinomas are associated with patients that have Miur-Torre syndrome.Muir-Torre syndrome places patients at risk for malignancy, especially GI/GU malignancies and they require more surveillance than the general population.A lipedematous scalp may be one way of presentation of sebaceous carcinoma, especially in regards to Muir-Torre patients.