Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer.

The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain (TEAD) family transcription factors. The YAP/TAZ-TEAD complex is a key regulator of cancer-specific transcriptional programs, which promote tumor progression in diverse types of cancer, including breast cancer. Despite intensive efforts, the YAP/TAZ-TEAD complex in cancer has remained largely undruggable due to an incomplete mechanistic understanding. Here, we report that nuclear phosphoinositides function as cofactors that mediate the binding of YAP/TAZ to TEADs. The enzymatic products of phosphoinositide kinases PIPKIα and IPMK, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P3), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P2 and PI(3,4,5)P3 attenuates YAP/TAZ interaction with the TEADs, the expression of YAP/TAZ target genes, and breast cancer cell motility. Although we could not conclusively exclude the possibility that other enzymatic products of IPMK such as inositol phosphates play a role in the mechanism, our results point to a previously unrecognized role of nuclear phosphoinositide signaling in control of YAP/TAZ activity and implicate this pathway as a potential therapeutic target in YAP/TAZ-driven breast cancer.

KSR1 regulates small-cell lung carcinoma tumor initiation and cisplatin resistance.

Small-cell lung cancer (SCLC) is designated a recalcitrant cancer due to its five-year relative survival rate of less than 7%. First line SCLC treatment has changed modestly in the last 40 years. The NeuroD1 subtype of SCLC (SCLC-N) commonly harbors MYC amplifications and other hallmarks of aggressive behavior. Finding novel therapeutic options that effectively eliminate residual disease observed after initial response to therapy is essential to improving SCLC patient outcome. Here we show that Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK signaling cascade is critical for clonogenicity and tumor initiation in vitro and in vivo in the highly aggressive, metastatic and therapy resistant NeuroD1 subtype of SCLC. Tumor-initiating cells (TICs) are reported as the sanctuary population within the bulk tumor responsible for therapeutic resistance and relapse. Previous studies concluded ERK activation was inhibitory to growth and tumor development. We show that signaling through KSR1 is conserved in SCLC-N and that it regulates tumor initiation through interaction with ERK. We further show that KSR1 mediates cisplatin resistance in SCLC-N cells. While 50% of control SCLC-N cells show resistance after 6 weeks of exposure to cisplatin, CRISPR/Cas9-mediated KSR1 knockout prevents resistance in >90% of SCLC-N cells. KSR1 KO also significantly enhances the ability of cisplatin to decrease SCLC-N TICs, indicating that targeting KSR1 might be selectively toxic to cells responsible for therapeutic resistance and tumor initiation. Thus, KSR1 function in SCLC-N serves as a novel model for understanding the role of KSR1-dependent signaling in normal and malignant tissues. These findings shed light on a key distinct protein responsible for regulation in SCLC-N tumors, and a potential subtype specific therapeutic target.

Albendazole inhibits colon cancer progression and therapy resistance by targeting ubiquitin ligase RNF20.

BACKGROUND: The repurposing of FDA-approved drugs for anti-cancer therapies is appealing due to their established safety profiles and pharmacokinetic properties and can be quickly moved into clinical trials. Cancer progression and resistance to conventional chemotherapy remain the key hurdles in improving the clinical management of colon cancer patients and associated mortality. METHODS: High-throughput screening (HTS) was performed using an annotated library of 1,600 FDA-approved drugs to identify drugs with strong anti-CRC properties. The candidate drug exhibiting most promising inhibitory effects in in-vitro studies was tested for its efficacy using in-vivo models of CRC progression and chemoresistance and patient derived organoids (PTDOs). RESULTS: Albendazole, an anti-helminth drug, demonstrated the strongest inhibitory effects on the tumorigenic potentials of CRC cells, xenograft tumor growth and organoids from mice. Also, albendazole sensitized the chemoresistant CRC cells to 5-fluorouracil (5-FU) and oxaliplatin suggesting potential to treat chemoresistant CRC. Mechanistically, Albendazole treatment modulated the expression of RNF20, to promote apoptosis in CRC cells by delaying the G2/M phase and suppressing anti-apoptotic-Bcl2 family transcription. CONCLUSIONS: Albendazole, an FDA approved drug, carries strong therapeutic potential to treat colon cancers which are aggressive and potentially resistant to conventional chemotherapeutic agents. Our findings also lay the groundwork for further clinical testing.

SOS1 and KSR1 modulate MEK inhibitor responsiveness to target resistant cell populations based on PI3K and KRAS mutation status.

KRAS is the most commonly mutated oncogene. Targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells within the heterogeneous tumor population and/or acquired mutations that reactivate receptor tyrosine kinase (RTK)/RAS signaling can lead to outgrowth of tumor-initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 (Son of Sevenless 1) or KSR1 (Kinase Suppressor of RAS 1) both enhances the efficacy of, and prevents resistance to, the MEK inhibitor trametinib in KRAS-mutated lung (LUAD) and colorectal (COAD) adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting spheroid-initiating cells in KRASG12/G13-mutated LUAD and COAD cell lines that lacked PIK3CA comutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to trametinib and BI-3406 combination therapy. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented drug-induced SIC upregulation and restored trametinib sensitivity across all tested KRAS mutant cell lines in both PIK3CA-mutated and PIK3CA wild-type cancers. Our findings demonstrate that vertical inhibition of RTK/RAS signaling is an effective strategy to prevent therapeutic resistance in KRAS-mutated cancers, but therapeutic efficacy is dependent on both the specific KRAS mutant and underlying comutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations.

Claudin-1 interacts with EPHA2 to promote cancer stemness and chemoresistance in colorectal cancer.

Therapy resistance is the primary problem in treating late-stage colorectal cancer (CRC). Claudins are frequently dysregulated in cancer, and several are being investigated as novel therapeutic targets and biomarkers. We have previously demonstrated that Claudin-1 (CLDN1) expression in CRC promotes epithelial-mesenchymal transition, metastasis, and resistance to anoikis. Here, we hypothesize that CLDN1 promotes cancer stemness and chemoresistance in CRC. We found that high CLDN1 expression in CRC is associated with cancer stemness and chemoresistance signaling pathways in patient datasets, and it promotes chemoresistance both in vitro and in vivo. Using functional stemness assays, proteomics, biophysical binding assays, and patient-derived organoids, we found that CLDN1 promotes properties of cancer stemness including CD44 expression, tumor-initiating potential, and chemoresistance through a direct interaction with ephrin type-A receptor 2 (EPHA2) tyrosine kinase. This interaction is dependent on the CLDN1 PDZ-binding motif, increases EPHA2 protein expression by inhibiting its degradation, and enhances downstream AKT signaling and CD44 expression to promote stemness and chemoresistance. These results suggest CLDN1 is a viable target for pharmacological intervention and/or biomarker development.

Differential methylation patterns in lean and obese non-alcoholic steatohepatitis-associated hepatocellular carcinoma.

BACKGROUND: Nonalcoholic fatty liver disease affects about 24% of the world's population and may progress to nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). While more common in those that are obese, NASH-HCC can develop in lean individuals. The mechanisms by which HCC develops and the role of epigenetic changes in the context of obesity and normal weight are not well understood. METHODS: In this study, we used previously generated mouse models of lean and obese HCC using a choline deficient/high trans-fat/fructose/cholesterol diet and a choline supplemented/high trans-fat/fructose/cholesterol diet, respectively, to evaluate methylation differences in HCC progression in lean versus obese mice. Differentially methylated regions were determined using reduced representation bisulfite sequencing. RESULTS: A larger number of differentially methylated regions (DMRs) were seen in NASH-HCC progression in the obese mice compared to the non-obese mice. No overlap existed in the DMRs with the largest methylation differences between the two models. In lean NASH-HCC, methylation differences were seen in genes involved with cancer progression and prognosis (including HCC), such as CHCHD2, FSCN1, and ZDHHC12, and lipid metabolism, including PNPLA6 and LDLRAP1. In obese NASH- HCC, methylation differences were seen in genes known to be associated with HCC, including RNF217, GJA8, PTPRE, PSAPL1, and LRRC8D. Genes involved in Wnt-signaling pathways were enriched in hypomethylated DMRs in the obese NASH-HCC. CONCLUSIONS: These data suggest that differential methylation may play a role in hepatocarcinogenesis in lean versus obese NASH. Hypomethylation of Wnt signaling pathway-related genes in obese mice may drive progression of HCC, while progression of HCC in lean mice may be driven through other signaling pathways, including lipid metabolism.

PGC-1ß and ERRα Promote Glutamine Metabolism and Colorectal Cancer Survival via Transcriptional Upregulation of PCK2.

BACKGROUND: Previous studies have shown that Peroxisome Proliferator-Activated Receptor Gamma, Coactivator 1 Beta (PGC-1ß) and Estrogen-Related Receptor Alpha (ERRα) are over-expressed in colorectal cancer and promote tumor survival. METHODS: In this study, we use immunoprecipitation of epitope tagged endogenous PGC-1ß and inducible PGC-1ß mutants to show that amino acid motif LRELL on PGC-1ß is responsible for the physical interaction with ERRα and promotes ERRα mRNA and protein expression. We use RNAsequencing to determine the genes regulated by both PGC-1ß & ERRα and find that mitochondrial Phosphoenolpyruvate Carboxykinase 2 (PCK2) is the gene that decreased most significantly after depletion of both genes. RESULTS: Depletion of PCK2 in colorectal cancer cells was sufficient to reduce anchorage-independent growth and inhibit glutamine utilization by the TCA cycle. Lastly, shRNA-mediated depletion of ERRα decreased anchorage-independent growth and glutamine metabolism, which could not be rescued by plasmid derived expression of PCK2. DISCUSSION: These findings suggest that transcriptional control of PCK2 is one mechanism used by PGC-1ß and ERRα to promote glutamine metabolism and colorectal cancer cell survival.

Differential progression of unhealthy diet-induced hepatocellular carcinoma in obese and non-obese mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) ranks first among liver diseases in Western countries. NAFLD is typically associated with obesity and diabetes, however it also develops in lean individuals without metabolic syndrome. The prevalence of lean NAFLD is 7 percent in the U.S. and 25-30 percent in some Asian countries. NAFLD starts with excess liver fat accumulation (NAFL), progresses to nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). The pathogenesis of lean NASH-HCC and how it differs from obese NASH-HCC is not well understood. METHODS: In this work, we generated a mouse model of lean and obese NASH-HCC using a choline deficient/high trans-fat/fructose/cholesterol diet and a choline supplemented/high trans-fat/fructose/cholesterol diet, respectively, to compare progression to NASH-HCC in lean versus obese mice. Comparisons were made at the organismal, histological, and molecular level by investigating fatty acid metabolism in the plasma of these mice. RESULTS: Obese mice showed more pronounced glucose intolerance and insulin resistance, higher levels of plasma cholesterol and triglycerides, and higher penetrance of NASH compared to lean mice. Despite the abnormal metabolic profile of obese mice, male obese and lean mice developed HCC with similar penetrance (53.3% and 53.8%, respectively), albeit lean mice showed faster tumor progression as evidenced by the larger tumor size and lower HCC-free survival. None of the female lean mice developed HCC, while 50% of female obese mice developed HCC. Both groups of mice showed a reduction in plasma polyunsaturated fatty acids (PUFAs), however, the levels were higher towards the endpoint in obese mice compared to lean mice. CONCLUSIONS: Unhealthy diet composition appears to drive progression to NASH-HCC rather than the organismal effects of obesity. PUFA levels may increase due to systemic inflammation in obese mice and act as suppressors of tumor progression, thus delaying HCC progression in obese mice compared to lean mice. These models could be used to further dissect the molecular pathogenesis of lean and obese NASH-HCC and address the mechanisms whereby PUFAs may be implicated in hepatocarcinogenesis.

Splenic and PB immune recovery in neoadjuvant treated gastrointestinal cancer patients.

In recent years, immune therapy, notably immune checkpoint inhibitors (ICI), in conjunction with chemotherapy and surgery has demonstrated therapeutic activity for some tumor types. However, little is known about the optimal combination of immune therapy with standard of care therapies and approaches. In patients with gastrointestinal (GI) cancers, especially pancreatic ductal adenocarcinoma (PDAC), preoperative (neoadjuvant) chemotherapy has increased the number of patients who can undergo surgery and improved their responses. However, most chemotherapy is immunosuppressive, and few studies have examined the impact of neoadjuvant chemotherapy (NCT) on patient immunity and/or the optimal combination of chemotherapy with immune therapy. Furthermore, the majority of chemo/immunotherapy studies focused on immune regulation in cancer patients have focused on postoperative (adjuvant) chemotherapy and are limited to peripheral blood (PB) and occasionally tumor infiltrating lymphocytes (TILs); representing a minority of immune cells in the host. Our previous studies examined the phenotype and frequencies of myeloid and lymphoid cells in the PB and spleens of GI cancer patients, independent of chemotherapy regimen. These results led us to question the impact of NCT on host immunity. We report herein, unique studies examining the splenic and PB phenotypes, frequencies, and numbers of myeloid and lymphoid cell populations in NCT treated GI cancer patients, as compared to treatment naïve cancer patients and patients with benign GI tumors at surgery. Overall, we noted limited immunological differences in patients 6 weeks following NCT (at surgery), as compared to treatment naive patients, supporting rapid immune normalization. We observed that NCT patients had a lower myeloid derived suppressor cells (MDSCs) frequency in the spleen, but not the PB, as compared to treatment naive cancer patients and patients with benign GI tumors. Further, NCT patients had a higher splenic and PB frequency of CD4+ T-cells, and checkpoint protein expression, as compared to untreated, cancer patients and patients with benign GI tumors. Interestingly, in NCT treated cancer patients the frequency of mature (CD45RO+) CD4+ and CD8+ T-cells in the PB and spleens was higher than in treatment naive patients. These differences may also be associated, in part with patient stage, tumor grade, and/or NCT treatment regimen. In summary, the phenotypic profile of leukocytes at the time of surgery, approximately 6 weeks following NCT treatment in GI cancer patients, are similar to treatment naive GI cancer patients (i.e., patients who receive adjuvant therapy); suggesting that NCT may not limit the response to immune intervention and may improve tumor responses due to the lower splenic frequency of MDSCs and higher frequency of mature T-cells.

KSR1- and ERK-dependent translational regulation of the epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here, we show that KSR1 and ERK promote EMT-like phenotype through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E- to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro, and reverses EMT-like phenotype, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1 and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E- to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT-like phenotype via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.

The majority of cancer deaths result from tumor cells spreading to other parts of the body via a process known as metastasis. 90% of all cancers originate in epithelial cells that line the inner and outer surface of organs in our bodies. Epithelial cells, however, are typically stationary and must undergo various chemical and physical changes to transform in to migratory cells that can invade other tissues. This transformation process alters the amount of protein cells use to interact with one another. For example, epithelial cells from the colon produce less of a protein called E-cadherin as they transition into migrating cancer cells and make another protein called N-cadherin instead. A protein called KSR1 is a key component of a signaling pathway that is responsible for generating the proteins colon cancer cells need to survive. But it is unknown which proteins KSR1 helps synthesize and whether it plays a role in the metastasis of colon cancer cells. To investigate this, Rao et al. studied the proteins generated by cancerous colon cells cultured in the laboratory, in the presence and absence of KSR1. The experiment showed that KSR1 increases the levels of a protein called EPSTI1, which colon cancer cells need to transform into migratory cells. Depleting KSR1 caused cancer cells to generate less EPSTI1 and to share more features with healthy cells, such as higher levels of E-cadherin on their surface and reduced mobility. Adding EPSTI1 to the cancer cells that lacked KSR1 restored the traits associated with metastasis, such as high levels of N-cadherin, and allowed the cells to move more easily. These findings suggest that KSR1 and EPSTI1 could be new drug targets for reducing, or potentially reversing, the invasive behavior of colon cancer cells. However, further investigation is needed to reveal how EPSTI1 is generated and how this protein helps colon cancer cells move and invade other tissues.

A Gene Expression High-Throughput Screen (GE-HTS) for Coordinated Detection of Functionally Similar Effectors in Cancer.

Genome-wide, loss-of-function screening can be used to identify novel vulnerabilities upon which specific tumor cells depend for survival. Functional Signature Ontology (FUSION) is a gene expression-based high-throughput screening (GE-HTS) method that allows researchers to identify functionally similar proteins, small molecules, and microRNA mimics, revealing novel therapeutic targets. FUSION uses cell-based high-throughput screening and computational analysis to match gene expression signatures produced by natural products to those produced by small interfering RNA (siRNA) and synthetic microRNA libraries to identify putative protein targets and mechanisms of action (MoA) for several previously undescribed natural products. We have used FUSION to screen for functional analogues to Kinase suppressor of Ras 1 (KSR1), a scaffold protein downstream of Ras in the Raf-MEK-ERK kinase cascade, and biologically validated several proteins with functional similarity to KSR1. FUSION incorporates bioinformatics analysis that may offer higher resolution of the endpoint readout than other screens which utilize Boolean outputs regarding a single pathway activation (i.e., synthetic lethal and cell proliferation). Challenges associated with FUSION and other high-content genome-wide screens include variation, batch effects, and controlling for potential off-target effects. In this review, we discuss the efficacy of FUSION to identify novel inhibitors and oncogene-induced changes that may be cancer cell-specific as well as several potential pitfalls within FUSION and best practices to avoid them.

High-throughput identification of protein functional similarities using a gene-expression-based siRNA screen.

A gene expression-based siRNA screen was used to evaluate functional similarity between genetic perturbations to identify functionally similar proteins. A siRNA library (siGenome library, Dharmacon) consisting of multiple siRNAs per gene that have been pooled in to one well per gene was arrayed in a 384-well format and used to individually target 14,335 proteins for depletion in HCT116 colon cancer cells. For each protein depletion, the gene expression of eight genes was quantified using the multiplexed Affymetrix Quantigene 2.0 assay in technical triplicate. As a proof of concept, six genes (BNIP3, NDRG1, ALDOC, LOXL2, ACSL5, BNIP3L) whose expression pattern reliably reflect the disruption of the molecular scaffold KSR1 were measured upon each protein depletion. The remaining two genes (PPIB and HPRT) are housekeeping genes used for normalization. The gene expression signatures from this screen can be used to estimate the functional similarity between any two proteins and successfully identified functional relationships for specific proteins such as KSR1 and more generalized processes, such as autophagy.

The Proteomic Landscape of Pancreatic Ductal Adenocarcinoma Liver Metastases Identifies Molecular Subtypes and Associations with Clinical Response.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease that can be separated into distinct subtypes based on molecular signatures. Identifying PDAC subtype-specific therapeutic vulnerabilities is necessary to develop precision medicine approaches to treat PDAC. EXPERIMENTAL DESIGN: A total of 56 PDAC liver metastases were obtained from the UNMC Rapid Autopsy Program and analyzed with quantitative proteomics. PDAC subtypes were identified by principal component analysis based on protein expression profiling. Proteomic subtypes were further characterized by the associated clinical information, including but not limited to survival analysis, drug treatment response, and smoking and drinking status. RESULTS: Over 3,960 proteins were identified and used to delineate four distinct PDAC microenvironment subtypes: (i) metabolic; (ii) progenitor-like; (iii) proliferative; and (iv) inflammatory. PDAC risk factors of alcohol and tobacco consumption correlate with subtype classifications. Enhanced survival is observed in FOLFIRINOX treated metabolic and progenitor-like subtypes compared with the proliferative and inflammatory subtypes. In addition, TYMP, PDCD6IP, ERAP1, and STMN showed significant association with patient survival in a subtype-specific manner. Gemcitabine-induced alterations in the proteome identify proteins, such as serine hydroxymethyltransferase 1, associated with drug resistance. CONCLUSIONS: These data demonstrate that proteomic analysis of clinical PDAC liver metastases can identify molecular signatures unique to disease subtypes and point to opportunities for therapeutic development to improve the treatment of PDAC.

MnTnBuOE-2-PyP treatment protects from radioactive iodine (I-131) treatment-related side effects in thyroid cancer.

Treatment of differentiated thyroid cancer often involves administration of radioactive iodine (I-131) for remnant ablation or adjuvant therapy. However, there is morbidity associated with I-131 therapy, which can result in both acute and chronic complications. Currently, there are no approved radioprotectors that can be used in conjunction with I-131 to reduce complications in thyroid cancer therapy. It is well known that the damaging effects of ionizing radiation are mediated, in part, by the formation of reactive oxygen species (ROS). A potent scavenger of ROS, Mn(III)meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP), has radioprotective and anti-tumor effects in various cancer models including head and neck, prostate, and brain tumors exposed to external beam radiation therapy. Female C57BL/6 mice were administered I-131 orally at doses of 0.0085-0.01 mCi/g (3.145 × 105 to 3.7 × 105 Bq) of body weight with or without MnTnBuOE-2-PyP. We measured acute external inflammation, blood cell counts, and collected thyroid tissue and salivary glands for histological examination. We found oral administration of I-131 caused an acute decrease in platelets and white blood cells, caused facial swelling, and loss of thyroid and salivary tissues. However, when MnTnBuOE-2-PyP was given during and after I-131 administration, blood cell counts remained in the normal range, less facial inflammation was observed, and the salivary glands were protected from radiation-induced killing. These data indicate that MnTnBuOE-2-PyP may be a potent radioprotector of salivary glands in thyroid cancer patients receiving I-131 therapy.

A Genome-wide Functional Signature Ontology Map and Applications to Natural Product Mechanism of Action Discovery.

Gene expression signature-based inference of functional connectivity within and between genetic perturbations, chemical perturbations, and disease status can lead to the development of actionable hypotheses for gene function, chemical modes of action, and disease treatment strategies. Here, we report a FuSiOn-based genome-wide integration of hypomorphic cellular phenotypes that enables functional annotation of gene network topology, assignment of mechanistic hypotheses to genes of unknown function, and detection of cooperativity among cell regulatory systems. Dovetailing genetic perturbation data with chemical perturbation phenotypes allowed simultaneous generation of mechanism of action hypotheses for thousands of uncharacterized natural products fractions (NPFs). The predicted mechanism of actions span a broad spectrum of cellular mechanisms, many of which are not currently recognized as "druggable." To enable use of FuSiOn as a hypothesis generation resource, all associations and analyses are available within an open source web-based GUI (http://fusion.yuhs.ac).

ERK-mediated TIMELESS expression suppresses G2/M arrest in colon cancer cells.

The cell cycle is under circadian regulation. Oncogenes can dysregulate circadian-regulated genes to disrupt the cell cycle, promoting tumor cell proliferation. As a regulator of G2/M arrest in response to DNA damage, the circadian gene Timeless Circadian Clock (TIMELESS) coordinates this connection and is a potential locus for oncogenic manipulation. TIMELESS expression was evaluated using RNASeq data from TCGA and by RT-qPCR and western blot analysis in a panel of colon cancer cell lines. TIMELESS expression following ERK inhibition was examined via western blot. Cell metabolic capacity, propidium iodide, and CFSE staining were used to evaluate the effect of TIMELESS depletion on colon cancer cell survival and proliferation. Cell metabolic capacity following TIMELESS depletion in combination with Wee1 or CHK1 inhibition was assessed. TIMELESS is overexpressed in cancer and required for increased cancer cell proliferation. ERK activation promotes TIMELESS expression. TIMELESS depletion increases γH2AX, a marker of DNA damage, and triggers G2/M arrest via increased CHK1 and CDK1 phosphorylation. TIMELESS depletion in combination with Wee1 or CHK1 inhibition causes an additive decrease in cancer cell metabolic capacity with limited effects in non-transformed human colon epithelial cells. The data show that ERK activation contributes to the overexpression of TIMELESS in cancer. Depletion of TIMELESS increases γH2AX and causes G2/M arrest, limiting cell proliferation. These results demonstrate a role for TIMELESS in cancer and encourage further examination of the link between circadian rhythm dysregulation and cancer cell proliferation.

RAS genes in colorectal carcinoma: pathogenesis, testing guidelines and treatment implications.

The RAS family is among the most commonly mutated genes in all human malignancies including colon cancer. In normal cells, RAS proteins act as a link in the intracellular signal transduction initiated by binding of growth factors to cell membrane receptors mediating cell survival. RAS isoproteins have great morphological similarities, but despite that, they are thought to have different functions in different tissues. RAS mutations, as supported by several studies including animal models, have a role in the development and progression of colorectal cancer. The detection of RAS mutations in patients with colorectal carcinoma, specifically KRAS and NRAS, has significant clinical implications. It is currently recommended that patients with colon cancer who are considered for antiepidermal growth factor receptor monoclonal antibodies, get RAS mutation testing since only those with wildtype-RAS genes benefit from such treatment. Despite decades of research, there is currently no effective and safe treatment that directly targets RAS-mutated neoplasms. Multiple therapeutic approaches directed against RAS mutations are currently experimental, including a promising immunotherapy study using T-cells in patients with metastatic colon cancer.

WDR5 supports colon cancer cells by promoting methylation of H3K4 and suppressing DNA damage.

BACKGROUND: KMT2/MLL proteins are commonly overexpressed or mutated in cancer and have been shown to support cancer maintenance. These proteins are responsible for methylating histone 3 at lysine 4 and promoting transcription and DNA synthesis; however, they are inactive outside of a multi-protein complex that requires WDR5. WDR5 has been implicated in cancer for its role in the COMPASS complex and its interaction with Myc; however, the role of WDR5 in colon cancer has not yet been elucidated. METHODS: WDR5 expression was evaluated using RT-qPCR and western blot analysis. Cell viability and colony forming assays were utilized to evaluate the effects of WDR5 depletion or inhibition in colon cancer cells. Downstream effects of WDR5 depletion and inhibition were observed by western blot. RESULTS: WDR5 is overexpressed in colon tumors and colon cancer cell lines at the mRNA and protein level. WDR5 depletion reduces cell viability in HCT116, LoVo, RKO, HCT15, SW480, SW620, and T84 colon cancer cells. Inhibition of the WDR5:KMT2/MLL interaction using OICR-9429 reduces cell viability in the same panel of cell lines albeit not to the same extent as RNAi-mediated WDR5 depletion. WDR5 depletion reduced H3K4Me3 and increased phosphorylation of H2AX in HCT116, SW620, and RKO colon cancer cells; however, OICR-9429 treatment did not recapitulate these effects in all cell lines potentially explaining the reduced toxicity of OICR-9429 treatment as compared to WDR5 depletion. WDR5 depletion also sensitized colon cancer cells to radiation-induced DNA damage. CONCLUSIONS: These data demonstrate a clear role for WDR5 in colon cancer and future studies should examine its potential to serve as a therapeutic target in cancer. Additional studies are needed to fully elucidate if the requirement for WDR5 is independent of or consistent with its role within the COMPASS complex. OICR-9429 treatment was particularly toxic to SW620 and T84 colon cancer cells, two cell lines without mutations in WDR5 and KMT2/MLL proteins suggesting COMPASS complex inhibition may be particularly effective in tumors lacking KMT2 mutations. Additionally, the ability of WDR5 depletion to amplify the toxic effects of radiation presents the possibility of targeting WDR5 to sensitize cells to DNA-damaging therapies.