An integrative gene expression signature analysis identifies CMS4 KRAS-mutated colorectal cancers sensitive to combined MEK and SRC targeted therapy.

BACKGROUND: Over half of colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors, has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms that are not well understood. This study aims to investigate MEK inhibitor-associated resistance signaling and identify subpopulation(s) of CRC patients who may be sensitive to biomarker-driven drug combination(s). METHODS: We classified 2250 primary and metastatic human CRC tumors by consensus molecular subtypes (CMS). For each tumor, we generated multiple gene expression signature scores measuring MEK pathway activation, MEKi "bypass" resistance, SRC activation, dasatinib sensitivity, EMT, PC1, Hu-Lgr5-ISC, Hu-EphB2-ISC, Hu-Late TA, Hu-Proliferation, and WNT activity. We carried out correlation, survival and other bioinformatic analyses. Validation analyses were performed in two independent publicly available CRC tumor datasets (n = 585 and n = 677) and a CRC cell line dataset (n = 154). RESULTS: Here we report a central role of SRC in mediating "bypass"-resistance to MEK inhibition (MEKi), primarily in cancer stem cells (CSCs). Our integrated and comprehensive gene expression signature analyses in 2250 CRC tumors reveal that MEKi-resistance is strikingly-correlated with SRC activation (Spearman P < 10-320), which is similarly associated with EMT (epithelial to mesenchymal transition), regional metastasis and disease recurrence with poor prognosis. Deeper analysis shows that both MEKi-resistance and SRC activation are preferentially associated with a mesenchymal CSC phenotype. This association is validated in additional independent CRC tumor and cell lines datasets. The CMS classification analysis demonstrates the strikingly-distinct associations of CMS1-4 subtypes with the MEKi-resistance and SRC activation. Importantly, MEKi + SRCi sensitivities are predicted to occur predominantly in the KRAS mutant, mesenchymal CSC-like CMS4 CRCs. CONCLUSIONS: Large human tumor gene expression datasets representing CRC heterogeneity can provide deep biological insights heretofore not possible with cell line models, suggesting novel repurposed drug combinations. We identified SRC as a common targetable node--an Achilles' heel--in MEKi-targeted therapy-associated resistance in mesenchymal stem-like CRCs, which may help development of a biomarker-driven drug combination (MEKi + SRCi) to treat problematic subpopulations of CRC.

Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis.

Autophagy is an evolutionarily conserved 'self-eating' process. Although the genes essential for autophagy (named Atg) have been identified in yeast, the molecular mechanism of how Atg proteins control autophagosome formation in mammalian cells remains to be elucidated. Here, we demonstrate that Bif-1 (also known as Endophilin B1) interacts with Beclin 1 through ultraviolet irradiation resistance-associated gene (UVRAG) and functions as a positive mediator of the class III PI(3) kinase (PI(3)KC3). In response to nutrient deprivation, Bif-1 localizes to autophagosomes where it colocalizes with Atg5, as well as microtubule-associated protein light chain 3 (LC3). Furthermore, loss of Bif-1 suppresses autophagosome formation. Although the SH3 domain of Bif-1 is sufficient for binding to UVRAG, both the BAR and SH3 domains are required for Bif-1 to activate PI(3)KC3 and induce autophagosome formation. We also observed that Bif-1 ablation prolongs cell survival under starvation conditions. Moreover, knockout of Bif-1 significantly enhances the development of spontaneous tumours in mice. These findings suggest that Bif-1 joins the UVRAG-Beclin 1 complex as a potential activator of autophagy and tumour suppressor.

The deubiquitylase USP7 is a novel cyclin F-interacting protein and regulates cyclin F protein stability.

Cyclin F, unlike canonical and transcriptional cyclins, does not bind or activate any cyclin-dependent kinases. Instead, it harbors an F-box motif and primarily functions as the substrate recognition subunit of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCFCyclin F. By targeting specific proteins for ubiquitin-mediated proteasomal degradation, cyclin F plays a critical role in the regulation of centrosomal duplication, DNA replication and repair, and maintenance of genomic stability. Cyclin F abundance and activity are tightly regulated throughout the cell cycle. However, the molecular mechanisms regulating cyclin F are scantily understood. Here, we identify the deubiquitylase USP7 as a novel cyclin F-interacting protein. We observe that USP7 stabilizes cyclin F protein and that this function is independent of the deubiquitylase activity of USP7. Additionally, our data suggest that USP7 is also involved in the regulation of cyclin F mRNA. Pharmacological inhibition of the deubiquitylase activity of USP7 resulted in downregulation of cyclin F mRNA.

Dasatinib inhibits migration and invasion in diverse human sarcoma cell lines and induces apoptosis in bone sarcoma cells dependent on SRC kinase for survival.

Sarcomas are rare malignant mesenchymal tumors for which there are limited treatment options. One potential molecular target for sarcoma treatment is the Src tyrosine kinase. Dasatinib (BMS-354825), a small-molecule inhibitor of Src kinase activity, is a promising cancer therapeutic agent with p.o. bioavailability. Dasatinib exhibits antitumor effects in cultured human cell lines derived from epithelial tumors, including prostate and lung carcinomas. However, the action of dasatinib in mesenchymally derived tumors has yet to be shown. Based on our previous findings of Src activation in human sarcomas, we evaluated the effects of dasatinib in 12 cultured human sarcoma cell lines derived from bone and soft tissue sarcomas. Dasatinib inhibited Src kinase activity at nanomolar concentrations in these sarcoma cell lines. Downstream components of Src signaling, including focal adhesion kinase and Crk-associated substrate (p130(CAS)), were also inhibited at similar concentrations. This inhibition of Src signaling was accompanied by blockade of cell migration and invasion. Moreover, apoptosis was induced in the osteosarcoma and Ewing's subset of bone sarcomas at nanomolar concentrations of dasatinib. Inhibition of Src protein expression by small interfering RNA also induced apoptosis, indicating that these bone sarcoma cell lines are dependent on Src activity for survival. These results show that dasatinib inhibits migration and invasion of diverse sarcoma cell types and selectively blocks the survival of bone sarcoma cells. Therefore, dasatinib may provide therapeutic benefit by preventing the growth and metastasis of sarcomas in patients.

Repurposing EGFR Inhibitor Utility in Colorectal Cancer in Mutant APC and TP53 Subpopulations.

BACKGROUND: EGFR is a major therapeutic target for colorectal cancer. Currently, extended RAS/RAF testing identifies only nonresponders to EGFR inhibitors (EGFRi). We aimed to develop a mutation signature that further refines drug-sensitive subpopulations to improve EGFRi outcomes. METHODS: A prespecified, 203-gene expression signature score measuring cetuximab sensitivity (CTX-S) was validated with two independent clinical trial datasets of cetuximab-treated patients with colorectal cancer (n = 44 and n = 80) as well as an in vitro dataset of 147 cell lines. The CTX-S score was then used to decipher mutated genes that predict EGFRi sensitivity. The predictive value of the identified mutation signature was further validated by additional independent datasets. RESULTS: Here, we report the discovery of a 2-gene (APC+TP53) mutation signature that was useful in identifying EGFRi-sensitive colorectal cancer subpopulations. Mutant APC+TP53 tumors were more predominant in left- versus right-sided colorectal cancers (52% vs. 21%, P = 0.0004), in microsatellite stable (MSS) versus microsatellite instable (MSI) cases (47% vs. 2%, P < 0.0001), and in the consensus molecular subtype 2 versus others (75% vs. 37%, P < 0.0001). Moreover, mutant APC+TP53 tumors had favorable outcomes in two cetuximab-treated patient-derived tumor xenograft (PDX) datasets (P = 0.0277, n = 52; P = 0.0008, n = 98). CONCLUSIONS: Our findings suggest that the APC and TP53 combination mutation may account for the laterality of EGFRi sensitivity and provide a rationale for refining treated populations. The results also suggest addition of APC+TP53 sequencing to extended RAS/RAF testing that may directly increase the response rates of EGFRi therapy in selected patients. IMPACT: These findings, if further validated through clinical trials, could also expand the utility of EGFRi therapies that are currently underutilized.

PTPRS drives adaptive resistance to MEK/ERK inhibitors through SRC.

PTPRS is the most commonly mutated receptor tyrosine phosphatase in colorectal cancer (CRC). PTPRS has been shown to directly affect ERK and regulate its activation and nuclear localization. Here we identify that PTPRS may play a significant role in developing adaptive resistance to MEK/ERK inhibitors (MEKi/ERKi) through SRC activation. Moreover, we demonstrate a new clinical approach to averting adaptive resistance through the use of the SRC inhibitor, dasatinib. Our data suggest the potential for dasatinib to enhance the efficacy of MEKi and ERKi by preventing adaptive resistance pathways operating through SRC.

Roscovitine inhibits differentiation and invasion in a three-dimensional skin reconstruction model of metastatic melanoma.

The aim of this study was to investigate the therapeutic potential of a cyclin-dependent kinase inhibitor, roscovitine, in cultured melanoma cells and a three-dimensional skin reconstruction model of metastatic melanoma. The modulatory effects of roscovitine on the growth and survival of normal melanocytes and cultured melanoma cell lines were tested. Additionally, we investigated the potential of roscovitine to regulate the growth and differentiation of a metastatic melanoma cell line (A375) in a three-dimensional skin reconstruction culture consisting of A375 cells admixed with normal human keratinocytes embedded within a collagen-constricted fibroblast matrix. We show that roscovitine is able to induce apoptosis in the melanoma cell lines A375, 888, and 624 but not in normal human cultured epithelial melanocytes. The degree of apoptosis within these cell lines correlated with the accumulation of p53 protein and concomitant reduction of X-linked inhibitor of apoptosis protein, with no change in the proteins Bcl-2 and survivin. We also found that roscovitine inhibited the growth and differentiation of A375 melanoma cells within the dermal layer of the skin. The results of this study show that roscovitine has the potential to inhibit the differentiation and invasion of metastatic melanoma and may be useful as a therapy for the treatment of patients with metastatic melanoma.

PTPRS Regulates Colorectal Cancer RAS Pathway Activity by Inactivating Erk and Preventing Its Nuclear Translocation.

Colorectal cancer (CRC) growth and progression is frequently driven by RAS pathway activation through upstream growth factor receptor activation or through mutational activation of KRAS or BRAF. Here we describe an additional mechanism by which the RAS pathway may be modulated in CRC. PTPRS, a receptor-type protein tyrosine phosphatase, appears to regulate RAS pathway activation through ERK. PTPRS modulates ERK phosphorylation and subsequent translocation to the nucleus. Native mutations in PTPRS, present in ~10% of CRC, may reduce its phosphatase activity while increasing ERK activation and downstream transcriptional signaling.

p21Cip1 nullizygosity increases tumor metastasis in irradiated mice.

p21(Cip1) is a cyclin-dependent kinase inhibitor whose abundance increases in cells exposed to radiation or other DNA-damaging agents. Such increases activate a G1 checkpoint, which allows time for DNA repair before S phase entry. By inhibiting cell cycle progression, p21(Cip1) potentially suppresses tumorigenesis, and in support, we show that p21(Cip1) heterozygous and nullizygous mice develop more tumors than do wild-type mice when exposed to a single dose of gamma-irradiation. Importantly, we also show that p21(Cip1) nullizygosity increases the incidence of metastatic tumors in irradiated mice. We suggest that p21(Cip1) is haploinsufficient for tumor suppression and functions as an antimetastatic agent.

Assessing needs and assets for building a regional network infrastructure to reduce cancer related health disparities.

Significant cancer health disparities exist in the United States and Puerto Rico. While numerous initiatives have been implemented to reduce cancer disparities, regional coordination of these efforts between institutions is often limited. To address cancer health disparities nation-wide, a series of regional transdisciplinary networks through the Geographic Management Program (GMaP) and the Minority Biospecimen/Biobanking Geographic Management Program (BMaP) were established in six regions across the country. This paper describes the development of the Region 3 GMaP/BMaP network composed of over 100 investigators from nine institutions in five Southeastern states and Puerto Rico to develop a state-of-the-art network for cancer health disparities research and training. We describe a series of partnership activities that led to the formation of the infrastructure for this network, recount the participatory processes utilized to develop and implement a needs and assets assessment and implementation plan, and describe our approach to data collection. Completion, by all nine institutions, of the needs and assets assessment resulted in several beneficial outcomes for Region 3 GMaP/BMaP. This network entails ongoing commitment from the institutions and institutional leaders, continuous participatory and engagement activities, and effective coordination and communication centered on team science goals.

Proliferative status regulates HDAC11 mRNA abundance in nontransformed fibroblasts.

Histone deacetylases (HDACs) are important determinants of gene transcription and other biological processes. HDAC11 is one of the least characterized HDACs and is the only member of the class IV HDAC family. Our studies examined the events that control the expression of the HDAC11 transcript. We show that platelet-derived growth factor (PDGF) rapidly reduces the abundance of HDAC11 mRNA when added to density-arrested Balb/c-3T3 cells, which are nontransformed fibroblasts. Reduction required mRNA and protein synthesis, but not AKT or ERK activity, and resulted from accelerated turnover of the HDAC11 transcript. Reduction was transient in cells receiving PDGF alone but sustained in cells receiving both PDGF and platelet-poor plasma, which together promote G0/G1 traverse and S phase entry. Plasma alone did not appreciably reduce HDAC11 mRNA abundance, nor did epidermal growth factor, insulin-like growth factor, or insulin. HDAC11 mRNA accumulated in Balb/c-3T3 cells exiting the cell cycle due to density-dependent growth inhibition or serum deprivation. Of note, HDAC11 mRNA did not accumulate in a spontaneously transformed Balb/c-3T3 clonal variant (clone 2) that does not density arrest. The HDAC11 promoter was active in Balb/c-3T3 but not clone 2 cells; inactivity in clone 2 cells did not result from methylation of CpG islands. Overexpression of HDAC11 inhibited the cell cycle progression of both transformed and nontransformed fibroblasts. Our studies identify the HDAC11 transcript as a PDGF target and show that HDAC11 mRNA abundance correlates inversely with proliferative status.

The Establishment of the First Cancer Tissue Biobank at a Hispanic-Serving Institution: A National Cancer Institute-Funded Initiative between Moffitt Cancer Center in Florida and the Ponce School of Medicine and Health Sciences in Puerto Rico.

Population-based studies are important to address emerging issues in health disparities among populations. The Partnership between the Moffitt Cancer Center (MCC) in Florida and the Ponce School of Medicine and Health Sciences (PSMHS) in Puerto Rico (the PSMHS-MCC Partnership) was developed to facilitate high-quality research, training, and community outreach focusing on the Puerto Rican population in the island and in the mainland, with funding from the National Cancer Institute. We report here the establishment of a Tissue Biobank at PSMHS, modeled after the MCC tissue biorepository, to support translational research projects on this minority population. This facility, the Puerto Rico Tissue Biobank, was jointly developed by a team of basic and clinical scientists from both institutions in close collaboration with the administrators and clinical faculty of the tissue accrual sites. The efforts required and challenges that needed to be overcome to establish the first functional, centralized cancer-related biobank in Puerto Rico, and to ensure that it continuously evolves to address new needs of this underserved Hispanic population, are described. As a result of the collaborative efforts between PSMHS and MCC, a tissue procurement algorithm was successfully established to acquire, process, store, and conduct pathological analyses of cancer-related biospecimens and their associated clinical-pathological data from Puerto Rican patients with cancer recruited at a tertiary hospital setting. All protocols in place are in accordance with standard operational procedures that ensure high quality of biological materials and patient confidentiality. The processes described here provide a model that can be applied to achieve the establishment of a functional biobank in similar settings.

Identification of Aurora-A as a direct target of E2F3 during G2/M cell cycle progression.

Aurora-A is a centrosome kinase and plays a pivotal role in G(2)/M cell cycle progression. Expression of Aurora-A is cell cycle-dependent. Levels of Aurora-A mRNA and protein are low in G(1)/S, accumulate during G(2)/M, and decrease rapidly after mitosis. Previous studies have shown regulation of the Aurora-A protein level during the cell cycle through the ubiquitin-proteasome pathway. However, the mechanism of transcriptional regulation of Aurora-A remains largely unknown. Here, we demonstrated that E2F3 modulates Aurora-A mRNA expression during the cell cycle. Ectopic expression of E2F3 induces Aurora-A expression. Stable knockdown of E2F3 decreases mRNA and protein levels of Aurora-A and delays G(2)/M entry. Further, E2F3 directly binds to Aurora-A promoter and stimulates the promoter activity. Deletion and mutation analyses of the Aurora-A promoter revealed that a region located 96-bp upstream of the transcription initiation site is critical for the activation of the promoter by E2F3. In addition, expression of E2F3 positively correlates with the protein level of Aurora-A in human ovarian cancer examined. These results indicate for the first time that Aurora-A is transcriptionally regulated by E2F3 during the cell cycle and that E2F3 is a causal factor for up-regulation of Aurora-A in a subset of human ovarian cancer. Thus, the E2F3-Aurora-A axis could be an important target for cancer intervention.

Loss of nuclear p21(Cip1/WAF1) during neoplastic progression to metastasis in gamma-irradiated p21 hemizygous mice.

p21(Cip1/WAF1) localizes to the nucleus in response to gamma-irradiation induced DNA damage and mediates a G(1) checkpoint arrest. Although gamma-irradiated p21(+/-) mice develop a broad spectrum of tumors, gamma-irradiated p21(-/-) mice develop significantly more metastatic cancers. To evaluate the expression of p21 in tissues prone or resistant to tumorigenesis as a function of gamma-irradiation, and to determine whether phenotypic loss of p21 heterozygosity occurs during tumor progression in p21(+/-) mice, tissues and tumors from gamma-irradiated mice were evaluated immunohistochemically. The percentage of tumors in p21(+/-) mice that were nuclear p21-positive declined with progression to metastasis (p<0.0001). Benign tumors were more often p21-positive and comprised of larger subsets of nuclear p21-positive cells than were malignant tumors of the same histopathological type, while metastatic cancers were nuclear p21-negative (p=0.0003). Even when a primary cancer was comprised of a subset of nuclear p21-positive cells, the metastatic foci of that same cancer were nuclear p21-negative. Mesenchymal tumors, though rare, were more likely metastatic than were epithelial tumors (p=0.0004), and these were invariably nuclear p21-negative. Prepubescent epithelial tissues from which most tumors later originated in mice with reduced p21 gene dosage (i.e., harderian gland, ovary, small intestine, and lung) were p21 expressive within 4 h of gamma-irradiation (p=0.0625), so that p21/Ki67 ratios increased post-gamma-irradiation (p=0.03). In contrast, p21 did not localize to nuclei of cortical thymocytes, a tissue where tumorigenesis was not augmented by reduced p21 gene dosage. Cellular subclones of malignant tumors, especially those of mesenchymal cell origin, which lack nuclear p21 may more readily acquire the genetic alterations of the metastatic phenotype.

Activation of p27Kip1 Expression by E2F1. A negative feedback mechanism.

The E2F1 transcription factor is a critical regulator of cell cycle due to its ability to promote S phase entry. However, E2F1 overexpression also sensitizes cells to apoptosis and E2F1-null mice are predisposed to tumor development, suggesting that it also has properties of a growth suppressor. E2F1 transcription function is regulated by interaction with hypophosphorylated pRb. Cdk inhibitors such as p16INK4a and p27Kip1 inhibit pRb phosphorylation by the cyclin D/Cdk4 and cyclin E/Cdk2 complexes, thus keeping E2F1 in an inactive state. We found that E2F1 binds to the p27 promoter in vivo and activates p27 mRNA and protein expression. Depletion of endogenous E2F1 by siRNA causes a reduction in basal p27 expression level. Inhibition of endogenous p27 expression by siRNA increases E2F1 transcriptional activity and permits accelerated cell cycle progression by exogenous E2F1. These observations suggest that induction of p27 acts as a negative feedback mechanism for E2F1 and may also contribute to other functions of E2F1.