PTE, a novel module to target Polycomb Repressive Complex 1 to the human cyclin D2 (CCND2) oncogene.

Polycomb group proteins are essential epigenetic repressors. They form multiple protein complexes of which two kinds, PRC1 and PRC2, are indispensable for repression. Although much is known about their biochemical properties, how mammalian PRC1 and PRC2 are targeted to specific genes is poorly understood. Here, we establish the cyclin D2 (CCND2) oncogene as a simple model to address this question. We provide the evidence that the targeting of PRC1 to CCND2 involves a dedicated PRC1-targeting element (PTE). The PTE appears to act in concert with an adjacent cytosine-phosphate-guanine (CpG) island to arrange for the robust binding of PRC1 and PRC2 to repressed CCND2 Our findings pave the way to identify sequence-specific DNA-binding proteins implicated in the targeting of mammalian PRC1 complexes and provide novel link between polycomb repression and cancer.

Chronic hypoxia favours adoption to a castration-resistant cell state in prostate cancer.

Predicting and treating recurrence in intermediate-risk prostate cancer patients remains a challenge despite having identified genomic instability [1] and hypoxia [2, 3] as risk factors. This underlies challenges in assigning the functional impact of these risk factors to mechanisms promoting prostate cancer progression. Here we show chronic hypoxia (CH), as observed in prostate tumours [4], leads to the adoption of an androgen-independent state in prostate cancer cells. Specifically, CH results in prostate cancer cells adopting transcriptional and metabolic alterations typical of castration-resistant prostate cancer cells. These changes include the increased expression of transmembrane transporters for the methionine cycle and related pathways leading to increased abundance of metabolites and expression of enzymes related to glycolysis. Targeting of the Glucose Transporter 1 (GLUT1) identified a dependency on glycolysis in androgen-independent cells. Overall, we identified a therapeutically targetable weakness in chronic hypoxia and androgen-independent prostate cancer. These findings may offer additional strategies for treatment development against hypoxic prostate cancer.

Focal overexpression of CEACAM6 contributes to enhanced tumourigenesis in head and neck cancer via suppression of apoptosis.

BACKGROUND: Overexpression of CEACAM6 has been reported for a number of malignancies. However, the mechanism of how CEACAM6 contributes to cancer formation and its role in head and neck squamous cell carcinoma (HNSCC) remains unclear. Therefore, we examined the role of CEACAM6 in head and neck squamous cell carcinoma (HNSCC). METHODS: CEACAM6 expression was examined in normal squamous epithelia as well as a number of patient HNSCC samples and tumours derived from HNSCC cell lines injected into NOD/SCID mice. CEACAM6 expression was manipulated in HNSCC cell lines by shRNA-mediated CEACAM6 knockdown or virally-delivered overexpression of CEACAM6. The role of CEACAM6 in tumour growth and chemotherapeutic sensitivity was then assessed in vivo and in vitro respectively. RESULTS: CEACAM6 expression was significantly increased in highly tumourigenic HNSCC cell lines when compared to poorly tumourigenic HNSCC cell lines. Moreover, HNSCC patient tumours demonstrated focal expression of CEACAM6. Functional investigation of CEACAM6, involving over-expression and knock down studies, demonstrated that CEACAM6 over-expression could enhance tumour initiating activity and tumour growth via activation of AKT and suppression of caspase-3 mediated cell death. CONCLUSION: We report that CEACAM6 is focally overexpressed in a large fraction of human HNSCCs in situ. We also show that over-expression of CEACAM6 increases tumour growth and tumour initiating activity by suppressing PI3K/AKT-dependent apoptosis of HNSCC in a xenotransplant model of HNSCC. Finally, our studies indicate that foci of CEACAM6 expressing cells are selectively ablated by treatment of xenotransplant tumours with pharmacological inhibitors of PI3K/AKT in vivo.

Tumor-initiating activity and tumor morphology of HNSCC is modulated by interactions between clonal variants within the tumor.

Tumor initiation (TI) in xenotransplantation models of head and neck squamous cell carcinoma (HNSCC) is an inefficient process. Poor TI could be due to (1) posttransplant cell loss, (2) a rare sub-population of cancer stem cells or (3) a requirement for specific cellular interactions, which rely on cell number. By tracking GFP-expressing HNSCC cells, we conclude that the posttransplant loss of cancer cells is minimal in the xenotransplant model. Furthermore, an examination of putative cancer stem cell markers (such as CD133, CD44, SP and label retention) in HNSCC cell lines revealed no correlation between marker expression and tumorigenicity. In addition, single-cell clones randomly isolated from HNSCC cell lines and then transplanted into mice were all capable of initiating tumors with efficiencies varying almost 34-fold. As the observed variation in the clones was both more and less tumorigenic than the parental cells, a combination of two clones, at suboptimal cell numbers for TI, was implanted into mice and was found to modulate the tumor-initiating activity, thus indicating that TI is dependent on a 'critical' number of cells and, for the first time, that interactions between clonal variants within tumors can modulate the overall tumor-initiating activity. Put in context with previous literature on tumorigenic activity, we believe that interactions between clonal variants within a tumor as well as (1) stromal interactions, (2) angiogenic activity, (3) immunocompetence and (4) cancer stem cells may all contribute to tumorigenic potential and the propensity for tumor growth and recurrence.

GLUT1 inhibition blocks growth of RB1-positive triple negative breast cancer.

Triple negative breast cancer (TNBC) is a deadly form of breast cancer due to the development of resistance to chemotherapy affecting over 30% of patients. New therapeutics and companion biomarkers are urgently needed. Recognizing the elevated expression of glucose transporter 1 (GLUT1, encoded by SLC2A1) and associated metabolic dependencies in TNBC, we investigated the vulnerability of TNBC cell lines and patient-derived samples to GLUT1 inhibition. We report that genetic or pharmacological inhibition of GLUT1 with BAY-876 impairs the growth of a subset of TNBC cells displaying high glycolytic and lower oxidative phosphorylation (OXPHOS) rates. Pathway enrichment analysis of gene expression data suggests that the functionality of the E2F pathway may reflect to some extent OXPHOS activity. Furthermore, the protein levels of retinoblastoma tumor suppressor (RB1) strongly correlate with the degree of sensitivity to GLUT1 inhibition in TNBC, where RB1-negative cells are insensitive to GLUT1 inhibition. Collectively, our results highlight a strong and targetable RB1-GLUT1 metabolic axis in TNBC and warrant clinical evaluation of GLUT1 inhibition in TNBC patients stratified according to RB1 protein expression levels.

Noncoding mutations target cis-regulatory elements of the FOXA1 plexus in prostate cancer.

Prostate cancer is the second most commonly diagnosed malignancy among men worldwide. Recurrently mutated in primary and metastatic prostate tumors, FOXA1 encodes a pioneer transcription factor involved in disease onset and progression through both androgen receptor-dependent and androgen receptor-independent mechanisms. Despite its oncogenic properties however, the regulation of FOXA1 expression remains unknown. Here, we identify a set of six cis-regulatory elements in the FOXA1 regulatory plexus harboring somatic single-nucleotide variants in primary prostate tumors. We find that deletion and repression of these cis-regulatory elements significantly decreases FOXA1 expression and prostate cancer cell growth. Six of the ten single-nucleotide variants mapping to FOXA1 regulatory plexus significantly alter the transactivation potential of cis-regulatory elements by modulating the binding of transcription factors. Collectively, our results identify cis-regulatory elements within the FOXA1 plexus mutated in primary prostate tumors as potential targets for therapeutic intervention.

Inactivation of glutathione peroxidase activity contributes to UV-induced squamous cell carcinoma formation.

Cutaneous squamous cell carcinomas (CSCC) are a common malignancy of keratinocytes that arise in sites of the skin exposed to excessive UV radiation. In the present study, we show that human SCC cell lines, preneoplastic solar keratoses (SK), and CSCC are associated with perturbations in glutathione peroxidase (GPX) activity and peroxide levels. Specifically, we found that two of three SKs and four of five CSCCs, in vivo, were associated with decreased GPX activity and all SKs and CSCCs were associated with an elevated peroxide burden. Given the association of decreased GPX activity with CSCC, we examined the basis for the GPX deficiency in the CSCCs. Our data indicated that GPX was inactivated by a post-translational mechanism and that GPX could be inactivated by increases in intracellular peroxide levels. We next tested whether the decreased peroxidase activity coupled with an elevated peroxidative burden might contribute to CSCC formation in vivo. This was tested in Gpx1(-/-) and Gpx2(-/-) mice exposed to solar-simulated UV radiation. These studies showed that Gpx2 deficiency predisposed mice to UV-induced CSCC formation. These results suggest that inactivation of GPX2 in human skin may be an early event in UV-induced SCC formation.

Loss of E2F7 expression is an early event in squamous differentiation and causes derepression of the key differentiation activator Sp1.

Squamous differentiation is controlled by key transcription factors such as Sp1 and E2F. We have previously shown that E2F1 can suppress transcription of the differentiation-specific gene, transglutaminase type 1 (TG1), by an indirect mechanism mediated by Sp1. Transient transfection of E2F1-E2F6 indicated that E2F-mediated reduction of Sp1 transcription was not responsible for E2F-mediated suppression of squamous differentiation. However, we found that E2F4 and E2F7, but not E2Fs 1, 2, 3, 5, or 6, could suppress the activation of the Sp1 promoter in differentiated keratinocytes (KCs). E2F4-mediated suppression could not be antagonized by E2Fs 1, 2, 3, 5, or 6 and was localized to a region of the human Sp1 promoter spanning -139 to + 35 bp. Chromatin immunoprecipitation analysis, as well as transient overexpression and short hairpin RNA knockdown experiments indicate that E2F7 binds to a unique binding site located between -139 and -119 bp of the Sp1 promoter, and knockdown of E2F7 in proliferating KCs leads to a derepression of Sp1 expression and the induction of TG1. In contrast, E2F4 knockdown in proliferating KCs did not alter Sp1 expression. These data indicate that loss of E2F7 during the initiation of differentiation leads to the derepression of Sp1 and subsequent transcription of differentiation-specific genes such as TG1.