Purpose: Autophagy is a critical survival pathway for cancer cells under conditions of nutrient or oxygen limitation, or cell stress. As a consequence of antiangiogenic therapy, solid tumors encounter hypoxia induction and imbalances in nutrient supply. We wished to determine the role of autophagy in protection of tumor cells from the effects of antiangiogenic therapy and chemotherapy. We examined the effect of inhibiting autophagy on hypoxic colon cancer cells in vitro and on bevacizumab-and oxaliplatin-treated mouse xenografts in vivo.Experimental Design: The autophagic response to hypoxia and DNA-damaging agents was assessed by fluorescent microscopic imaging, autophagy-related gene expression, and by electron microscopic ultrastructural analysis. Pharmacologic and molecular approaches to autophagy inhibition were taken in a panel of colon cancer cell lines. Mouse xenograft models were treated with combinations of oxaliplatin, bevacizumab, and chloroquine to assess effects on tumor growth reduction and on pharmacodynamic markers of autophagy inhibition.Results: Autophagy was induced in colon cancer models by exposure to both hypoxia and oxaliplatin. Inhibition of autophagy, either with chloroquine or by downregulation of beclin1 or of ATG5, enhanced sensitivity to oxaliplatin under normal and hypoxic conditions in a synergistic manner. Both bevacizumab and oxaliplatin treatments activate autophagy in HT29 murine xenografts. The addition of chloroquine to bevacizumab-based treatment provided greater tumor control in concert with evidence of autophagy inhibition.Conclusions: These findings implicate autophagy as a mechanism of resistance to antiangiogenic therapies and support investigation of inhibitory approaches in the management of this disease.

Mechanisms regulating DNA repair processes remain incompletely defined. Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target.

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Emerging evidence indicates the deubiquitinase USP22 regulates transcriptional activation and modification of target substrates to promote pro-oncogenic phenotypes. Here, in vivo characterization of tumor-associated USP22 upregulation and unbiased interrogation of USP22-regulated functions in vitro demonstrated critical roles for USP22 in prostate cancer. Specifically, clinical datasets validated that USP22 expression is elevated in prostate cancer, and a novel murine model demonstrated a hyperproliferative phenotype with prostate-specific USP22 overexpression. Accordingly, upon overexpression or depletion of USP22, enrichment of cell-cycle and DNA repair pathways was observed in the USP22-sensitive transcriptome and ubiquitylome using prostate cancer models of clinical relevance. Depletion of USP22 sensitized cells to genotoxic insult, and the role of USP22 in response to genotoxic insult was further confirmed using mouse adult fibroblasts from the novel murine model of USP22 expression. As it was hypothesized that USP22 deubiquitylates target substrates to promote protumorigenic phenotypes, analysis of the USP22-sensitive ubiquitylome identified the nucleotide excision repair protein, XPC, as a critical mediator of the USP22-mediated response to genotoxic insult. Thus, XPC undergoes deubiquitylation as a result of USP22 function and promotes USP22-mediated survival to DNA damage. Combined, these findings reveal unexpected functions of USP22 as a driver of protumorigenic phenotypes and have significant implications for the role of USP22 in therapeutic outcomes. Significance:The studies herein present a novel mouse model of tumor-associated USP22 overexpression and implicate USP22 in modulation of cellular survival and DNA repair, in part through regulation of XPC.

Loss of the retinoblastoma (RB) tumor suppressor protein is a critical step in reprogramming biological networks that drive cancer progression, although mechanistic insight has been largely limited to the impact of RB loss on cell cycle regulation. Here, isogenic modeling of RB loss identified disease stage-specific rewiring of E2F1 function, providing the first-in-field mapping of the E2F1 cistrome and transcriptome after RB loss across disease progression. Biochemical and functional assessment using both in vitro and in vivo models identified an unexpected, prominent role for E2F1 in regulation of redox metabolism after RB loss, driving an increase in the synthesis of the antioxidant, glutathione, specific to advanced disease. These E2F1-dependent events resulted in protection from reactive oxygen species (ROS) in response to therapeutic intervention. On balance, these findings reveal novel pathways through which RB loss promotes cancer progression and highlight potentially new nodes of intervention for treating RB-deficient cancers.Research.