Tumors defective in homologous recombination rely on oxidative metabolism: relevance to treatments with PARP inhibitors

Mitochondrial metabolism and the generation of reactive oxygen species (ROS) contribute to the acquisition of DNA mutations and genomic instability in cancer. How genomic instability influences the metabolic capacity of cancer cells is nevertheless poorly understood. Here, we show that homologous recombination-defective (HRD) cancers rely on oxidative metabolism to supply NAD+ and ATP for poly(ADP-ribose) polymerase (PARP)-dependent DNA repair mechanisms. Studies in breast and ovarian cancer HRD models depict a metabolic shift that includes enhanced expression of the oxidative phosphorylation (OXPHOS) pathway and its key components and a decline in the glycolytic Warburg phenotype. Hence, HRD cells are more sensitive to metformin and NAD+ concentration changes. On the other hand, shifting from an OXPHOS to a highly glycolytic metabolism interferes with the sensitivity to PARP inhibitors (PARPi) in these HRD cells. This feature is associated with a weak response to PARP inhibition in patient-derived xenografts, emerging as a new mechanism to determine PARPi sensitivity. This study shows a mechanistic link between two major cancer hallmarks, which in turn suggests novel possibilities for specifically treating HRD cancers with OXPHOS inhibitors

p55, the Drosophila Ortholog of RbAp46/RbAp48, Is Required for the Repression of dE2F2/RBF-Regulated Genes

Many proteins have been proposed to be involved in retinoblastoma protein (pRB)-mediated repression, but it is largely uncertain which cofactors are essential for pRB to repress endogenous E2F-regulated promoters. Here we have taken advantage of the stream-lined Drosophila dE2F/RBF pathway, which has only two E2Fs (dE2F1 and dE2F2), and two pRB family members (RBF1 and RBF2). With RNA interference (RNAi), we depleted potential corepressors and looked for the elevated expression of groups of E2F target genes that are known to be directly regulated by RBF1 and RBF2. Previous studies have implicated histone deacetylase (HDAC) and SWI/SNF chromatin-modifying complexes in pRB-mediated repression. However, our results fail to support the idea that the SWI/SNF proteins are required for RBF-mediated repression and suggest that a requirement for HDAC activities is likely to be limited to a subset of targets. We found that the chromatin assembly factor p55/dCAF-1 is essential for the repression of dE2F2-regulated targets. The removal of p55 deregulated the expression of E2F targets that are normally repressed by dE2F2/RBF1 and dE2F2/RBF2 complexes in a cell cycle-independent manner but had no effect on the expression of E2F targets that are normally coupled with cell proliferation. The results indicate that the mechanisms of RBF regulation at these two types of E2F targets are different and suggest that p55, and perhaps p55's mammalian orthologs RbAp46 and RbAp48, have a conserved function in repression by pRB-related proteins

VEGFR3 Inhibition Chemosensitizes Ovarian Cancer Stemlike Cells through Down-Regulation of BRCA1 and BRCA2

In ovarian cancer, loss of BRCA gene expression in tumors is associated with improved response to chemotherapy and increased survival. A means to pharmacologically downregulate BRCA gene expression could improve the outcomes of patients with BRCA wild-type tumors. We report that vascular endothelial growth factor receptor 3 (VEGFR3) inhibition in ovarian cancer cells is associated with decreased levels of both BRCA1 and BRCA2. Inhibition of VEGFR3 in ovarian tumor cells was associated with growth arrest. CD133+ ovarian cancer stemlike cells were preferentially susceptible to VEGFR3-mediated growth inhibition. VEGFR3 inhibition–mediated down-regulation of BRCA gene expression reversed chemotherapy resistance and restored chemosensitivity in resistant cell lines in which a BRCA2 mutation had reverted to wild type. Finally, we demonstrate that tumor-associated macrophages are a primary source of VEGF-C in the tumor microenvironment. Our studies suggest that VEGFR3 inhibition may be a pharmacologic means to downregulate BRCA genes and improve the outcomes of patients with BRCA wild-type tumors

Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2

RBF1, a Drosophila pRB family homolog, is required for cell cycle arrest and the regulation of E2F-dependent transcription. Here, we describe the properties of RBF2, a second family member. RBF2 represses E2F transcription and is present at E2F-regulated promoters. Analysis of in vivo protein complexes reveals that RBF1 and RBF2 interact with different subsets of E2F proteins. dE2F1, a potent transcriptional activator, is regulated specifically by RBF1. In contrast, RBF2 binds exclusively to dE2F2, a form of E2F that functions as a transcriptional repressor. We find that RBF2-mediated repression requires dE2F2. More over, RBF2 and dE2F2 act synergistically to antagonize dE2F1-mediated activation, and they co-operate to block S phase progression in transgenic animals. The network of interactions between RBF1 or RBF2 and dE2F1 or dE2F2 reveals how the activities of these proteins are integrated. These results suggest that there is a remarkable degree of symmetry in the arrangement of E2F and RB family members in mammalian cells and in Drosophila

INHBA(+) cancer-associated fibroblasts generate an immunosuppressive tumor microenvironment in ovarian cancer.

Effective targeting of cancer-associated fibroblasts (CAFs) is hindered by the lack of specific biomarkers and a poor understanding of the mechanisms by which different populations of CAFs contribute to cancer progression. While the role of TGFβ in CAFs is well-studied, less attention has been focused on a structurally and functionally similar protein, Activin A (encoded by INHBA). Here, we identified INHBA(+) CAFs as key players in tumor promotion and immunosuppression. Spatiotemporal analyses of patient-matched primary, metastatic, and recurrent ovarian carcinomas revealed that aggressive metastatic tumors enriched in INHBA(+) CAFs were also enriched in regulatory T cells (Tregs). In ovarian cancer mouse models, intraperitoneal injection of the Activin A neutralizing antibody attenuated tumor progression and infiltration with pro-tumorigenic subsets of myofibroblasts and macrophages. Downregulation of INHBA in human ovarian CAFs inhibited pro-tumorigenic CAF functions. Co-culture of human ovarian CAFs and T cells revealed the dependence of Treg differentiation on direct contact with INHBA(+) CAFs. Mechanistically, INHBA/recombinant Activin A in CAFs induced the autocrine expression of PD-L1 through SMAD2-dependent signaling, which promoted Treg differentiation. Collectively, our study identified an INHBA(+) subset of immunomodulatory pro-tumoral CAFs as a potential therapeutic target in advanced ovarian cancers which typically show a poor response to immunotherapy

Glucose deprivation elicits phenotypic plasticity via ZEB1-mediated expression of NNMT.

Glucose is considered the primary energy source for all cells, and some cancers are addicted to glucose. Here, we investigated the functional consequences of chronic glucose deprivation in serous ovarian cancer cells. We found that cells resistant to glucose starvation (glucose-restricted cells) demonstrated increased metabolic plasticity that was dependent on NNMT (Nicotinamide N-methyltransferase) expression. We further show that ZEB1 induced NNMT, rendered cells resistant to glucose deprivation and recapitulated metabolic adaptations and mesenchymal gene expression observed in glucose-restricted cells. NNMT depletion reversed metabolic plasticity in glucose-restricted cells and prevented de novo formation of glucose-restricted colonies. In addition to its role in glucose independence, we found that NNMT was required for other ZEB1-induced phenotypes, such as increased migration. NNMT protein levels were also elevated in metastatic and recurrent tumors compared to matched primary carcinomas, while normal ovary and fallopian tube tissue had no detectable NNMT expression. Our studies define a novel ZEB1/NNMT signaling axis, which elicits mesenchymal gene expression, as well as phenotypic and metabolic plasticity in ovarian cancer cells upon chronic glucose starvation. Understanding the causes of cancer cell plasticity is crucial for the development of therapeutic strategies to counter intratumoral heterogeneity, acquired drug resistance and recurrence in high-grade serous ovarian cancer (HGSC)

Screening cell mechanotype by parallel microfiltration.

Cell mechanical phenotype or 'mechanotype' is emerging as a valuable label-free biomarker. For example, marked changes in the viscoelastic characteristics of cells occur during malignant transformation and cancer progression. Here we describe a simple and scalable technique to measure cell mechanotype: this parallel microfiltration assay enables multiple samples to be simultaneously measured by driving cell suspensions through porous membranes. To validate the method, we compare the filtration of untransformed and HRas(V12)-transformed murine ovary cells and find significantly increased deformability of the transformed cells. Inducing epithelial-to-mesenchymal transition (EMT) in human ovarian cancer cells by overexpression of key transcription factors (Snail, Slug, Zeb1) or by acquiring drug resistance produces a similar increase in deformability. Mechanistically, we show that EMT-mediated changes in epithelial (loss of E-Cadherin) and mesenchymal markers (vimentin induction) correlate with altered mechanotype. Our results demonstrate a method to screen cell mechanotype that has potential for broader clinical application

Are Epithelial Ovarian Cancers of the Mesenchymal Subtype Actually Intraperitoneal Metastases to the Ovary?

Primary ovarian high-grade serous carcinoma (HGSC) has been classified into 4 molecular subtypes: Immunoreactive, Proliferative, Differentiated, and Mesenchymal (Mes), of which the Mes subtype (Mes-HGSC) is associated with the worst clinical outcomes. We propose that Mes-HGSC comprise clusters of cancer and associated stromal cells that detached from tumors in the upper abdomen/omentum and disseminated in the peritoneal cavity, including to the ovary. Using comparative analyses of multiple transcriptomic data sets, we provide the following evidence that the phenotype of Mes-HGSC matches the phenotype of tumors in the upper abdomen/omentum: (1) irrespective of the primary ovarian HGSC molecular subtype, matched upper abdominal/omental metastases were typically of the Mes subtype, (2) the Mes subtype was present at the ovarian site only in patients with concurrent upper abdominal/omental metastases and not in those with HGSC confined to the ovary, and (3) ovarian Mes-HGSC had an expression profile characteristic of stromal cells in the upper abdominal/omental metastases. We suggest that ovarian Mes-HGSC signifies advanced intraperitoneal tumor dissemination to the ovary rather than a subtype of primary ovarian HGSC. This is consistent with the presence of upper abdominal/omental disease, suboptimal debulking, and worst survival previously reported in patients with ovarian Mes-HGSC compared to other molecular subtypes

A COL11A1-correlated pan-cancer gene signature of activated fibroblasts for the prioritization of therapeutic targets.

Although cancer-associated fibroblasts (CAFs) are viewed as a promising therapeutic target, the design of rational therapy has been hampered by two key obstacles. First, attempts to ablate CAFs have resulted in significant toxicity because currently used biomarkers cannot effectively distinguish activated CAFs from non-cancer associated fibroblasts and mesenchymal progenitor cells. Second, it is unclear whether CAFs in different organs have different molecular and functional properties that necessitate organ-specific therapeutic designs. Our analyses uncovered COL11A1 as a highly specific biomarker of activated CAFs. Using COL11A1 as a 'seed', we identified co-expressed genes in 13 types of primary carcinoma in The Cancer Genome Atlas. We demonstrated that a molecular signature of activated CAFs is conserved in epithelial cancers regardless of organ site and transforming events within cancer cells, suggesting that targeting fibroblast activation should be effective in multiple cancers. We prioritized several potential pan-cancer therapeutic targets that are likely to have high specificity for activated CAFs and minimal toxicity in normal tissues

A COL11A1-correlated pan-cancer gene signature of activated fibroblasts for the prioritization of therapeutic targets.

Although cancer-associated fibroblasts (CAFs) are viewed as a promising therapeutic target, the design of rational therapy has been hampered by two key obstacles. First, attempts to ablate CAFs have resulted in significant toxicity because currently used biomarkers cannot effectively distinguish activated CAFs from non-cancer associated fibroblasts and mesenchymal progenitor cells. Second, it is unclear whether CAFs in different organs have different molecular and functional properties that necessitate organ-specific therapeutic designs. Our analyses uncovered COL11A1 as a highly specific biomarker of activated CAFs. Using COL11A1 as a 'seed', we identified co-expressed genes in 13 types of primary carcinoma in The Cancer Genome Atlas. We demonstrated that a molecular signature of activated CAFs is conserved in epithelial cancers regardless of organ site and transforming events within cancer cells, suggesting that targeting fibroblast activation should be effective in multiple cancers. We prioritized several potential pan-cancer therapeutic targets that are likely to have high specificity for activated CAFs and minimal toxicity in normal tissues