Epithelial ovarian cancers that are nonhomologous recombination deficient, as well as those that are recurrent and in a platinum‐resistant state, have limited therapeutic options. The objectives of ...this study were to characterize the mechanism of action and investigate the therapeutic potential of a small molecule, VDX‐111, against ovarian cancer. We examined the ability of VDX‐111 to inhibit the growth of a panel of ovarian cancer cell lines, focusing on BRCA wild‐type lines. We found that VDX‐111 causes a dose‐dependent loss of cell viability across ovarian cancer cell lines. Reverse phase protein array (RPPA) analysis was used to identify changes in cell signaling in response to VDX‐111 treatment. An RPPA analysis performed on cells treated with VDX‐111 detected changes in cell signaling related to autophagy and necroptosis. Immunoblots of OVCAR3 and SNU8 cells confirmed a dose‐dependent increase in LC3A/B and RIPK1. Incucyte live cell imaging was used to measure cell proliferation and death in response to VDX‐111 alone and with inhibitors of apoptosis, necroptosis, and autophagy. Annexin/PI assays suggested predominantly nonapoptotic cell death, while real‐time kinetic imaging of cell growth indicated the necroptosis inhibitor, necrostatin‐1, attenuates VDX‐111‐induced loss of cell viability, suggesting a necroptosis‐dependent mechanism. Furthermore, VDX‐111 inhibited tumor growth in patient‐derived xenograft and syngeneic murine models. In conclusion, the cytotoxic effects of VDX‐111 seen in vitro and in vivo appear to occur in a necroptosis‐dependent manner and may promote an antitumor immune response.
Porcupine O-acyltransferase (PORCN) is considered essential for Wnt secretion and signaling. However, we observed that PORCN inhibition does not phenocopy the effects of WNT4 knockdown in ...WNT4-dependent breast cancer cells. This suggests a unique relationship between PORCN and WNT4 signaling. To examine the role of PORCN in WNT4 signaling, here we overexpressed WNT4 or WNT3A in breast cancer, ovarian cancer, and fibrosarcoma cell lines. Conditioned media from these lines and co-culture systems were used to assess the dependence of Wnt secretion and activity on the critical Wnt secretion proteins PORCN and Wnt ligand secretion (WLS) mediator. We observed that WLS is universally required for Wnt secretion and paracrine signaling. In contrast, the dependence of WNT3A secretion and activity on PORCN varied across the cell lines, and WNT4 secretion was PORCN-independent in all models. Surprisingly, WNT4 did not exhibit paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we identified cell-autonomous Wnt signaling activation by WNT4 and WNT3A, independent of PORCN or Wnt secretion. The PORCN-independent, cell-autonomous Wnt signaling demonstrated here may be critical in WNT4-driven cellular contexts or in those that are considered to have dysfunctional Wnt signaling.
High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) allows for high resolution, genome-wide mapping of RNA-binding proteins. This methodology is frequently used ...to validate predicted targets of microRNA binding, as well as direct targets of other RNA-binding proteins. Hence, the accuracy and sensitivity of binding site identification is critical.
We found that substantial mispriming during reverse transcription results in the overrepresentation of sequences complementary to the primer used for reverse transcription. Up to 45 % of peaks in publicly available HITS-CLIP libraries are attributable to this mispriming artifact, and the majority of libraries have detectable levels of mispriming. We also found that standard techniques for validating microRNA-target interactions fail to differentiate between artifactual peaks and physiologically relevant peaks.
Here, we present a modification to the HITS-CLIP protocol that effectively eliminates this artifact and improves the sensitivity and complexity of resulting libraries.
miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally ...focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA–target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA–mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.
High-grade serous ovarian cancer (HGSOC) is the deadliest ovarian cancer histotype due in-part to the lack of therapeutic options for chemotherapy-resistant disease. PARP inhibitors (PARPi) represent ...a targeted treatment. However, PARPi resistance is becoming a significant clinical challenge. There is an urgent need to overcome resistance mechanisms to extend disease-free intervals. We established isogeneic PARPi-sensitive and -resistant HGSOC cell lines. In three PARPi-resistant models, there is a significant increase in AP-1 transcriptional activity and DNA repair capacity. Using RNA-sequencing and an shRNA screen, we identified activating transcription factor 6 (ATF6) as a mediator of AP-1 activity, DNA damage response, and PARPi resistance. In publicly available datasets, ATF6 expression is elevated in HGSOC and portends a poorer recurrence-free survival. In a cohort of primary HGSOC tumors, higher ATF6 expression significantly correlated to PARPi resistance. In PARPi-resistant cell lines and a PDX model, inhibition of a known ATF6 regulator, p38, attenuated AP-1 activity and RAD51 foci formation, enhanced DNA damage, significantly inhibited tumor burden, and reduced accumulation of nuclear ATF6.
This study highlights that a novel p38-ATF6-mediated AP-1 signaling axis contributes to PARPi resistance and provides a clinical rationale for combining PARPi and AP-1 signaling inhibitors.
High-grade serous ovarian cancer is the deadliest gynecologic malignancy due to progression to resistant disease. Claudin-4 is classically defined as a tight junction protein and is often associated ...with epithelial cancers. Claudin-4 is aberrantly expressed in nearly 70% of all ovarian cancer tumors and conveys a worse overall prognosis. Elevated claudin-4 expression correlates to increased DNA repair activity and resistance to DNA damaging agents. PARP inhibitors are emerging as an effective therapeutic option for patients with ovarian cancer and function by promoting DNA damage. The study examines the relationship between claudin-4 expression and the response to PARP inhibitors using both genetic and pharmacologic inhibition of claudin-4 in in vitro and ex vivo models of ovarian cancer to examine DNA repair markers and functional activity. Genetic inhibition of claudin-4 results in the downregulation of several DNA damage repair effectors, including 53BP1 and XRCC1. Claudin-4 knockdown did not change homology-directed repair but inhibited nonhomologous end-joining and reduced 53BP1 foci formation. In 15 primary ovarian cancer tumors, higher claudin-4 expression significantly correlated to a dampened PARP inhibitor-mediated antiproliferation response. Further, claudin-4 inhibition in high claudin-4 tumors sensitized tumor sections to PARP inhibition. These data highlight that claudin-4 expression in ovarian cancer tumors could serve as both a marker of PARP inhibitor response and a therapeutic target to improve PARP inhibitor response.
Ovarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian carcinoma (HGSC) subtype accounts for almost 70% of ...ovarian cancers and is the deadliest. HGSC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. HGSC survival in peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). Most anoikis resistant mechanisms are dependent on microenvironment interactions with cell surface-associated proteins, such as integrins and receptor tyrosine kinases (RTKs). We previously identified the gene CASC4 as a driver of anoikis resistance. CASC4 is predicted to be a Golgi-associated protein that may regulate protein trafficking to the plasma membrane, but CASC4 is largely uncharacterized in literature; thus, we sought to determine how CASC4 confers anoikis resistance to HGSC cells. Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we cultured three human HGSC cell lines (PEO1, CaOV3, OVCAR3), and a murine HGSC cell line, (ID8) with shRNA-mediated CASC4 knockdowns (CASC4 KD) in suspension, to recapitulate the peritoneal fluid environment in vitro. CASC4 KD significantly inhibited cell proliferation and colony formation ability, and increased apoptosis. A Reverse Phase Protein Assay (RPPA) showed that CASC4 KD resulted in a broad re-programming of membrane-associated proteins. Specifically, CASC4 KD led to decreased protein levels of the RTK Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways, leading us to hypothesize that CASC4 drives HGSC survival through mediating recycling and trafficking of EGFR. Indeed, loss of CASC4 led to a decrease in both EGFR membrane localization, reduced turnover of EGFR, and increased EGFR ubiquitination. Moreover, a syngeneic ID8 murine model of ovarian cancer showed that knocking down CASC4 leads to decreased tumor burden and dissemination.
Patients harboring germline breast cancer susceptibility genes 1 and 2 (BRCA1/2) mutations are predisposed to developing breast, pancreatic, and ovarian cancers. BRCA2 plays a critical role in ...homologous recombination (HR) DNA repair and deleterious mutations in
confer sensitivity to PARP inhibition. Recently, the PARP inhibitors olaparib and rucaparib were FDA approved for the treatment of metastatic breast cancer and patients with recurrent ovarian cancer with mutations in BRCA1/2. Despite their initial antitumor activity, the development of resistance limits the clinical utility of PARP inhibitor therapy. Multiple resistance mechanisms have been described, including reversion mutations that restore the reading frame of the
gene. In this study, we generated olaparib- and rucaparib-resistant
-mutant Capan1 cell lines. We did not detect secondary reversion mutations in the olaparib- or rucaparib-resistant clones. Several of the resistant clones had gene duplication and amplification of the mutant
allele, with a corresponding increase in expression of a truncated BRCA2 protein. In addition, HR-mediated DNA repair was rescued, as evidenced by the restoration of RAD51 foci formation. Using mass spectrometry, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), as an interacting partner of truncated BRCA2. RNAi-mediated knockdown of
or
was sufficient to resensitize cells to olaparib. The results demonstrate that independent of a
reversion, mutation amplification of a mutant-carrying
contributes to PARP inhibitor resistance.
Euchromatic histone-lysine-N-methyltransferases 1 and 2 (EHMT1/2, aka GLP/G9A) catalyze dimethylation of histone H3 lysine 9 (H3K9me2) and have roles in epigenetic silencing of gene expression. ...EHMT1/2 also have direct roles in DNA repair and are implicated in chemoresistance in several cancers. Resistance to chemotherapy and PARP inhibitors (PARPi) is a major cause of mortality in high-grade serous ovarian carcinoma (HGSOC), but the contribution of the epigenetic landscape is unknown.
To identify epigenetic mechanisms of PARPi resistance in HGSOC, we utilized unbiased exploratory techniques, including RNA-Seq and mass spectrometry profiling of histone modifications. Compared to sensitive cells, PARPi-resistant HGSOC cells display a global increase of H3K9me2 accompanied by overexpression of EHMT1/2. EHMT1/2 overexpression was also observed in a PARPi-resistant in vivo patient-derived xenograft (PDX) model. Genetic or pharmacologic disruption of EHMT1/2 sensitizes HGSOC cells to PARPi. Cell death assays demonstrate that EHMT1/2 disruption does not increase PARPi-induced apoptosis. Functional DNA repair assays show that disruption of EHMT1/2 ablates homologous recombination (HR) and non-homologous end joining (NHEJ), while immunofluorescent staining of phosphorylated histone H2AX shows large increases in DNA damage. Propidium iodide staining and flow cytometry analysis of cell cycle show that PARPi treatment increases the proportion of PARPi-resistant cells in S and G2 phases, while cells treated with an EHMT1/2 inhibitor remain in G1. Co-treatment with PARPi and EHMT1/2 inhibitor produces an intermediate phenotype. Immunoblot of cell cycle regulators shows that combined EHMT1/2 and PARP inhibition reduces expression of specific cyclins and phosphorylation of mitotic markers. These data suggest DNA damage and altered cell cycle regulation as mechanisms of sensitization. RNA-Seq of PARPi-resistant cells treated with EHMT1/2 inhibitor showed significant gene expression changes enriched in pro-survival pathways that remain unexplored in the context of PARPi resistance, including PI3K, AKT, and mTOR.
This study demonstrates that disrupting EHMT1/2 sensitizes HGSOC cells to PARPi, and suggests a potential mechanism through DNA damage and cell cycle dysregulation. RNA-Seq identifies several unexplored pathways that may alter PARPi resistance. Further study of EHMT1/2 and regulated genes will facilitate development of novel therapeutic strategies to successfully treat HGSOC.
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