The Yes1-associated transcriptional regulator-mastermind-like transcriptional co-activator 2 (YAP1-MAML2 [YM]) fusion protein arises from an intrachromosomal inversion and is implicated in various cancers. However, the oncogenic role of the endogenous YM fusion protein remained undefined. In this study, we employed YM-positive ES-2 ovarian cancer cells as a model to explore the roles of the YM fusion in cancer initiation and maintenance. The YM fusion protein localizes to nuclear speckles and contains bifunctional domains: the YAP1 N-terminal domain interacts with transcriptional enhanced associate domain (TEAD) transcription factors, while the MAML2 C-terminal domain activates YAP1/TEAD-driven transcription. YM exhibited transforming activity, as shown by its ability to induce focus formation in immortalized epithelial cells. YM depletion reduced cancer cell proliferation and survival both in vitro and in xenograft tumor models. This effect was correlated with a downregulation of YAP1/TEAD-driven genes essential for cellular proliferation and survival, as revealed by transcriptomic analysis. Importantly, YM-positive cancer cells were sensitive to YAP1/TEAD-targeted pharmacologic inhibition. Collectively, these findings establish the YM fusion as a critical driver of oncogenesis and a promising therapeutic target for cancers harboring the YM fusion.

Abstract Homeobox C8 (HOXC8) is a transcription factor preferentially overexpressed in a large percentage of lung adenocarcinoma (LUAD). To investigate the function of HOXC8 in LUAD, we showed that knockdown of HOXC8 led to massive LUAD cell death in a mechanism of pyroptosis because both YVAD, a caspase-1 (CASP1) inhibitor, and disulfiram, which prevents gasdemin D (GSDMD) pore formation, blocked cell death caused by HOXC8 depletion. Intriguingly, ASC and NLRP3, components of canonic inflammasome, were not involved in pyroptosis occurring in HOXC8-depleted cells. Instead, we found that silencing HOXC8 led to a marked increase in CASP1 abundance. As forced expression of CASP1 is sufficient to induce CASP1 activation and pyroptosis, we reason that HOXC8 deters pyroptosis by suppressing the level of CASP1. Moreover, we revealed that knockdown of HOXC8 augmented CASP1 transcription involving in HDAC1/2. In fact, HOXC8 and HDAC1 were in the same immunocomplex and the presence of HOXC8 is required for the recruitment of HDAC1 to CASP1 promoter. Since HOXC8 also binds CASP1 promoter, we conclude that HOXC8 negatively regulates CASP1 expression by drafting HDAC1/2 to CASP1. This study suggests that HOXC8 participates LUAD development by controlling CASP1 expression and pyroptosis. Citation Format: Lei Sun, Ravi Padia, Ozlem Calbay, Cheng Chi, Mahmuda Akter, Lingtao Jin, Shuang Huang. HOXC8 epigenetically controls caspse-1 expression and pyroptosis in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6004.

DNA ligase (LIG) I and IIIα finalize base excision repair (BER) by sealing a nick product after nucleotide insertion by DNA polymerase (pol) β at the downstream steps. We previously demonstrated that a functional interplay between polβ and BER ligases is critical for efficient repair, and polβ mismatch or oxidized nucleotide insertions confound the final ligation step. Yet, how targeting downstream enzymes with small molecule inhibitors could affect this coordination remains unknown. Here, we report that DNA ligase inhibitors, L67 and L82-G17, slightly enhance hypersensitivity to oxidative stress-inducing agent, KBrO3, in polβ+/+ cells more than polβ-/- null cells. We showed less efficient ligation after polβ nucleotide insertions in the presence of the DNA ligase inhibitors. Furthermore, the mutations at the ligase inhibitor binding sites (G448, R451, A455) of LIG1 significantly affect nick DNA binding affinity and nick sealing efficiency. Finally, our results demonstrated that the BER ligases seal a gap repair intermediate by the effect of polβ inhibitor that diminishes gap filling activity. Overall, our results contribute to understand how the BER inhibitors against downstream enzymes, polβ, LIG1, and LIGIIIα, could impact the efficiency of gap filling and subsequent nick sealing at the final steps leading to the formation of deleterious repair intermediates.

Canonical pyroptosis is type of programmed cell death depending on active caspase-1, and the inflammasome carries out caspase-1 activation. Here, we showed that docosahexaenoic acid (DHA) induced ovarian cancer cell deaths in caspase-1-dependent manner. DHA increased caspase-1 activity and led to interleukin-1β secretion and gasdermin D cleavage while disulfiram inhibited DHA-induced cell death, suggesting that DHA triggered pyroptosis. Intriguingly, ASC, the molecule recruiting caspase-1 to inflammasome for activation, was dispensable for DHA-induced pyroptosis. Instead, we observed remarkable elevation in caspase-1 abundance concurrent with the activation of caspase-1 in DHA-treated cells. As ectopically overexpressing caspase-1 resulted in robust amount of active caspase-1, we reason that DHA activates caspase-1 and pyroptosis through the generation of excessive amount of caspase-1 protein. Mechanistically, DHA increased caspase-1 by specifically accelerating caspase-1 protein synthesis via the p38MAPK/Mnk1 signaling pathway. We have uncovered an unknown pyroptosis mechanism in which caspase-1-dependent pyroptosis can occur without the participation of ASC/inflammasome.

Fibroblast activation protein (FAP) is tumor-specific and plays an important role in tumorigenecity. However, agents against its enzymatic activity or extracellular presence were unsuccessful in the clinic for undefined reasons. Here we show that FAP expression is higher in advanced ovarian cancer and is only detected in invasive ovarian cancer cells. Silencing FAP induces apoptosis and FAP’s enzymatic activity is dispensable for cell survival. To elucidate the cause of apoptosis, we find that NF-κB activity is diminished when FAP is depleted and BIRC5 (survivin) acts downstream of FAP-NF-κB axis to promote cell survival. To uncover the link between FAP and NF-κB activation, we reveal that PRKDC (DNA-PK, DNA-dependent protein kinase) forms complex with FAP and is required for NF-κB activation and cell survival. Remarkably, FAP-PRKDC interaction occurs only in lipid rafts, and depleting FAP prevents lipid raft localization of PRKDC. Given the known ability of PRKDC to direct NF-κB activation, these results suggest that FAP recruits PRKDC in lipid rafts for NF-κB activation. FAP’s non-enzymatic role and functioning from lipid rafts for cell survival also offer an explanation on the failure of past FAP-targeted therapies. Finally, we demonstrate that EpCAM aptamer-delivered FAP siRNA impeded intraperitoneal xenograft development of ovary tumors.

Docosahexaenoic acid (DHA) is a natural compound, which exhibits anti-cancer capability in various cancer types in experimental models. Similar to previous reports in other cancer cell types, we demonstrated that DHA inhibited ovarian cancer cell growth by inducing cell death. In our model, distinct from the effect of DHA on other cancer cell types, we revealed that DHA-induced cell death was only slightly blocked by apoptosis inhibitor (caspase 3 inhibitor DEVD). We characterized the mechanism associated with DHA-led cell death and found that DHA-induced cell death was effectively blocked by caspase 1 inhibitor, indicating that DHA kills ovarian cancer cells by inducing pyroptosis. This mechanism was further supported by the observation that Disulfiram, a Gasdermin D inhibitor, prevented DHA-induced cell death and DHA treatment leads to the cleavage of Gasdermin D. Surprisingly, known inflammasome inhibitors did not affect DHA-induced cell death and Gasdermin D cleavage, ruling out the involvement of the inflammasome for caspase 1 activation. Instead, DHA remarkably elevated the amount of both caspase I and cleaved caspase 1 in ovarian cancer cells. This suggests that DHA activates caspase 1 by augmenting the abundance of caspase 1. Furthermore, we showed that DHA increased the level of intracellular reactive oxygen species (ROS). Use of ROS inhibitor (NAC) abrogated DHA-induced pyroptosis. This study uncovers pyroptosis as the mechanism for DHA-induced ovarian cancer cell death and reveals a previously unknown mechanism for caspase 1 activation. Citation Format: Ozlem Calbay, Shuang Huang. Inflammasome-independent pyroptosis mediates DHA-led ovarian cancer cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3005.

Epithelial–mesenchymal transition (EMT) is implicated in tumor metastasis and therapeutic resistance. It remains a challenge to target cancer cells that have undergone EMT. The Snail family of key EMT-inducing transcription factors directly binds to and transcriptionally represses not only epithelial genes but also a myriad of additional genomic targets that may carry out significant biological functions. Therefore, we reasoned that EMT inherently causes various concomitant phenotypes, some of which may create targetable vulnerabilities for cancer treatment. In the present study, we found that Snail transcription factors bind to the promoters of multiple genes encoding subunits of the AMP-activated protein kinase (AMPK) complex, and expression of AMPK genes was markedly downregulated by EMT. Accordingly, high AMPK expression in tumors correlated with epithelial cell markers and low AMPK expression in tumors was strongly associated with adverse prognosis. AMPK is the principal sensor of cellular energy status. In response to energy stress, AMPK is activated and critically reprograms cellular metabolism to restore energy homeostasis and maintain cell survival. We showed that activation of AMPK by energy stress was severely impaired by EMT. Consequently, EMT cancer cells became hypersensitive to a variety of energy stress conditions and primarily underwent pyroptosis, a regulated form of necrotic cell death. Collectively, the study suggests that EMT impedes the activation of AMPK signaling induced by energy stress and sensitizes cancer cells to pyroptotic cell death under energy stress conditions. Therefore, while EMT promotes malignant progression, it concurrently induces collateral vulnerabilities that may be therapeutically exploited.

MicroRNAs (miRNAs) are actively involved in the progression and metastasis of ovarian cancer. Here we show that miR-203b–3p is one of the miRNAs whose expression is diminished in advanced ovarian cancer (Stage III/IV). Introducing miR-203b-3p into ovarian cancer cells suppressed cell migration, in vitro invasion and peritoneal metastatic colonization. With the aid of cytokine array and modified Cross-Linking, Ligation, and Sequencing of Hybrids (qCLASH), we identified C-X-C motif chemokine ligand 1 (CXCL1) mRNA as a target of miR-203b-3p. Recombinant CXCL1 largely restored cell migration/invasion and CXCL1 neutralizing antibody blocked cell migration/invasion. Intriguingly, miR-203b-3p targets CXCL1 in an unconventional manner: 1) miR-203b-3p targets the 5′-untranslated region (UTR) and protein coding region of CXCL1 mRNA and 2) seed sequences in miR-203b-3p are not the conventional nucleotides 2 to 8 observed for most of miRNA/target complementation. Finally, we show that epithelial cell adhesion molecule (EpCAM) aptamer can effectively deliver miR-203b-3p into ovarian cancer cells and EpCAM aptamer-delivered miR-203b-3p impeded peritoneal metastatic colonization and prolonged lifespan of tumor-bearing mice. In summary, our findings provide a novel mechanism in which attenuated miR-203b-3p expression sustains CXCL1 abundance and hence ovarian cancer progression. Importantly, we suggest that EpCAM aptamer-delivered miR-203b-3p may be exploited for therapeutic purpose against advanced ovarian cancer.

PRKCI, the gene for protein kinase Cι (PKCι), is frequently amplified in ovarian cancer and recent studies have shown that PKCι participates in ovary tumorigenesis. However, it is unknown whether PKCι is differentially involved in the growth/survival between PRKCI-amplified and non-amplified ovarian cancer cells. In this study, we analyzed ovarian cancer patient dataset and revealed that PRKCI is the only PKC family member significantly amplified in ovarian cancer and PRKCI amplification is associated with higher PKCι expression. Using a panel of ovarian cancer cell lines, we found that abundance of PKCι is generally associated with PRKCI amplification. Interestingly, silencing PKCι led to apoptosis in PRKCI-amplified ovarian cancer cells but not in those without PRKCI amplification, thus indicating an oncogenic addiction to PKCɩ in PRKCI-amplified cells. Since small-molecule inhibitors characterized to selectively block atypical PKCs did not offer selectivity nor sensitivity in PRKCI-amplified ovarian cancer cells and were even cytotoxic to non-cancerous ovary surface or fallopian tube epithelial cells, we designed an EpCAM aptamer-PKCι siRNA chimera (EpCAM-siPKCι aptamer). EpCAM-siPKCι aptamer not only effectively induced apoptosis of PRKCI-amplified ovarian cancer cells but also greatly deterred intraperitoneal tumor development in xenograft mouse model. This study has demonstrated a precision medicine-based strategy to target a subset of ovarian cancer that contains PRKCI amplification and shown that the EpCAM aptamer-delivered PKCι siRNA may be used to suppress such tumors.