Lung cancer is considered one of the most frequent causes of cancer-related death worldwide and Non-Small Cell Lung Cancer (NSCLC) accounts for 80% of all lung cancer cases. Autophagy is a cellular process responsible for the recycling of damaged organelles and protein aggregates. Transforming growth factor beta-1 (TGFβ) is involved in Epithelial to Mesenchymal Transition (EMT) and autophagy induction in different cancer models and plays an important role in the pathogenesis of NSCLC. It is not clear how autophagy can regulate EMT in NSCLC cells. In the present study, we have investigated the regulatory role of autophagy in EMT induction in NSCLC and show that TGFβ can simultaneously induce both autophagy and EMT in the NSCL lines A549 and H1975. Upon chemical inhibition of autophagy using Bafilomycin-A1, the expression of the mesenchymal marker vimentin and N-cadherin was reduced. Immunoblotting and immunocytochemistry (ICC) showed that the mesenchymal marker vimentin was significantly downregulated upon TGFβ treatment in ATG7 knockdown cells when compared to corresponding cells treated with scramble shRNA (negative control), while E-cadherin was unchanged. Furthermore, autophagy inhibition (Bafilomycin A1 and ATG7 knockdown) decreased two important mesenchymal functions, migration and contraction, of NSCLC cells upon TGFβ treatment. This study identified a crucial role of autophagy as a potential positive regulator of TGFβ-induced EMT in NSCLC cells and identifies inhibitors of autophagy as promising new drugs in antagonizing the role of EMT inducers, like TGFβ, in the clinical progression of NSCLC.

Poly(ADP‐ribose) polymerase 1 inhibitors alone or in combination with DNA damaging agents are promising clinical drugs in the treatment of cancer. However, there is a need to understand the molecular mechanisms of resistance to PARP1 inhibitors. Expression of HMGA2 in cancer is associated with poor prognosis for patients. Here, we investigated the novel relationship between HMGA2 and PARP1 in DNA damage‐induced PARP1 activity. We used human triple‐negative breast cancer and fibrosarcoma cell lines to demonstrate that HMGA2 colocalizes and interacts with PARP1. High cellular HMGA2 levels correlated with increased DNA damage‐induced PARP1 activity, which was dependent on functional DNA‐binding AT‐hook domains of HMGA2. HMGA2 inhibited PARP1 trapping to DNA and counteracted the cytotoxic effect of PARP inhibitors. Consequently, HMGA2 decreased caspase 3/7 induction and increased cell survival upon treatment with the alkylating methyl methanesulfonate alone or in combination with the PARP inhibitor AZD2281 (olaparib). HMGA2 increased mitochondrial oxygen consumption rate and spare respiratory capacity and increased NAMPT levels, suggesting metabolic support for enhanced PARP1 activity upon DNA damage. Our data showed that expression of HMGA2 in cancer cells reduces sensitivity to PARP inhibitors and suggests that targeting HMGA2 in combination with PARP inhibition may be a promising new therapeutic approach.

Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in 2-dimensional (2D) and 3-dimensional (3D) culture, we investigated the mechanisms of simvastatin's myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on Geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both Farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced more cell death in RH30 cells compared to C2C12 in 3D culture model with similar effects on autophagy flux as in 2D culture. Overall our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin myotoxicity is different in 2D versus 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatininduced cell death in myoblasts.

Introduction Clear cell ovarian cancer (CCC) accounts for 5-11% of ovarian cancers in North America with a higher frequency reported in cohorts from Japan. CCC usually arises directly from endometriosis, which is associated with oxidative stress. The latter plays a critical role in the pathogenesis of both endometriosis and CCC. CCC is inherently resistant to standard chemotherapies, and the outcomes for patients with advanced stage CCC have not changed in several decades. Therefore, there is an urgent need for new treatment options for advanced CCC. CCC possess a distinct genetic profile; >50% harbour ARID1A mutations typically resulting in loss of protein function and often with co-occurrence of PIK3CA mutation or gene amplification leading to PI3K/AKT pathway hyper-activation. ARID1A mutations leads to accumulation of ROS, decrease in GSH due to downregulation of SLC7A11 and related reduction in the GSH precursor cystine thereby rendering cancer cells highly dependent on oxidative phosphorylation. EO3001 is a small molecule drug candidate with selective activity against ARID1A-deficient cell lines in vitro. It has been shown that EO3001 directly binds and inhibits FDX-1 function to block iron-sulfur cluster formation in complex I, a critical component of the mitochondrial electron transport chain. Iron-sulfur clusters play a critical role in the oxidation-reduction reactions of electron transport in mitochondria relied on by cancer cells that have made an adaptive shift from glycolysis to high mitochondrial dependence. Complex I plays a role in redox control and the biosynthesis of macromolecules and nucleic acids necessary for cell proliferation. It is suggested that these complex I-dependent events contribute to tumor formation, resistance to cell death, and metastasis of cancer cells in part by causing an increase in ROS levels Methods We generated isogenic ovarian cancer cell lines (RMG-1 and OVCA438, -/+ ARID1A loss) using CRISPR/Cas9. We will assess therapeutic effects of EO3001 on cells’ tumorigenic potential in vitro -under ambient and stress conditions, notably endometriotic cyst content which derives malignant transformation, and in vivo on cell viability, cell proliferation, ROS levels, migration, invasion, and metastasis. We will use the organoids modeling system -using primary endometrial cells harboring ARID1A mutations- to assess the impact of EO3001 on organoid growth and response to stress conditions and evaluate the effect of EO3001 on cancer metastesis by the ex vivo pulmonary metastasis assay (PuMA). Conclusions Exploiting the vulnerability in reliance on OXPHOS in ARID1A-deficient CCC using EO3001 might represent a promising strategy for the treatment of these patients as well as patients harboring other ARID1A-deficient malignancies. Citation Format: Amal M. EL-Naggar, Yuchen Ding, Lucy Li, Forouh Kalantari, Jeffrey Bacha, Dennis Brown, David Huntsman. Investigating the therapeutic efficacy of EO3001 in clear cell carcinoma of the ovary. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4967.

Introduction: Clear cell carcinoma of the ovary (CCC) is the 2nd most common ovarian cancer and is histologically and clinically distinct from other subtypes. Late stage CCC have a worse prognosis than other ovarian cancer histotypes as they are inherently resistant to the standard platinum/taxane chemotherapy. Deep endometriosis of the ovary, known as endometrioma or chocolate cyst, is the most common precursor for CCC. However, how transformation from endometriosis to CCC and, in particular, the important role of the hypoxic and ROS-rich microenvironment of endometriotic cysts are not well understood. As the molecular mechanisms pertinent to the genesis and progression of CCC are largely unknown, there are few, if any, therapeutic strategies for patients with advanced stage disease. Identifying factors that shape the development, progression and metastasis of CCC which can be targeted therapeutically could have tremendous potential to improve outcomes in this disease. Our recent findings identified cystathionine gamma-lyase (CTH), a key enzyme in the transsulfuration pathway, as a marker of Mullerian tract derived ciliated cells and CCC of both the ovary and uterus regardless of which mutations are present. Also, CTH is highly expressed both in CCC and the endometriosis adjacent to this cancer. Whether and how the transsulfuration pathway, notably CTH, enables CCC to adapt to the hostile microenvironment of an endometriotic cyst and ultimately to promote metastasis remain unanswered. Methods: We generated CTH knockout (KO) cells using CRISPR/Cas9. We assessed effects of CTH loss in vitro -under ambient and stress conditions- and in vivo on cell viability, cell proliferation, ROS levels, migration, invasion, and metastasis. Further, we used an organoid model system to assess the impact of CTH loss in primary endometrial cells on organoid growth and response to stress conditions including exposure to endometriotic cyst contents. Results: Our in-vitro, in-vivo data as well as data derived from the organoid modelling system show that CTH is critical for adaptive response to hypoxia. Further, it underpins the growth of CCC cells in-vitro and the growth of CTH in a mouse model of CCC. These data highly indicate that expression of the transsulfuration pathway enzyme CTH enables cells to survive in endometriotic cysts then upon transformation both marks CCC and potentiates tumor progression and metastasis. Conclusion: Targeting CTH in CCC and potentially other cancers might represent a novel and impactful therapeutic approach. Citation Format: Amal M. El-Naggar, Yuchen Ding, Genny Trigo-Gonzalez, Lucy Li, Shary Chen, Busra Turgu, Forouh Kalantari, Rodrigo Vallejos, Kiran Parmar, Cindy Shen, Gian Luca Negri, Paul Yong, Gregg Morin, David G. Huntsman. On the verge of metastasis: CTH defines a cell-state-dependent adaptive response to microenvironmental stresses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2482.