Identification of myoferlin as a mitochondria-associated membranes component required for calcium signaling in PDAC cell lines.

BACKGROUND: Pancreatic ductal adenocarcinoma is an aggressive cancer type with one of the lowest survival rates due to late diagnosis and the absence of effective treatments. A better understanding of PDAC biology will help researchers to discover the Achilles' heel of cancer cells. In that regard, our research team investigated the function of an emerging oncoprotein known as myoferlin. Myoferlin is overexpressed in PDAC and its silencing/targeting has been shown to affect cancer cell proliferation, migration, mitochondrial dynamics and metabolism. Nevertheless, our comprehension of myoferlin functions in cells remains limited. In this study, we aimed to understand the molecular mechanism linking myoferlin silencing to mitochondrial dynamics. METHODS: Experiments were performed on two pancreas cancer cell lines, Panc-1 and MiaPaCa-2. Myoferlin localization on mitochondria was evaluated by immunofluorescence, proximity ligation assay, and cell fractionation. The presence of myoferlin in mitochondria-associated membranes was assessed by cell fractionation and its function in mitochondrial calcium transfer was evaluated using calcium flow experiments, proximity ligation assays, co-immunoprecipitation, and timelapse fluorescence microscopy in living cells. RESULTS: Myoferlin localization on mitochondria was investigated. Our results suggest that myoferlin is unlikely to be located on mitochondria. Instead, we identified myoferlin as a new component of mitochondria-associated membranes. Its silencing significantly reduces the mitochondrial calcium level upon stimulation, probably through myoferlin interaction with the inositol 1,4,5-triphosphate receptors 3. CONCLUSIONS: For the first time, myoferlin was specifically demonstrated to be located in mitochondria-associated membranes where it participates to calcium flow. We hypothesized that this function explains our previous results on mitochondrial dynamics. This study improves our comprehension of myoferlin localization and function in cancer biology.

Resistance to Gemcitabine in Pancreatic Cancer Is Connected to Methylglyoxal Stress and Heat Shock Response.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor prognosis. Gemcitabine is the first-line therapy for PDAC, but gemcitabine resistance is a major impediment to achieving satisfactory clinical outcomes. This study investigated whether methylglyoxal (MG), an oncometabolite spontaneously formed as a by-product of glycolysis, notably favors PDAC resistance to gemcitabine. We observed that human PDAC tumors expressing elevated levels of glycolytic enzymes together with high levels of glyoxalase 1 (GLO1), the major MG-detoxifying enzyme, present with a poor prognosis. Next, we showed that glycolysis and subsequent MG stress are triggered in PDAC cells rendered resistant to gemcitabine when compared with parental cells. In fact, acquired resistance, following short and long-term gemcitabine challenges, correlated with the upregulation of GLUT1, LDHA, GLO1, and the accumulation of MG protein adducts. We showed that MG-mediated activation of heat shock response is, at least in part, the molecular mechanism underlying survival in gemcitabine-treated PDAC cells. This novel adverse effect of gemcitabine, i.e., induction of MG stress and HSR activation, is efficiently reversed using potent MG scavengers such as metformin and aminoguanidine. We propose that the MG blockade could be exploited to resensitize resistant PDAC tumors and to improve patient outcomes using gemcitabine therapy.

Acidosis-induced regulation of adipocyte G0S2 promotes crosstalk between adipocytes and breast cancer cells as well as tumor progression.

Bidirectional interactions between cancer cells and their microenvironment govern tumor progression. Among the stromal cells in this microenvironment, adipocytes have been reported to upregulate cancer cell migration and invasion by producing fatty acids. Conversely, cancer cells alter adipocyte phenotype notably via increased lipolysis. We aimed to identify the mechanisms through which cancer cells trigger adipocyte lipolysis and evaluate the functional consequences on cancer progression. Here, we show that cancer cell-induced acidification of the extracellular medium strongly promotes preadipocyte lipolysis through a mechanism that does not involve lipophagy but requires adipose triglyceride lipase (ATGL) activity. This increased lipolysis is triggered mainly by attenuation of the G0/G1 switch gene 2 (G0S2)-induced inhibition of ATGL. G0S2-mediated regulation in preadipocytes affects their communication with breast cancer cells, modifying the phenotype of the cancer cells and increasing their resistance to chemotherapeutic agents in vitro. Furthermore, we demonstrate that the adipocyte-specific overexpression of G0S2 impairs mammary tumor growth and lung metastasis formation in vivo. Our results highlight the importance of acidosis in cancer cell-adipocyte crosstalk and identify G0S2 as the main regulator of cancer-induced lipolysis, regulating tumor establishment and spreading.

Human papillomavirus E6/E7 oncoproteins promote radiotherapy-mediated tumor suppression by globally hijacking host DNA damage repair.

Rationale: Whatever the mucosa primary infected, HPV-positive cancers are traditionally associated with a favorable outcome, attributable to a high sensitivity to radiation therapy. However, the direct impact of viral E6/E7 oncoproteins on the intrinsic cellular radiosensitivity (and, globally, on host DNA repair) remains mostly speculative. Methods: Using several isogenic cell models expressing HPV16 E6 and/or E7, the effect of viral oncoproteins on global DNA damage response was first investigated by in vitro/in vivo approaches. The binary interactome of each individual HPV oncoprotein with factors involved in the various host DNA damage/repair mechanisms was then precisely mapped by Gaussia princeps luciferase complementation assay (and validated by co-immunoprecipitation). The stability/half-life of protein targets for HPV E6 and/or E7 as well as their subcellular localizations were determined. At last, the host genome integrity following E6/E7 expression and the synergy between radiotherapy and compounds targeting DNA repair were analyzed. Results: We first showed that the sole expression of one viral oncoprotein from HPV16 was able to significantly increase the sensitivity to irradiation of cells without affecting their basal viability parameters. In total, 10 novel targets (CHEK2, CLK2, CLK2/3, ERCC3, MNAT1, PER1, RMI1, RPA1, UVSSA and XRCC6) for E6 and 11 (ALKBH2, CHEK2, DNA2, DUT, ENDOV, ERCC3, PARP3, PMS1, PNKP, POLDIP2 and RBBP8) for E7 were identified. Importantly, not degraded following their interaction with E6 or E7, these proteins have been shown to be less linked to host DNA and to colocalize with HPV replication foci, denoting their crucial implication in viral life cycle. Finally, we found that E6/E7 oncoproteins globally jeopardize host genome integrity, increase the cellular sensitivity to DNA repair inhibitors and enhance their synergy with radiotherapy. Conclusion: Taken together, our findings provide a molecular insight into the direct hijacking of host DNA damage/repair responses by HPV oncoproteins, demonstrate the significant impact of this phenomenon on both intrinsic cellular radiosensitivity and host DNA integrity and suggest novel connected therapeutic vulnerabilities.

Paladin, overexpressed in colon cancer, is required for actin polymerisation and liver metastasis dissemination.

INTRODUCTION: Colorectal cancer remains a public health issue and most colon cancer patients succumb to the development of metastases. Using a specific protocol of pressure-assisted interstitial fluid extrusion to recover soluble biomarkers, we identified paladin as a potential colon cancer liver metastases biomarker. METHODS: Using shRNA gene knockdown, we explored the biological function of paladin in colon cancer cells and investigated the phospho-proteome within colon cancer cells. We successively applied in vitro migration assays, in vivo metastasis models and co-immunoprecipitation experiments. RESULTS: We discovered that paladin is required for colon cancer cell migration and metastasis, and that paladin depletion altered the phospho-proteome within colon cancer cells. Data are available via ProteomeXchange with identifier PXD030803. Thanks to immunoprecipitation experiments, we demonstrated that paladin, was interacting with SSH1, a phosphatase involved in colon cancer metastasis. Finally, we showed that paladin depletion in cancer cells results in a less dynamic actin cytoskeleton. CONCLUSIONS: Paladin is an undervalued protein in oncology. This study highlights for the first time that, paladin is participating in actin cytoskeleton remodelling and is required for efficient cancer cell migration.

Myoferlin targeting triggers mitophagy and primes ferroptosis in pancreatic cancer cells.

Myoferlin, an emerging oncoprotein, has been associated with a low survival in several cancer types including pancreas ductal adenocarcinoma where it controls mitochondria structure and respiratory functions. Owing to the high susceptibility of KRAS-mutated cancer cells to iron-dependent cell death, ferroptosis, and to the high iron content in mitochondria, we investigated the relation existing between mitochondrial integrity and iron-dependent cell death. We discovered that myoferlin targeting with WJ460 pharmacological compound triggered mitophagy and ROS accumulation culminating with lipid peroxidation and apoptosis-independent cell death. WJ460 caused a reduction of the abundance of ferroptosis core regulators xc- cystine/glutamate transporter and GPX-4. Mitophagy inhibitor Mdivi1 and iron chelators inhibited the myoferlin-related ROS production and restored cell growth. Additionally, we reported a synergic effect between ferroptosis inducers, erastin and RSL3, and WJ460.

Cancer-Associated Fibroblast Diversity Shapes Tumor Metabolism in Pancreatic Cancer.

Despite extensive research, the 5-year survival rate of pancreatic cancer (PDAC) patients remains at only 9%. Patients often show poor treatment response, due partly to a highly complex tumor microenvironment (TME). Cancer-associated fibroblast (CAF) heterogeneity is characteristic of the pancreatic TME, where several CAF subpopulations have been identified, such as myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen presenting CAFs (apCAFs). In PDAC, cancer cells continuously adapt their metabolism (metabolic switch) to environmental changes in pH, oxygenation, and nutrient availability. Recent advances show that these environmental alterations are all heavily driven by stromal CAFs. CAFs and cancer cells exchange cytokines and metabolites, engaging in a tight bidirectional crosstalk, which promotes tumor aggressiveness and allows constant adaptation to external stress, such as chemotherapy. In this review, we summarize CAF diversity and CAF-mediated metabolic rewiring, in a PDAC-specific context. First, we recapitulate the most recently identified CAF subtypes, focusing on the cell of origin, activation mechanism, species-dependent markers, and functions. Next, we describe in detail the metabolic crosstalk between CAFs and tumor cells. Additionally, we elucidate how CAF-driven paracrine signaling, desmoplasia, and acidosis orchestrate cancer cell metabolism. Finally, we highlight how the CAF/cancer cell crosstalk could pave the way for new therapeutic strategies.

Myoferlin Is a Yet Unknown Interactor of the Mitochondrial Dynamics' Machinery in Pancreas Cancer Cells.

Pancreas ductal adenocarcinoma is one of the deadliest cancers where surgery remains the main survival factor. Mitochondria were described to be involved in tumor aggressiveness in several cancer types including pancreas cancer. We have previously reported that myoferlin controls mitochondrial structure and function, and demonstrated that myoferlin depletion disturbs the mitochondrial dynamics culminating in a mitochondrial fission. In order to unravel the mechanism underlying this observation, we explored the myoferlin localization in pancreatic cancer cells and showed a colocalization with the mitochondrial dynamic machinery element: mitofusin. This colocalization was confirmed in several pancreas cancer cell lines and in normal cell lines as well. Moreover, in pancreas cancer cell lines, it appeared that myoferlin interacted with mitofusin. These discoveries open-up new research avenues aiming at modulating mitofusin function in pancreas cancer.

Human colon cancer cells highly express myoferlin to maintain a fit mitochondrial network and escape p53-driven apoptosis.

Colon adenocarcinoma is the third most commonly diagnosed cancer and the second deadliest one. Metabolic reprogramming, described as an emerging hallmark of malignant cells, includes the predominant use of glycolysis to produce energy. Recent studies demonstrated that mitochondrial electron transport chain inhibitor reduced colon cancer tumour growth. Accumulating evidence show that myoferlin, a member of the ferlin family, is highly expressed in several cancer types, where it acts as a tumour promoter and participates in the metabolic rewiring towards oxidative metabolism. In this study, we showed that myoferlin expression in colon cancer lesions is associated with low patient survival and is higher than in non-tumoural adjacent tissue. Human colon cancer cells silenced for myoferlin exhibit a reduced oxidative phosphorylation activity associated with mitochondrial fission leading, ROS accumulation, decreased cell growth, and increased apoptosis. We observed the triggering of a DNA damage response culminating to a cell cycle arrest in wild-type p53 cells. The use of a p53 null cell line or a compound able to restore p53 activity (Prima-1) reverted the effects induced by myoferlin silencing, confirming the involvement of p53. The recent identification of a compound interacting with a myoferlin C2 domain and bearing anticancer potency identifies, together with our demonstration, this protein as a suitable new therapeutic target in colon cancer.

Myoferlin controls mitochondrial structure and activity in pancreatic ductal adenocarcinoma, and affects tumor aggressiveness.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death. Therapeutic options remain very limited and are based on classical chemotherapies. Energy metabolism reprogramming appears as an emerging hallmark of cancer and is considered a therapeutic target with considerable potential. Myoferlin, a ferlin family member protein overexpressed in PDAC, is involved in plasma membrane biology and has a tumor-promoting function. In the continuity of our previous studies, we investigated the role of myoferlin in the context of energy metabolism in PDAC. We used selected PDAC tumor samples and PDAC cell lines together with small interfering RNA technology to study the role of myoferlin in energetic metabolism. In PDAC patients, we showed that myoferlin expression is negatively correlated with overall survival and with glycolytic activity evaluated by 18F-deoxyglucose positron emission tomography. We found out that myoferlin is more abundant in lipogenic pancreatic cancer cell lines and is required to maintain a branched mitochondrial structure and a high oxidative phosphorylation activity. The observed mitochondrial fission induced by myoferlin depletion led to a decrease of cell proliferation, ATP production, and autophagy induction, thus indicating an essential role of myoferlin for PDAC cell fitness. The metabolic phenotype switch generated by myoferlin silencing could open up a new perspective in the development of therapeutic strategies, especially in the context of energy metabolism.