Browsing by Author "Tokat, Ünal Metin"

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Open Access
Alterations of ceramide synthesis induce PD-L1 internalization and signaling to regulate tumor metastasis and immunotherapy response
(Cell Press, 2024-08-27) Wofford, Wyatt; Kim, Jisun; Kim, Dosung; Janneh, Alhaji H.; Lee, Han Gyul; Atılgan, Cansu; Oleinik, Natalia; Kassir, Mohamed Faisal; Saatçi, Özge; Chakraborty, Paramita; Tokat, Ünal Metin; Gencer, Salih; Howley, Breege; Howe, Philip; Mehrotra, Shikhar; Şahin, Özgür; Öğretmen, Besim
Programmed death ligand 1, PD-L1 (CD274), facilitates immune evasion and exerts pro-survival functions in cancer cells. Here, we report a mechanism whereby internalization of PD-L1 in response to alterations of bioactive lipid/ceramide metabolism by ceramide synthase 4 (CerS4) induces sonic hedgehog (Shh) and transforming growth factor b receptor signaling to enhance tumor metastasis in triple-negative breast cancers (TNBCs), exhibiting immunotherapy resistance. Mechanistically, data showed that internalized PD-L1 interacts with an RNA-binding protein, caprin-1, to stabilize Shh/TGFBR1/Wnt mRNAs to induce b-catenin signaling and TNBC growth/metastasis, consistent with increased infiltration of FoxP3+ + regulatory T cells and resistance to immunotherapy. While mammary tumors developed in MMTV-PyMT/CerS4-/-- /- were highly metastatic, targeting the Shh/PD-L1 axis using sonidegib and anti-PD-L1 antibody vastly decreased tumor growth and metastasis, consistent with the inhibition of PD-L1 internalization and Shh/Wnt signaling, restoring anti-tumor immune response. These data, validated in clinical samples and databases, provide a mechanism-based therapeutic strategy to improve immunotherapy responses in metastatic TNBCs.
Open Access
Evaluation of the relationship between aromatase/sirtuin1 interaction and miRNA expression in human neuroblastoma cells
(Bentham Science Publishers, 2022-10-21) Kartal, Yasemin; Tokat, Ünal Metin; Uğur, Pelin Kelicen; Yılmaz, Serkan; Karahan, Sevilay; Budak, Murat Timur
Background: Changes in activation/inhibition of Sirtuin-1 (SIRT1) and aromatase play an important role in a plethora of diseases. MicroRNAs (miRNAs) modulate multiple molecular pathways and affect a substantial number of physiological and pathological processes. Objective: The aim of this study was to investigate any possible interaction between aromatase and SIRT1 in SH-SY5Y cells and to see how there is a connection between this interaction and miRNA expression, if there is an interaction. Methods: In this study, cells were incubated in serum-deprived media for 6, 12, and 24 h. Aromatase and SIRT1 expressions were evaluated by Western blot. The IC50 concentration of SIRT1 activator (SRT1720), SIRT1 inhibitor (EX527), and aromatase inhibitors (letrozole and fadrozole) was determined by the XTT method. Then, CYP19A1 and SIRT1 levels were evaluated in the presence of SIRT1 siRNA or IC50 values for each activator/inhibitor. Finally, CYP19A1, SIRT1 expression and miRNA target gene were assessed with bioinformatic approaches. Results: Aromatase and SIRT1 protein levels were significantly elevated in the cells incubated at 24 h in serum-deprived media (p ≤ 0.05). SIRT1 also positively regulated CYP19A1 in SH-SY5Y cells in media with/without FBS. Serum deprivation depending on time course caused changes in the oxidant/ antioxidant system. While oxidative stress index tended to decrease in the absence of FBS at 24 h compared to the control, it showed a significant decrease at 48 h in a serum-deprived manner (p ≤ 0.001). As a result of bioinformatics analysis, we determined 3 miRNAs that could potentially regulate SIRT1 and CYP19A1. hsa-miR-27a-3p and hsa-miR-181a-5p correlated in terms of their expressions at 24 h compared to 12 h, and there was a significant decrease in the expression of these miRNAs. On the contrary, the expression of hsa-miR-30c-5p significantly increased at 24 h compared to 12 h. Conclusion: Considering the results, a direct link between aromatase and SIRT1 was observed in human neuroblastoma cells. The identification of key miRNAs, hsa-miR-27a-3p, hsa-miR-30c-5p, and hsa-miR-181a-5p targeting both aromatase and SIRT1, provides an approach with novel insights on neurology-associated diseases.
Open Access
HSP90 inhibitors induce GPNMB cell-surface expression by modulating lysosomal positioning and sensitize breast cancer cells to glembatumumab vedotin
(Nature Publishing Group, 2022-02-02) Biondini, M.; Kiepas, A.; El-Houjeiri, L; Annis, M.G.; Hsu, B.E.; Fortier, A.M.; Morin, G.; Martina, J.A.; Sirois, I.; Aguilar-Mahecha, A.; Gruosso, T.; McGuirk, S; Rose, A.A.N.; Tokat, Ünal Metin; Johnson, R.M.; Şahin, Ö.; Bareke, E.; St-Pierre, J.; Park, M.; Basik, M.; Majewski, J.; Puertollano, R.; Pause, A.; Huang, S.; Keler, T.; Siegel, P.M.
Transmembrane glycoprotein NMB (GPNMB) is a prognostic marker of poor outcome in patients with triple-negative breast cancer (TNBC). Glembatumumab Vedotin, an antibody drug conjugate targeting GPNMB, exhibits variable efficacy against GPNMB-positive metastatic TNBC as a single agent. We show that GPNMB levels increase in response to standard-of-care and experimental therapies for multiple breast cancer subtypes. While these therapeutic stressors induce GPNMB expression through differential engagement of the MiTF family of transcription factors, not all are capable of increasing GPNMB cell-surface localization required for Glembatumumab Vedotin inhibition. Using a FACS-based genetic screen, we discovered that suppression of heat shock protein 90 (HSP90) concomitantly increases GPNMB expression and cell-surface localization. Mechanistically, HSP90 inhibition resulted in lysosomal dispersion towards the cell periphery and fusion with the plasma membrane, which delivers GPNMB to the cell surface. Finally, treatment with HSP90 inhibitors sensitizes breast cancers to Glembatumumab Vedotin in vivo, suggesting that combination of HSP90 inhibitors and Glembatumumab Vedotin may be a viable treatment strategy for patients with metastatic TNBC.
Open Access
Identification of actionable drug targets in triple-negative breast cancer
(2022-09) Tokat, Ünal Metin
Triple-negative breast cancer (TNBC) is the most aggressive subtype of the breast cancer, representing 15-20% of all cases. Due to a lack of clinically available/approved targeted therapies, TNBC patients are still in desperate need of chemotherapy. Although TNBC patients initially have relatively high rates of response to chemotherapy, de novo and acquired resistance constitute a major problem, leading to worsened patient outcomes. Considering the initial high response rates and broad availability and affordability of the chemotherapy agents, it is of great utility to explore the molecular mechanisms driving or contributing to de novo and acquired chemoresistance. Therefore, I developed 9 acquired resistant cell lines and 3 acquired resistant xenografts and harnessed 4 de novo TNBC chemoresistance models to identify the resistance-driving factors for each chemotherapy class/agent. Following characterization of these models in comparison to their parental counterparts at protein level, I determined few candidate proteins involved in chemoresistance. By combining experimental and bioinformatic approaches, I showed translational potential of i) p38 and JNK MAPK, and PI3K/AKT inhibitors, and ER stress inducers in de novo and acquired resistance against antimetabolite chemotherapy agents such as gemcitabine and fluorouracil; ii) dual EGFR/HER2, EGFR, SRC and CDK1 inhibitors against an antimicrotubule agent, paclitaxel; iii) FAK and dual PI3K/mTOR inhibitors against an alkylating chemotherapy agent, cisplatin, and iv) FAK, SRC and PI3K/AKT inhibitors against a topoisomerase II inhibitor, doxorubicin. I further observed that molecular underpinnings of de novo and acquired resistance were largely overlapping albeit not the same. We have also investigated cross chemotherapy resistance patterns in each acquired cell line model, which can guide second- or third-line treatment selection. As a result, I present a rational approach to exploit chemotherapy-small molecule inhibitor combinations for previously untreated or treated TNBC patients. In other words, using low-dose chemotherapy and targeted therapy combinations we provide promising preclinical in vitro data for future in vivo testing.
Open Access
Targeting LINC00152 activates cAMP/Ca²⁺/ferroptosis axis and overcomes tamoxifen resistance in ER+ breast cancer
(Nature Publishing Group, 2024-06-15) Saatçi, Özge; Alam, Rashedul; Huynh-Dam, Kim-Tuyen; Işık, Aynur; Üner, Meral; Belder, Nevin; Ersan, Pelin Gülizar; Tokat, Ünal Metin; Ulukan, Bürge; Çetin, Metin; Çalışır, Kübra; Gedik, Mustafa Emre; Bal, Hilal; Şener Şahin, Özlem; Riazalhosseini, Yasser; Thieffry, Denis; Gautheret, Daniel; Ogretmen, Besim; Aksoy, Sercan; Üner, Ayşegül; Akyol, Aytekin; Şahin, Özgür
Tamoxifen has been the mainstay therapy to treat early, locally advanced, and metastatic estrogen receptor-positive (ER + ) breast cancer, constituting around 75% of all cases. However, the emergence of resistance is common, necessitating the identification of novel therapeutic targets. Here, we demonstrated that long-noncoding RNA LINC00152 confers tamoxifen resistance by blocking tamoxifen-induced ferroptosis, an iron-mediated cell death. Mechanistically, inhibiting LINC00152 reduces the mRNA stability of phosphodiesterase 4D (PDE4D), leading to activation of the cAMP/PKA/CREB axis and increased expression of the TRPC1 Ca²⁺ channel. This causes cytosolic Ca²⁺ overload and generation of reactive oxygen species (ROS) that is, on the one hand, accompanied by downregulation of FTH1, a member of the iron sequestration unit, thus increasing intracellular Fe²⁺ levels; and on the other hand, inhibition of the peroxidase activity upon reduced GPX4 and xCT levels, in part by cAMP/CREB. These ultimately restore tamoxifen-dependent lipid peroxidation and ferroptotic cell death, which are reversed upon chelating Ca²⁺ or overexpressing GPX4 or xCT. Overexpressing PDE4D reverses LINC00152 inhibition-mediated tamoxifen sensitization by de-activating the cAMP/Ca²⁺/ferroptosis axis. Importantly, high LINC00152 expression is significantly correlated with high PDE4D/low ferroptosis and worse survival in multiple cohorts of tamoxifen- or tamoxifen-containing endocrine therapy-treated ER+ breast cancer patients. Overall, we identified LINC00152 inhibition as a novel mechanism of tamoxifen sensitization via restoring tamoxifen-dependent ferroptosis upon destabilizing PDE4D, increasing cAMP and Ca²⁺ levels, thus leading to ROS generation and lipid peroxidation. Our findings reveal LINC00152 and its effectors as actionable therapeutic targets to improve clinical outcome in refractory ER+ breast cancer.
Open Access
Targeting lysyl oxidase (LOX) overcomes chemotherapy resistance in triple negative breast cancer
(Nature Research, 2020) Saatçi, Ö.; Kaymak, A.; Raza, Umar; Ersan, Pelin G.; Akbulut, Özge; Banister, C. E.; Sikirzhytski, V.; Tokat, Ünal Metin; Aykut, Gamze; Ansari, Suhail A.; Tatlı-Doğan, H.; Doğan, M.; Jandaghi, P.; Işık, A.; Gündoğdu, F.; Kösemehmetoğlu, K.; Dizdar, Ö.; Aksoy, S.; Akyol, A.; Üner, A.; Buckhaults, P. J.; Riazalhosseini, Y.; Şahin, Özgür
Chemoresistance is a major obstacle in triple negative breast cancer (TNBC), the most aggressive breast cancer subtype. Here we identify hypoxia-induced ECM re-modeler, lysyl oxidase (LOX) as a key inducer of chemoresistance by developing chemoresistant TNBC tumors in vivo and characterizing their transcriptomes by RNA-sequencing. Inhibiting LOX reduces collagen cross-linking and fibronectin assembly, increases drug penetration, and downregulates ITGA5/FN1 expression, resulting in inhibition of FAK/Src signaling, induction of apoptosis and re-sensitization to chemotherapy. Similarly, inhibiting FAK/Src results in chemosensitization. These effects are observed in 3D-cultured cell lines, tumor organoids, chemoresistant xenografts, syngeneic tumors and PDX models. Re-expressing the hypoxia-repressed miR-142-3p, which targets HIF1A, LOX and ITGA5, causes further suppression of the HIF-1α/LOX/ITGA5/FN1 axis. Notably, higher LOX, ITGA5, or FN1, or lower miR-142-3p levels are associated with shorter survival in chemotherapy-treated TNBC patients. These results provide strong pre-clinical rationale for developing and testing LOX inhibitors to overcome chemoresistance in TNBC patients.
Open Access
Toxic PARP trapping upon cAMP-induced DNA damage reinstates the efficacy of endocrine therapy and CDK4/6 inhibitors in treatment-refractory ER+ breast cancer
(Nature Research, 2023-11-02) Saatci, O.; Cetin, M; Uner, M.; Tokat, Ünal Metin; Chatzistamou, I.; Ersan, P. G.; Montaudon, E.; Akyol, A.; Aksoy, S.; Uner, A.; Marangoni, E.; Sajish, M.
Resistance to endocrine therapy and CDK4/6 inhibitors, the standard of care (SOC) in estrogen receptor-positive (ER+) breast cancer, greatly reduces patient survival. Therefore, elucidating the mechanisms of sensitivity and resistance to SOC therapy and identifying actionable targets are urgently needed. Here, we show that SOC therapy causes DNA damage and toxic PARP1 trapping upon generation of a functional BRCAness (i.e., BRCA1/2 deficiency) phenotype, leading to increased histone parylation and reduced H3K9 acetylation, resulting in transcriptional blockage and cell death. Mechanistically, SOC therapy downregulates phosphodiesterase 4D (PDE4D), a novel ER target gene in a feedforward loop with ER, resulting in increased cAMP, PKA-dependent phosphorylation of mitochondrial COXIV-I, ROS generation and DNA damage. However, during SOC resistance, an ER-to-EGFR switch induces PDE4D overexpression via c-Jun. Notably, combining SOC with inhibitors of PDE4D, EGFR or PARP1 overcomes SOC resistance irrespective of the BRCA1/2 status, providing actionable targets for restoring SOC efficacy. © 2023, The Author(s).