TARGETING RIBOSOMAL P70S6K1 AS A POTENTIAL ANTI-TUMORIGENIC THERAPEUTIC APPROACH AGAINST TRIPLE-NEGATIVE BREAST CANCER DEVELOPED IN TYPE II DIABETIC CONDITION

Abstract

The triple-negative breast cancer (TNBC), characterized by the absence of expression of hormone receptors and epidermal growth factor receptor-2 amplification, is usually the most malignant and aggressive mammary epithelial tumor among other breast cancer subtypes. TNBC corresponds to 15-20% of all breast cancers and is known for its poor clinical outcome. Therefore, targeted therapy is a real challenge for TNBC management, making chemotherapy the main treatment modality. Type 2 diabetes (T2D) is considered a difficult-to-manage metabolic syndrome due to uncontrolled high glucose level resulting from insufficient insulin production or resistance. Interestingly, several epidemiological studies have shown that T2D is considered an independent risk factor for breast cancer development, especially TNBC. In vitro investigation revealed that the ribosomal protein S6 kinase beta-1(p70SK1) is overphosphorylated in glycated albumin (i.e., advanced glycation end products, AGEs)-treated TNBC MDA-MB-231 cells, suggesting phospho- xii p70S6K1 as a potential biomarker for TNBC tissues from T2D patients. Using immunohistochemical staining, overexpression of ribosomal protein p70S6K1 was confirmed in invasive ductal carcinoma (IDC) tissues from T2D patients compared to their non-diabetic counterpart. Moreover, phospho-p70S6K1 plays a key role in protein synthesis, tumor progression, and drug resistance as well. Therefore, targeting p70S6K1 could be a promising therapeutic target to reduce TNBC progression and overcome chemoresistance to improve the outcomes of T2D patients diagnosed with TNBC. In this work we have two main objectives: First, the investigation of the main function of p70S6K1 in TNBC using MDA-MB-231 cells treated with glycated albumin. We established siRNA transfection technology for p70S6K1 gene downregulation using MDA-MB-231 cells as a TNBC model, and we applied the pharmacological inhibitor PF- 4708671 to block p70S6K1 activity, in the presence of glycated albumin. Cancer cell invasion, the main cell event involved in tumor progression, was determined using Transwell® inserts and the expression levels of activated signaling proteins and cancer- related proteins using Western blot technology. Second, the determination of the role of p70S6K1 in acquired chemoresistance of glycated albumin-pretreated MDA-MB-231 cells towards cisplatin. The combined treatment of PF-4708671 and cisplatin at different incubation treatment periods and their comparison with cisplatin alone in glycated albumin-pretreated MDA-MB-231 cells were performed and evaluated. We used cell viability assay for cytotoxicity assessment, dead cell detection employing SytoxTM dye and mitochondria permeability transition pore opening (mPTPO) activity was evaluated under confocal microscope. The pro-apoptotic protein Bax and autophagy-related proteins (i.e., xiii

ATG5, LCB3) were determined using Western blot technology. Our results showed that p70S6K1 knockdown and PF-4708671-treated cells inhibited GA-induced TNBC MDA- MB-231 cell invasion, protein S6 (p70S6K1 downstream effector) and oncogenic ERK1/2 phosphorylation, and cancer-related protein expression, including galectin-3. Moreover, in the context of chemoresistance, we observed overphosphorylation of mTOR and downstream targets p70S6K1 and 4EBP1 in GA-treated MDA-MB-231 cells. PF-4708671 pretreatment decreased the phosphorylation of p70S6K1 and 4EBP1 in GA-pretreated cells. Regarding cellular toxicity, GA treatment suppressed cisplatin efficacy. However, PF-4708671 administration prior to cisplatin showed a significant impact on reversing cellular resistance to cisplatin sensitivity in GA-pretreated MDA-MB-231 cells. This effect of PF-4708671 treatment led to high dead cell percentage, upregulation of Bax and LC3B, and increased mPTPO activity. To conclude, p70S6K1 activity blockade could play a potential therapeutic strategy for TNBC patients with T2D, and its combination treatment with chemotherapy may overcome chemoresistance and improve patient outcomes. This study also presented galectin-3 as a potential prognostic biomarker for invasive BC progression in T2D patients, which will need to be validated in larger cohorts.