Comparative genomics incorporating translocation renal cell carcinoma mouse model reveals molecular mechanisms of tumorigenesis.

Translocation renal cell carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed clear cell RCC driver) disrupted nephrogenesis and glomerular development, causing neonatal death, while the clear cell RCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as alveolar soft part sarcoma) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an epithelial-mesenchymal transition. Electron microscopy of tRCC tumors showed lysosome expansion, and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models (ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3, RBM10-TFE3, and MALAT1-TFEB) disclosed significant convergence in canonical pathways (cell cycle, lysosome, and mTORC1) and less established pathways such as Myc, E2F, and inflammation (IL-6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc.). Therapeutic trials (adjusted for human drug exposures) showed antitumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis, including the cell of origin, and characterizes diverse mouse models available for research.

Heterotypic signaling of cancer-associated fibroblasts in shaping the cancer cell drug resistance.

The context-dependent reciprocal interaction between the cancer cells and surrounding fibroblasts is imperative for regulating malignant potential, metabolic reprogramming, immunosuppression, and ECM deposition. However, recent evidence also suggests that cancer-associated fibroblasts induce chemoresistance in cancer cells to various anticancer regimens. Because of the protumorigenic function of cancer-associated fibroblasts, these stromal cell types have emerged as fascinating therapeutic targets for cancer. However, this notion was recently challenged by studies that targeted cancer-associated fibroblasts and highlighted the underlying heterogeneity by identifying a subset of these cells with tumor-restricting functions. Hence, it is imperative to understand the heterogeneity and heterotypic signaling of cancer-associated fibroblasts to target tumor-promoting signaling processes by sparing tumor-restricting ones. In this review, we discuss the heterogeneity and heterotypic signaling of cancer-associated fibroblasts in shaping drug resistance and also list the cancer-associated fibroblast-targeting therapeutics.

HPV18 oncoproteins driven expression of PKM2 reprograms HeLa cell metabolism to maintain aerobic glycolysis and viability.

The molecular basis of human papillomavirus (HPV)-mediated cellular immortalization and malignant transformation has illustrated an indispensable role of viral E6/E7-oncoproteins. However, the impact of viral-oncoproteins on the metabolic phenotype of cancer cells remains ambiguous. We showed silencing of HPV18-encoded E6/E7-oncoprotein significantly reduced glucose consumption, lactate production, ATP level and viability. Silencing of HPV18-encoded E6/E7 in HeLa cells significantly down-regulated expression and activity of HK1, HK2, LDHA, and LDHB. Interestingly, there was an increased pyruvate kinase activity due to switch in expression from PKM2 isoform to PKM1. The switch in favor of alternatively spliced isoform PKM1, was regulated by viral-E6/E7-oncoprotein by inhibiting the c-Myc/hnRNP-axis. Further, the near absence of the PKM1 protein despite an adequate amount of PKM1 mRNA in HeLa cells was due to its proteasomal degradation. Our results suggests HPV18-encoded E6/E7 driven preferential expression of PKM2 is essential to support aerobic glycolysis and cell proliferation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00776-w.

Silibinin induces metabolic crisis in triple-negative breast cancer cells by modulating EGFR-MYC-TXNIP axis: potential therapeutic implications.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with limited treatment modalities and poor prognosis. Metabolic reprogramming in cancer is considered a hallmark of therapeutic relevance. Here, we report disruption of metabolic reprogramming in TNBC cells by silibinin via modulation of EGFR-MYC-TXNIP signaling. Metabolic assays combined with LC-MS-based metabolomics revealed inhibition of glycolysis and other key biosynthetic pathways by silibinin, to induce metabolic catastrophe in TNBC cells. Silibinin-induced metabolic suppression resulted in decreased cell biomass, proliferation, and stem cell properties. Mechanistically, we identify EGFR-MYC-TXNIP as an important regulator of TNBC metabolism and mediator of inhibitory effects of silibinin. Highlighting the clinical relevance of our observations, the analysis of METABRIC dataset revealed deregulation of EGFR-MYC-TXNIP axis in TNBC and association of EGFRhigh -MYChigh -TXNIPlow signature with aggressive glycolytic metabolism and poor disease-specific and metastasis-free survival. Importantly, combination treatment of silibinin or 2-deoxyglucose (glycolysis inhibitor) with paclitaxel synergistically inhibited proliferation of TNBC cells. Together, our results highlight the importance of EGFR-MYC-TXNIP axis in regulating TNBC metabolism, demonstrate the anti-TNBC activity of silibinin, and argue in favor of targeting metabolic vulnerabilities of TNBC, at least in combination with mainstay chemotherapeutic drugs, to effectively treat TNBC patients.

Role of ectopically expressed mtDNA encoded cytochrome c oxidase subunit I (MT-COI) in tumorigenesis.

Somatic mutations within mitochondrial DNA (mtDNA) encoded cytochrome c oxidase subunit I (MT-CO1 or MT-COI) are frequent in various cancer types. In addition, perturbation from orchestrated expression of mitochondrial DNA encoded genes is also associated with complex disorders, including cancer. Since codon bias and the mitochondrial translation system restricts functional characterization of over-expressed wild type or mutant mitochondrial DNA encoded genes, the codon optimization and artificial synthesis of entire MT-CO1 allowed us to over-express the wild type and one of its deleterious mutants into the mitochondria of the transfected cells. Ectopically expressed MT-CO1 was observed to efficiently express and localized to mitochondria but showed high level of aggregation under denaturing condition. Over-expression of wild type or mutant variant of MT-CO1 promoted anchorage dependent and independent proliferation potential in in-vitro experiments and introduced the cancer cell metabolic phenotype of high glucose uptake and lactate release. Reactive oxygen species generated in cells over-expressing MT-CO1 variants acted as key effectors mediating differential expression of apoptosis and DNA damage pathway related genes. High ROS generated also down-regulated the expression of global regulators of gene expression, DNMT3A and DNMT3B. The down-regulated expression of DNMTs co-related with differential methylation of the CpG islands in the promoter region of a select set of studied genes, in a manner to promote pro-cancerous phenotype. Apart from assigning the mechanistic role to the MT-CO1 variants and their perturbed expression in cancer development, the present study provides novel insights into the functional role of somatic mutations within MT-CO1 promoting cancer phenotype.

MicroRNA in lung cancer: role, mechanisms, pathways and therapeutic relevance.

Lung cancer is the cardinal cause of cancer-related deaths with restricted recourse of therapy throughout the world. Clinical success of therapies is not very promising due to - late diagnosis, limited therapeutic tools, relapse and the development of drug resistance. Recently, small ∼20-24 nucleotides molecules called microRNAs (miRNAs) have come into the limelight as they play outstanding role in the process of tumorigenesis by regulating cell cycle, metastasis, angiogenesis, metabolism and apoptosis. miRNAs essentially regulate gene expression via post-transcriptional regulation of mRNA. Nevertheless, few studies have conceded the role of miRNAs in activation of gene expression. A large body of data generated by numerous studies is suggestive of their tumor-suppressing, oncogenic, diagnostic and prognostic biomarker roles in lung cancer. They have also been implicated in regulating cancer cell metabolism and resistance or sensitivity towards chemotherapy and radiotherapy. Further, miRNAs have also been convoluted in regulation of immune checkpoints - Programmed death 1 (PD-1) and its ligand (PD-L1). These molecules play a significant role in tumor immune escape leading to the generation of a microenvironment favouring tumor growth and progression. Therefore, it is imperative to explore the expression of miRNA and understand its relevance in lung cancer and development of anti-cancer strategies (anti - miRs, miR mimics and micro RNA sponges). In view of the above, the role of miRNA in lung cancer has been dissected and the associated mechanisms and pathways are discussed in this review.

Curcumin decreases Warburg effect in cancer cells by down-regulating pyruvate kinase M2 via mTOR-HIF1α inhibition.

Warburg effect is an emerging hallmark of cancer cells with pyruvate kinase M2 (PKM2) as its key regulator. Curcumin is an extensively-studied anti-cancer compound, however, its role in affecting cancer metabolism remains poorly understood. Herein, we show that curcumin inhibits glucose uptake and lactate production (Warburg effect) in a variety of cancer cell lines by down-regulating PKM2 expression, via inhibition of mTOR-HIF1α axis. Stable PKM2 silencing revealed that PKM2 is required for Warburg effect and proliferation of cancer cells. PKM2 over-expression abrogated the effects of curcumin, demonstrating that inhibition of Warburg effect by curcumin is PKM2-mediated. High PKM2 expression correlated strongly with poor overall survival in cancer, suggesting the requirement of PKM2 in cancer progression. The study unravels novel PKM2-mediated inhibitory effect of curcumin on metabolic capacities of cancer cells. To the best of our knowledge, this is the first study linking curcumin with PKM2-driven cancer glycolysis, thus, providing new perspectives into the mechanism of its anticancer activity.

Posttranslational Modifications of Pyruvate Kinase M2: Tweaks that Benefit Cancer.

Cancer cells rewire metabolism to meet biosynthetic and energetic demands. The characteristic increase in glycolysis, i.e., Warburg effect, now considered as a hallmark, supports cancer in various ways. To attain such metabolic reshuffle, cancer cells preferentially re-express the M2 isoform of pyruvate kinase (PKM2, M2-PK) and alter its quaternary structure to generate less-active PKM2 dimers. The relatively inactive dimers cause the accumulation of glycolytic intermediates that are redirected into anabolic pathways. In addition, dimeric PKM2 also benefits cancer cells through various non-glycolytic moonlight functions, such as gene transcription, protein kinase activity, and redox balance. A large body of data have shown that several distinct posttranslation modifications (PTMs) regulate PKM2 in a way that benefits cancer growth, e.g., formation of PKM2 dimers. This review discusses the recent advancements in our understanding of various PTMs and the benefits they impart to the sustenance of cancer. Understanding the PTMs in PKM2 is crucial to assess their therapeutic potential and to design novel anticancer strategies.

Pyruvate kinase M knockdown-induced signaling via AMP-activated protein kinase promotes mitochondrial biogenesis, autophagy, and cancer cell survival.

Preferential expression of the low-activity (dimeric) M2 isoform of pyruvate kinase (PK) over its constitutively active splice variant M1 isoform is considered critical for aerobic glycolysis in cancer cells. However, our results reported here indicate co-expression of PKM1 and PKM2 and their possible physical interaction in cancer cells. We show that knockdown of either PKM1 or PKM2 differentially affects net PK activity, viability, and cellular ATP levels of the lung carcinoma cell lines H1299 and A549. The stable knockdown of PK isoforms in A549 cells significantly reduced the cellular ATP level, whereas in H1299 cells the level of ATP was unaltered. Interestingly, the PKM1/2 knockdown in H1299 cells activated AMP-activated protein kinase (AMPK) signaling and stimulated mitochondrial biogenesis and autophagy to maintain energy homeostasis. In contrast, knocking down either of the PKM isoforms in A549 cells lacking LKB1, a serine/threonine protein kinase upstream of AMPK, failed to activate AMPK and sustain energy homeostasis and resulted in apoptosis. Moreover, in a similar genetic background of silenced PKM1 or PKM2, the knocking down of AMPKα1/2 catalytic subunit in H1299 cells induced apoptosis. Our findings help explain why previous targeting of PKM2 in cancer cells to control tumor growth has not met with the expected success. We suggest that this lack of success is because of AMPK-mediated energy metabolism rewiring, protecting cancer cell viability. On the basis of our observations, we propose an alternative therapeutic strategy of silencing either of the PKM isoforms along with AMPK in tumors.

Additive Effect of Plasma Rich in Growth Factors With Guided Tissue Regeneration in Treatment of Intrabony Defects in Patients With Chronic Periodontitis: A Split-Mouth Randomized Controlled Clinical Trial.

BACKGROUND: Periodontal regeneration can be defined as complete restoration of lost periodontal tissues to their original architecture and function. A variety of treatment modalities have been proposed to achieve it. Plasma rich in growth factors (PRGF) is a concentrated suspension of growth factors that promotes restoration of lost periodontal tissues. The objective of the present study is to assess the effect of PRGF associated with guided tissue regeneration (GTR) versus GTR only in the treatment of intrabony defects (IBDs) in patients with chronic periodontitis (CP). METHODS: Patients with CP (n = 14) with 42 contralateral 2- and 3-walled defects were randomly assigned to test (PRGF+GTR) and control (GTR alone) treatment groups. Clinical and radiographic assessments performed at baseline and after 6 months were: 1) gingival index (GI), 2) probing depth (PD), 3) clinical attachment level (CAL), 4) radiologic defect depth, and 5) bone fill. RESULTS: Comparison of parameters measured at baseline and after 6 months showed mean PD reduction of 3.37 ± 1.62 mm in the control group (P <0.001) and 4.13 ± 1.59 mm in the test group (P <0.001). There was a significant difference in mean change in CAL (P <0.001) in the control group (5.42 ± 1.99) and the test group (5.99 ± 1.77). Mean change in GI was 1.89 ± 0.32 and 1.68 ± 0.58 in the control group and test group, respectively, and the difference was statistically significant (P <0.001). When compared between groups, clinical parameters did not show any statistically significant variations. Mean radiographic bone fill was 1.06 ± 0.81 and 1.0 ± 0.97 in the control group and test group, respectively. However, the difference was not statistically significant. CONCLUSIONS: PRGF with GTR, as well as GTR alone, was effective in improving clinical and radiographic parameters of patients with CP at the 6-month follow-up. There was no additive effect of PRGF when used along with GTR in the treatment of IBDs in patients with CP in terms of both clinical and radiologic outcomes.

DNA Methyltransferase1 (DNMT1) Isoform3 methylates mitochondrial genome and modulates its biology.

Here we demonstrate localization of the isoform3 of DNA Methyltransferase1 (DNMT1) enzyme to mitochondria, instead of isoform1 as reported earlier. The fused DNMT1-isoform1, reported earlier to localize in mitochondria, surprisingly showed its exclusive presence inside the nucleus after its ectopic expression; and failed to localize in mitochondria. On the other hand, ectopically expressed DNMT1-isoform3 targeted itself to mitochondria and subsequently methylated CpG regions in the mitochondrial genome. In addition, overexpression of DNMT1-isoform3 affected mitochondrial biology and regulated its function. Under different conditions of oxidative and nutritional stress, this isoform was down-regulated, resulting in hypomethylation of mitochondrial genome. Our study reveals how DNMT1-isoform3, instead of isoform1, is responsible for mtDNA methylation, influencing its biology.