Systematic analysis reveals a functional role for STAMBPL1 in the epithelial-mesenchymal transition process across multiple carcinomas.

BACKGROUND: Deubiquitinating enzymes (DUBs) are linked to cancer progression and dissemination, yet less is known about their regulation and impact on epithelial-mesenchymal transition (EMT). METHODS: An integrative translational approach combining systematic computational analyses of The Cancer Genome Atlas cancer cohorts with CRISPR genetics, biochemistry and immunohistochemistry methodologies to identify and assess the role of human DUBs in EMT. RESULTS: We identify a previously undiscovered biological function of STAM-binding protein like 1 (STAMBPL1) deubiquitinase in the EMT process in lung and breast carcinomas. We show that STAMBPL1 expression can be regulated by mutant p53 and that its catalytic activity is required to affect the transcription factor SNAI1. Accordingly, genetic depletion and CRISPR-mediated gene knockout of STAMBPL1 leads to marked recovery of epithelial markers, SNAI1 destabilisation and impaired migratory capacity of cancer cells. Reversely, STAMBPL1 expression reprogrammes cells towards a mesenchymal phenotype. A significant STAMBPL1-SNAI1 co-signature was observed across multiple tumour types. Importantly, STAMBPL1 is highly expressed in metastatic tissues compared to matched primary tumour of the same lung cancer patient and its expression predicts poor prognosis. CONCLUSIONS: Our study provides a novel concept of oncogenic regulation of a DUB and presents a new role and predictive value of STAMBPL1 in the EMT process across multiple carcinomas.

miR-100-5p confers resistance to ALK tyrosine kinase inhibitors Crizotinib and Lorlatinib in EML4-ALK positive NSCLC.

Lung cancer causes the highest number of cancer-related deaths worldwide. Resistance to therapy is a major clinical issue contributing to the poor prognosis of lung cancer. In recent years, targeted therapy has become a concept where subgroups of non-small cell lung cancer (NSCLC) with genetically altered receptor tyrosine kinases are targeted by tyrosine kinase inhibitors (TKIs). One such subgroup harbors a gene fusion of echinoderm microtubule-associated protein-like 4 (EML4) with anaplastic lymphoma kinase (ALK). Although most NSCLC patients with EML4-ALK fusions initially respond to ALK TKI-therapy they eventually develop resistance. While ALK kinase domain mutations contribute to ALK TKI-refractoriness, they are only present in a fraction of all ALK TKI-resistant tumors. In this study we sought to explore a possible involvement of microRNAs (miRNAs) in conferring resistance to ALK TKIs in ALK TKI-refractory NSCLC cell lines. We subjected our ALK TKI-refractory cancer cells along with parental cancer cells to systematic miRNA expression arrays. Furthermore, ALK TKI-refractory cancer cells were exposed to a synthetic miRNA inhibitory Locked Nucleic Acid (LNA)-library in the presence of ALK TKIs Crizotinib or Lorlatinib. The outcome of the combined approaches uncovered miR-100-5p to confer resistance to Crizotinib and Lorlatinib in EML4-ALK NSCLC cells and to be a potential therapeutic target in drug resistance.

USP10 regulates the stability of the EMT-transcription factor Slug/SNAI2.

Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism governing the switch of cells from an epithelial to a motile mesenchymal-like state. This transdifferentiation is regulated by key transcription factors, including Slug. The stability and function of Slug can be regulated by multiple mechanisms, including ubiquitin-mediated post-translational modifications. Here, by using a genome wide siRNA screen for human deubiquitinating enzymes (DUBs), we identified USP10 as a deubiquitinase for Slug in cancer cells. USP10 interacts with Slug and mediates its degradation by the proteasome. Importantly, USP10 is concomitantly highly expressed with Slug in cancer biopsies. Genetic knockdown of USP10 leads to suppressed Slug levels with a decreased expression of the mesenchymal marker Vimentin. Further, it reduces the migratory capacity of cancer cells. Reversely, overexpression of USP10 elevates the level of both Slug and Vimentin. Our study identifies USP10 as a regulator of the EMT-transcription factor Slug and cell migration.

Isolation of Autophagy Competent Lysosomes from Cancer Cells by Differential Large-Scale Multilayered Density Gradient Centrifugations.

Accurate isolation of functional and intact lysosomes enables the quantification and analyses of abundances, dynamic changes and enrichment levels of lysosomal content, allowing specific lysosomal investigations induced by autophagy. In this protocol chapter, we describe detailed practical instructions and advices for an efficacious lysosomal enrichment and isolation procedure by differential multilayered density gradient centrifugations using human cancer cell lines. By this method, intact and autophagy competent lysosomes can be isolated from cancer cells based on their distinct density and obtained fractions can further be analyzed for functional lysosomal assays, as well as for protein or metabolic loads to identify select spatiotemporal changes by comparative quantitative measurement. This method has been used to enrich lysosomes from a variety of cancer cells with activated chaperone-mediated autophagy, but can be optimized for other cell lines and tissues for multiple autophagy-induced conditions.

Quantitative proteomic analysis of temporal lysosomal proteome and the impact of the KFERQ-like motif and LAMP2A in lysosomal targeting.

Autophagic pathways are regulated mechanisms that play important roles in lysosome-mediated cellular degradation. Yet, the contribution of different autophagic pathways in lysosomal targeting, and characterization of the extent and specificity in their degradome remains largely uncharacterized. By undertaking a multiplex quantitative mass spectrometry approach, we have previously analyzed the lysosomal proteome during chaperone-mediated autophagy (CMA)-stimulated conditions in cancer cells. Here, we have extended our multiplex quantitative mass spectrometry and bioinformatics analysis on the proteome from isolated lysosomes to gain a comprehensive view of the temporal enriched lysosomal content upon non-macroautophagy-activated conditions. In parallel, we describe the functional dependency of LAMP2A on, and to what degree the presence of KFERQ-like motifs in proteins influences, their lysosomal targeting. These findings establish a framework for a better understanding of the degradome mediated by autophagic pathways beyond macroautophagy, and present characterization of the impact of LAMP2A in lysosomal targeting in cancer cells.Abbreviations: CMA: chaperone-mediated autophagy; ER: endoplasmic reticulum; EIF4A1: eukaryotic translation initiation factor 4A1; eMI: endosomal microautophagy; FC: fold change; GO: gene ontology; ISR: integrated stress response; LAMP2A: lysosomal associated membrane protein 2A; MA: macroautophagy; MI: microautophagy; MS: mass spectrometry; PCA: principal component analysis; TAX1BP1: Tax1 binding protein 1.

The deubiquitinase JOSD2 is a positive regulator of glucose metabolism.

Cancer cells undergo complex metabolic alterations. The mechanisms underlying the tuning of cancer metabolism are under active investigation. Here, we identify the uncharacterized deubiquitinase JOSD2 as a positive regulator of cancer cell proliferation by displaying comprehensive effects on glucose catabolism. We found that JOSD2 directly controls a metabolic enzyme complex that includes Aldolase A, Phosphofructokinase-1 and Phosphoglycerate dehydrogenase, in vitro and in vivo. Further, JOSD2 expression, but not a catalytically inactive mutant, deubiquitinates and stabilizes the enzyme complex, thereby enhancing their activities and the glycolytic rate. This represents a selective JOSD2 feature that is not shared among other Machado-Joseph disease DUBs or observed in nontransformed cells. JOSD2 deficiency displays cytostatic effects and reduces glycolysis in a broad spectrum of tumor cells of distinct origin and its expression correlates with poor prognosis in non-small cell lung cancer. Overall, our study provides evidence for a previously unknown biological mechanism in which JOSD2 integrates glucose and serine metabolism with potential therapeutic implications.

Antithetic hTERT Regulation by Androgens in Prostate Cancer Cells: hTERT Inhibition Is Mediated by the ING1 and ING2 Tumor Suppressors.

The human telomerase is a key factor during tumorigenesis in prostate cancer (PCa). The androgen receptor (AR) is a key drug target controlling PCa growth and regulates hTERT expression, but is described to either inhibit or to activate. Here, we reveal that androgens repress and activate hTERT expression in a concentration-dependent manner. Physiological low androgen levels activate, while, notably, supraphysiological androgen levels (SAL), used in bipolar androgen therapy (BAT), repress hTERT expression. We confirmed the SAL-mediated gene repression of hTERT in PCa cell lines, native human PCa samples derived from patients treated ex vivo, as well as in cancer spheroids derived from androgen-dependent or castration resistant PCa (CRPC) cells. Interestingly, chromatin immuno-precipitation (ChIP) combined with functional assays revealed a positive (pARE) and a negative androgen response element (nARE). The nARE was narrowed down to 63 bp in the hTERT core promoter region. AR and tumor suppressors, inhibitor of growth 1 and 2 (ING1 and ING2, respectively), are androgen-dependently recruited. Mechanistically, knockdown indicates that ING1 and ING2 mediate AR-regulated transrepression. Thus, our data suggest an oppositional, biphasic function of AR to control the hTERT expression, while the inhibition of hTERT by androgens is mediated by the AR co-repressors ING1 and ING2.

Targetome analysis of chaperone-mediated autophagy in cancer cells.

Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of select soluble proteins. Nearly one-third of the soluble proteins are predicted to be recognized by this pathway, yet only a minor fraction of this proteome has been identified as CMA substrates in cancer cells. Here, we undertook a quantitative multiplex mass spectrometry approach to study the proteome of isolated lysosomes in cancer cells during CMA-activated conditions. By integrating bioinformatics analyses, we identified and categorized proteins of multiple cellular pathways that were specifically targeted by CMA. Beyond verifying metabolic pathways, we show that multiple components involved in select biological processes, including cellular translation, was specifically targeted for degradation by CMA. In particular, several proteins of the translation initiation complex were identified as bona fide CMA substrates in multiple cancer cell lines of distinct origin and we show that CMA suppresses cellular translation. We further show that the identified CMA substrates display high expression in multiple primary cancers compared to their normal counterparts. Combined, these findings uncover cellular processes affected by CMA and reveal a new role for CMA in the control of translation in cancer cells. Abbreviations: 6-AN: 6-aminonicotinamide; ACTB: actin beta; AR7: atypical retinoid 7; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; CTS: cathepsins; DDX3X: DEAD-box helicase 3 X-linked; EEF2: eukaryotic translation elongation factor 2; EIF4A1: eukaryotic translation initiation factor 4A1; EIF4H: eukaryotic translation initiation factor 4H; GEO: Gene Expression Omnibus; GO: Gene Ontology; GSEA: gene set enrichment analysis; HK2: hexokinase 2; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; LAMP: lysosomal-associated membrane protein; LDHA: lactate dehydrogenase A; NES: normalized enrichment score; NFKBIA: NFKB inhibitor alpha; PCA: principle component analysis; PQ: paraquat; S.D.: standard deviation; SUnSET: surface sensing of translation; TMT: tandem mass tags; TOMM40/TOM40: translocase of outer mitochondrial membrane 40.