The exostosin glycosyltransferase 1/STAT3 axis is a driver of breast cancer aggressiveness.

The epithelial-mesenchymal transition (EMT) program is crucial for transforming carcinoma cells into a partially mesenchymal state, enhancing their chemoresistance, migration, and metastasis. This shift in cell state is tightly regulated by cellular mechanisms that are not yet fully characterized. One intriguing EMT aspect is the rewiring of the proteoglycan landscape, particularly the induction of heparan sulfate proteoglycan (HSPG) biosynthesis. This proteoglycan functions as a co-receptor that accelerates cancer-associated signaling pathways through its negatively-charged residues. However, the precise mechanisms through which EMT governs HSPG biosynthesis and its role in cancer cell plasticity remain elusive. Here, we identified exostosin glycosyltransferase 1 (EXT1), a central enzyme in HSPG biosynthesis, to be selectively upregulated in aggressive tumor subtypes and cancer cell lines, and to function as a key player in breast cancer aggressiveness. Notably, ectopic expression of EXT1 in epithelial cells is sufficient to induce HSPG levels and the expression of known mesenchymal markers, subsequently enhancing EMT features, including cell migration, invasion, and tumor formation. Additionally, EXT1 loss in MDA-MB-231 cells inhibits their aggressiveness-associated traits such as migration, chemoresistance, tumor formation, and metastasis. Our findings reveal that EXT1, through its role in HSPG biosynthesis, governs signal transducer and activator of transcription 3 (STAT3) signaling, a known regulator of cancer cell aggressiveness. Collectively, we present the EXT1/HSPG/STAT3 axis as a central regulator of cancer cell plasticity that directly links proteoglycan synthesis to oncogenic signaling pathways.

The glutathione peroxidase 8 (GPX8)/IL-6/STAT3 axis is essential in maintaining an aggressive breast cancer phenotype.

One of the emerging hallmarks of cancer illustrates the importance of metabolic reprogramming, necessary to synthesize the building blocks required to fulfill the high demands of rapidly proliferating cells. However, the proliferation-independent instructive role of metabolic enzymes in tumor plasticity is still unclear. Here, we provide evidence that glutathione peroxidase 8 (GPX8), a poorly characterized enzyme that resides in the endoplasmic reticulum, is an essential regulator of tumor aggressiveness. We found that GPX8 expression was induced by the epithelial-mesenchymal transition (EMT) program. Moreover, in breast cancer patients, GPX8 expression significantly correlated with known mesenchymal markers and poor prognosis. Strikingly, GPX8 knockout in mesenchymal-like cells (MDA-MB-231) resulted in an epithelial-like morphology, down-regulation of EMT characteristics, and loss of cancer stemness features. In addition, GPX8 knockout significantly delayed tumor initiation and decreased its growth rate in mice. We found that these GPX8 loss-dependent phenotypes were accompanied by the repression of crucial autocrine factors, in particular, interleukin-6 (IL-6). In these cells, IL-6 bound to the soluble receptor (sIL6R), stimulating the JAK/STAT3 signaling pathway by IL-6 trans-signaling mechanisms, so promoting cancer aggressiveness. We observed that in GPX8 knockout cells, this signaling mechanism was impaired as sIL6R failed to activate the JAK/STAT3 signaling pathway. Altogether, we present the GPX8/IL-6/STAT3 axis as a metabolic-inflammatory pathway that acts as a robust regulator of cancer cell aggressiveness.

Integrated genomics approach to identify biologically relevant alterations in fewer samples.

BACKGROUND: Several statistical tools have been developed to identify genes mutated at rates significantly higher than background, indicative of positive selection, involving large sample cohort studies. However, studies involving smaller sample sizes are inherently restrictive due to their limited statistical power to identify low frequency genetic variations. RESULTS: We performed an integrated characterization of copy number, mutation and expression analyses of four head and neck cancer cell lines - NT8e, OT9, AW13516 and AW8507 - by applying a filtering strategy to prioritize for genes affected by two or more alterations within or across the cell lines. Besides identifying TP53, PTEN, HRAS and MET as major altered HNSCC hallmark genes, this analysis uncovered 34 novel candidate genes altered. Of these, we find a heterozygous truncating mutation in Nuclear receptor binding protein, NRBP1 pseudokinase gene, identical to as reported in other cancers, is oncogenic when ectopically expressed in NIH-3 T3 cells. Knockdown of NRBP1 in an oral carcinoma cell line bearing NRBP1 mutation inhibit transformation and survival of the cells. CONCLUSIONS: In overall, we present the first comprehensive genomic characterization of four head and neck cancer cell lines established from Indian patients. We also demonstrate the ability of integrated analysis to uncover biologically important genetic variation in studies involving fewer or rare clinical specimens.

DPYSL2 interacts with JAK1 to mediate breast cancer cell migration.

The intricate neuronal wiring during development requires cytoskeletal reorganization orchestrated by signaling cues. Because cytoskeletal remodeling is a hallmark of cell migration, we investigated whether metastatic cancer cells exploit axon guidance proteins to migrate. Indeed, in breast cancer patients, we found a significant correlation between mesenchymal markers and the expression of dihydropyrimidinase-like 2 (DPYSL2), a regulator of cytoskeletal dynamics in growing axons. Strikingly, DPYSL2 knockout in mesenchymal-like breast cancer cells profoundly inhibited cell migration, invasion, stemness features, tumor growth rate, and metastasis. Next, we decoded the molecular mechanism underlying this phenomenon and revealed an interaction between DPYSL2 and Janus kinase 1 (JAK1). This binding is crucial for activating signal transducer and activator of transcription 3 (STAT3) and the subsequent expression of vimentin, the promigratory intermediate filament. These findings identify DPYSL2 as a molecular link between oncogenic signaling pathways and cytoskeletal reorganization in migrating breast cancer cells.

Depletion of Fumarate Hydratase, an Essential TCA Cycle Enzyme, Drives Proliferation in a Two-Step Model.

Fumarate hydratase (FH) is an evolutionary conserved TCA cycle enzyme that reversibly catalyzes the hydration of fumarate to L-malate and has a moonlight function in the DNA damage response (DDR). Interestingly, FH has a contradictory cellular function, as it is pro-survival through its role in the TCA cycle, yet its loss can drive tumorigenesis. Here, we found that in both non-cancerous (HEK-293T) and cancerous cell lines (HepG2), the cell response to FH loss is separated into two distinct time frames based on cell proliferation and DNA damage repair. During the early stages of FH loss, cell proliferation rate and DNA damage repair are inhibited. However, over time the cells overcome the FH loss and form knockout clones, indistinguishable from WT cells with respect to their proliferation rate. Due to the FH loss effect on DNA damage repair, we assumed that the recovered cells bear adaptive mutations. Therefore, we applied whole-exome sequencing to identify such mutated genes systematically. Indeed, we identified recurring mutations in genes belonging to central oncogenic signaling pathways, such as JAK/STAT3, which we validated in impaired FH-KO clones. Intriguingly, we demonstrate that these adaptive mutations are responsible for FH-KO cell proliferation under TCA cycle malfunction.

Large-Scale Differential Gene Expression Transcriptomic Analysis Identifies a Metabolic Signature Shared by All Cancer Cells.

Cancer-dependent metabolic rewiring is often manifested by selective expression of enzymes essential for the transformed cells' viability. However, the metabolic variations between normal and transformed cells are not fully characterized, and therefore, a systematic analysis will result in the identification of unknown cellular mechanisms crucial for tumorigenesis. Here, we applied differential gene expression transcriptome analysis to examine the changes in metabolic gene profiles between a wide range of normal tissues and cancer samples. We found that, in contrast to normal tissues which exhibit a tissue-specific expression profile, cancer samples are more homogenous despite their diverse origins. This similarity is due to a "proliferation metabolic signature" (PMS), composed of 158 genes (87 upregulated and 71 downregulated gene sets), where 143 are common to all proliferative cells but 15 are cancer specific. Intriguingly, the PMS gene set is enriched for genes encoding rate-limiting enzymes, and its upregulated set with genes associated with poor patient outcome and essential genes. Among these essential genes is ribulose-5-phosphate-3-epimerase (RPE), which encodes a pentose phosphate pathway enzyme and whose role in cancer is still unclear. Collectively, we identified a set of metabolic genes that can serve as novel cancer biomarkers and potential targets for drug development.

Modeling WWOX Loss of Function in vivo: What Have We Learned?

The WW domain-containing oxidoreductase (WWOX) gene encompasses a common fragile sites (CFS) known as FRA16D, and is implicated in cancer. WWOX encodes a 46kDa adaptor protein, which contains two N-terminal WW-domains and a catalytic domain at its C-terminus homologous to short-chain dehydrogenase/reductase (SDR) family proteins. A high sequence conservation of WWOX orthologues from insects to rodents and ultimately humans suggest its significant role in physiology and homeostasis. Indeed, data obtained from several animal models including flies, fish, and rodents demonstrate WWOX in vivo requirement and that its deregulation results in severe pathological consequences including growth retardation, post-natal lethality, neuropathy, metabolic disorders, and tumorigenesis. Altogether, these findings set WWOX as an essential protein that is necessary to maintain normal cellular/physiological homeostasis. Here, we review and discuss lessons and outcomes learned from modeling loss of WWOX expression in vivo.