Role of aberrant PI3K pathway activation in gallbladder tumorigenesis

The PI3K/AKT pathway governs a plethora of cellular processes, including cell growth, proliferation, and metabolism, in response to growth factors and cytokines. By acting as a unique lipid phosphatase converting phosphatidylinositol-3,4,5,-trisphosphate (PIP3) to phosphatidylinositol-4,5,-bisphosphate (PIP2), phosphatase and tensin homolog (PTEN) acts as the major cellular suppressor of PI3K signaling and AKT activation. Recently, PI3K mutations and loss/mutation of PTEN have been characterized in human gallbladder tumors; whether aberrant PTEN/PI3K pathway plays a causal role in gallbladder carcinogenesis, however, remains unknown. Herein we show that in mice, deregulation of PI3K/AKT signaling is sufficient to transform gallbladder epithelial cells and trigger fully penetrant, highly proliferative gallbladder tumors characterized by high levels of phospho-AKT. Histopathologically, these mouse tumors faithfully resemble human adenomatous gallbladder lesions. The identification of PI3K pathway deregulation as both an early event in the neoplastic transformation of the gallbladder epithelium and a main mechanism of tumor growth in Pten heterozygous and Pten mutant mouse models provides a new framework for studying in vivo the efficacy of target therapies directed against the PI3K pathway, as advanced metastatic tumors are often addicted to “trunkular” mutations

Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

Short hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting \u3e 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs

The RNA Binding Protein ESRP1 Fine-Tunes the Expression of Pluripotency-Related Factors in Mouse Embryonic Stem Cells

In pluripotent stem cells, there is increasing evidence for crosstalk between post-transcriptional and transcriptional networks, offering multifold steps at which pluripotency can be controlled. In addition to well-studied transcription factors, chromatin modifiers and miRNAs, RNA-binding proteins are emerging as fundamental players in pluripotency regulation. Here, we report a new role for the RNA-binding protein ESRP1 in the control of pluripotency. Knockdown of Esrp1 in mouse embryonic stem cells induces, other than the well-documented epithelial to mesenchymal-like state, also an increase in expression of the core transcription factors Oct4, Nanog and Sox2, thereby enhancing self-renewal of these cells. Esrp1-depleted embryonic stem cells displayed impaired early differentiation in vitro and formed larger teratomas in vivo when compared to control embryonic stem cells. We also show that ESRP1 binds to Oct4 and Sox2 mRNAs and decreases their polysomal loading. ESRP1 thus acts as a physiological regulator of the finely-tuned balance between self-renewal and commitment to a restricted developmental fate. Importantly, both mouse and human epithelial stem cells highly express ESRP1, pinpointing the importance of this RNA-binding protein in stem cell biology

A novel type of cellular senescence that can be enhanced in mouse models and human tumor xenografts to suppress prostate tumorigenesis

Irreversible cell growth arrest, a process termed cellular senescence, is emerging as an intrinsic tumor suppressive mechanism. Oncogene-induced senescence is thought to be invariably preceded by hyperproliferation, aberrant replication, and activation of a DNA damage checkpoint response (DDR), rendering therapeutic enhancement of this process unsuitable for cancer treatment. We previously demonstrated in a mouse model of prostate cancer that inactivation of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (Pten) elicits a senescence response that opposes tumorigenesis. Here, we show that Pten-loss-induced cellular senescence (PICS) represents a senescence response that is distinct from oncogene-induced senescence and can be targeted for cancer therapy. Using mouse embryonic fibroblasts, we determined that PICS occurs rapidly after Pten inactivation, in the absence of cellular proliferation and DDR. Further, we found that PICS is associated with enhanced p53 translation. Consistent with these data, we showed that in mice p53-stabilizing drugs potentiated PICS and its tumor suppressive potential. Importantly, we demonstrated that pharmacological inhibition of PTEN drives senescence and inhibits tumorigenesis in vivo in a human xenograft model of prostate cancer. Taken together, our data identify a type of cellular senescence that can be triggered in nonproliferating cells in the absence of DNA damage, which we believe will be useful for developing a 'pro-senescence' approach for cancer prevention and therapy

A co-clinical approach identifies mechanisms and potential therapies for androgen deprivation resistance in prostate cancer

Here we report an integrated analysis that leverages data from treatment of genetic mouse models of prostate cancer along with clinical data from patients to elucidate new mechanisms of castration resistance. We show that castration counteracts tumor progression in a Pten-loss driven mouse model of prostate cancer through the induction of apoptosis and proliferation block. Conversely, this response is bypassed upon deletion of either Trp53 or Lrf together with Pten, leading to the development of castration resistant prostate cancer (CRPC). Mechanistically, the integrated acquisition of data from mouse models and patients identifies the expression patterns of XAF1-XIAP/SRD5A1 as a predictive and actionable signature for CRPC. Importantly, we show that combined inhibition of XIAP, SRD5A1, and AR pathways overcomes castration resistance. Thus, our co-clinical approach facilitates stratification of patients and the development of tailored and innovative therapeutic treatments

Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma

Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma

Fatty acid synthase mediates EGFR palmitoylation in EGFR mutated non‐small cell lung cancer

Abstract Metabolic reprogramming is widely known as a hallmark of cancer cells to allow adaptation of cells to sustain survival signals. In this report, we describe a novel oncogenic signaling pathway exclusively acting in mutated epidermal growth factor receptor (EGFR) non‐small cell lung cancer (NSCLC) with acquired tyrosine kinase inhibitor (TKI) resistance. Mutated EGFR mediates TKI resistance through regulation of the fatty acid synthase (FASN), which produces 16‐C saturated fatty acid palmitate. Our work shows that the persistent signaling by mutated EGFR in TKI‐resistant tumor cells relies on EGFR palmitoylation and can be targeted by Orlistat, an FDA‐approved anti‐obesity drug. Inhibition of FASN with Orlistat induces EGFR ubiquitination and abrogates EGFR mutant signaling, and reduces tumor growths both in culture systems and in vivo. Together, our data provide compelling evidence on the functional interrelationship between mutated EGFR and FASN and that the fatty acid metabolism pathway is a candidate target for acquired TKI‐resistant EGFR mutant NSCLC patients

Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencingin aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas