Targeting NPC1 in Renal Cell Carcinoma.

Rapidly proliferating cancer cells have a greater requirement for cholesterol than normal cells. Tumor cells are largely dependent on exogenous lipids given that their growth requirements are not fully met by endogenous pathways. Our current study shows that ccRCC cells have redundant mechanisms of cholesterol acquisition. We demonstrate that all major lipoproteins (i.e., LDL, HDL, and VLDL) have a comparable ability to support the growth of ccRCC cells and are equally effective in counteracting the antitumor activities of TKIs. The intracellular trafficking of exogenous lipoprotein-derived cholesterol appears to be distinct from the movement of endogenously synthesized cholesterol. De novo synthetized cholesterol is transported from the endoplasmic reticulum directly to the plasma membrane and to the acyl-CoA: cholesterol acyltransferase, whereas lipoprotein-derived cholesterol is distributed through the NPC1-dependent endosomal trafficking system. Expression of NPC1 is increased in ccRCC at mRNA and protein levels, and high expression of NPC1 is associated with poor prognosis. Our current findings show that ccRCC cells are particularly sensitive to the inhibition of endolysosomal cholesterol export and underline the therapeutic potential of targeting NPC1 in ccRCC.

Examining the Effect of PARP-1 Inhibitors on Transcriptional Activity of Androgen Receptor in Prostate Cancer Cells.

Since the early 1940s, androgen ablation has been the cornerstone of treatment for prostate cancer (PC). Importantly, androgen receptor (AR) signaling is vital not only for the initiation of PC, which is initially androgen-dependent, but also for castration-resistant disease. Recent studies demonstrated clear promise of the poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors for targeting prostate cancer cells harboring mutations in DNA damage-repair genes. In addition, it has been established that PARP-1 inhibition suppresses growth of AR-positive prostate cancer cells in cell and animal models. Thus, prostate cancer represents a particularly promising disease site for targeting PARP-1, given that both DNA repair and AR-mediated transcription depend on PARP-1 function. Here, we describe the development and use of cell-based assay to evaluate the impact of PARP-1 inhibitors on the AR signaling in prostate cancer cells.

Acetyl-CoA Counteracts the Inhibitory Effect of Antiandrogens on Androgen Receptor Signaling in Prostate Cancer Cells.

The commonly used therapeutic management of PC involves androgen deprivation therapy (ADT) followed by treatment with AR signaling inhibitors (ARSI). However, nearly all patients develop drug-resistant disease, with a median progression-free survival of less than 2 years in chemotherapy-naïve men. Acetyl-coenzyme A (acetyl-CoA) is a central metabolic signaling molecule with key roles in biosynthetic processes and cancer signaling. In signaling, acetyl-CoA serves as the acetyl donor for acetylation, a critical post-translational modification. Acetylation affects the androgen receptor (AR) both directly and indirectly increasing expression of AR dependent genes. Our studies reveal that PC cells respond to the treatment with ARSI by increasing expression of ATP-citrate lyase (ACLY), a major enzyme responsible for cytosolic acetyl-CoA synthesis, and up-regulation of acetyl-CoA intracellular levels. Inhibition of ACLY results in a significant suppression of ligand-dependent and -independent routes of AR activation. Accordingly, the addition of exogenous acetyl-CoA, or its precursor acetate, augments AR transcriptional activity and diminishes the anti-AR activity of ARSI. Taken together, our findings suggest that PC cells respond to antiandrogens by increasing activity of the acetyl-coA pathway in order to reinstate AR signaling.

Prognostic role and biologic features of Musashi-2 expression in colon polyps and during colorectal cancer progression.

BACKGROUND: The RNA-binding protein Musashi-2 (MSI2) controls the translation of proteins that support stem cell identity and lineage determination and is associated with progression in some cancers. We assessed MSI2 as potential clinical biomarker in colorectal cancer (CRC) and tubulovillous adenoma (TA) of colon mucosa. METHODS: We assessed 125 patients, of whom 20 had polyps of the colon (TAs), and 105 had CRC. Among 105 patients with CRC, 45 had stages I-III; among metastatic CRC (mCRC) patients, 31 had synchronous and 29 metachronous liver metastases. We used immunohistochemistry to measure MSI2 expression in matching specimens of normal tissue versus TAs, primary CRC tumors, and metastases, correlating expression to clinical outcomes. We analyzed the biological effects of depleting MSI2 expression in human CRC cells. RESULTS: MSI2 expression was significantly elevated in polyps versus primary tissue, and further significantly elevated in primary tumors and metastases. MSI2 expression correlated with decreased progression free survival (PFS) and overall survival (OS), higher tumor grade, and right-side localization (p = 0.004) of tumors. In metastases, high MSI2 expression correlated with E-cadherin expression. Knockdown of MSI2 in CRC cells suppressed proliferation, survival and clonogenic capacity, and decreased expression of TGFß1, E-cadherin, and ZO1. CONCLUSION: Elevated expression of MSI2 is associated with pre-cancerous TAs in the colonic mucosa, suggesting it is an early event in transformation. MSI2 expression is further elevated during CRC progression, and associated with poor prognosis. Depletion of MSI2 reduces CRC cell growth. These data imply a causative role of MSI2 overexpression at multiple stages of CRC formation and progression.

Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC).

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.

CRISPR/Cas9 genome-wide loss-of-function screening identifies druggable cellular factors involved in sunitinib resistance in renal cell carcinoma.

BACKGROUND: Multi-targeted tyrosine kinase inhibitors (TKIs) are the standard of care for patients with advanced clear cell renal cell carcinoma (ccRCC). However, a significant number of ccRCC patients are primarily refractory to targeted therapeutics, showing neither disease stabilisation nor clinical benefits. METHODS: We used CRISPR/Cas9-based high-throughput loss of function (LOF) screening to identify cellular factors involved in the resistance to sunitinib. Next, we validated druggable molecular factors that are synthetically lethal with sunitinib treatment using cell and animal models of ccRCC. RESULTS: Our screening identified farnesyltransferase among the top hits contributing to sunitinib resistance in ccRCC. Combined treatment with farnesyltransferase inhibitor lonafarnib potently augmented the anti-tumour efficacy of sunitinib both in vitro and in vivo. CONCLUSION: CRISPR/Cas9 LOF screening presents a promising approach to identify and target cellular factors involved in the resistance to anti-cancer therapeutics.

Robust CRISPR/Cas9-Mediated Tissue-Specific Mutagenesis Reveals Gene Redundancy and Perdurance in Drosophila.

Tissue-specific loss-of-function (LOF) analysis is essential for characterizing gene function. Here, we present a simple, yet highly efficient, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. We describe convenient toolkits for making enhancer-driven Cas9 lines and multi-gRNAs that are optimized for mutagenizing somatic cells. We demonstrate that insertions or deletions in coding sequences more reliably cause somatic mutations than DNA excisions induced by two gRNAs. We further show that enhancer-driven Cas9 is less cytotoxic yet results in more complete LOF than Gal4-driven Cas9 in larval sensory neurons. Finally, CRISPR-TRiM efficiently unmasks redundant soluble N-ethylmaleimide-sensitive factor attachment protein receptor gene functions in neurons and epidermal cells. Importantly, Cas9 transgenes expressed at different times in the neuronal lineage reveal the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISPR tools can be applied to analyze tissue-specific gene function in many biological processes.