TRPM1 promotes tumor progression in acral melanoma by activating the Ca2+/CaMKIIδ/AKT pathway.

INTRODUCTION: Acral melanoma is a predominant and aggressive subtype of melanoma in non-Caucasian populations. There is a lack of genotype-driven therapies for over 50% of patients. TRPM1 (transient receptor potential melastatin 1), a nonspecific cation channel, is mainly expressed in retinal bipolar neurons and skin. Nonetheless, the function of TRPM1 in melanoma progression is poorly understood. OBJECTIVES: We investigated the association between TRPM1 and acral melanoma progression and revealed the molecular mechanisms by which TRPM1 promotes tumor progression and malignancy. METHODS: TRPM1 expression and CaMKII phosphorylation in tumor specimens were tested by immunohistochemistry analysis and scored by two independent investigators. The functions of TRPM1 and CaMKII were assessed using loss-of-function and gain-of-function approaches and examined by western blotting, colony formation, cell migration and invasion, and xenograft tumor growth assays. The effects of a CaMKII inhibitor, KN93, were evaluated using both in vitro cell and in vivo xenograft mouse models. RESULTS: We revealed that TRPM1 protein expression was positively associated with tumor progression and shorter survival in patients with acral melanoma. TRPM1 promoted AKT activation and the colony formation, cell mobility, and xenograft tumor growth of melanoma cells. TRPM1 elevated cytosolic Ca2+ levels and activated CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) to promote the CaMKIIδ/AKT interaction and AKT activation. The functions of TRPM1 in melanoma cells were suppressed by a CaMKII inhibitor, KN93. Significant upregulation of phospho-CaMKII levels in acral melanomas was related to increased expression of TRPM1. An acral melanoma cell line with high expression of TRPM1, CA11, was isolated from a patient to show the anti-tumor activity of KN93 in vitro and in vivo. CONCLUSIONS: TRPM1 promotes tumor progression and malignancy in acral melanoma by activating the Ca2+/CaMKIIδ/AKT pathway. CaMKII inhibition may be a potential therapeutic strategy for treating acral melanomas with high expression of TRPM1.

CHK2 activation contributes to the development of oxaliplatin resistance in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC), the most common cancer type, causes high morbidity and mortality. Patients who develop drug resistance to oxaliplatin-based regimens have short overall survival. Thus, identifying molecules involved in the development of oxaliplatin resistance is critical for designing therapeutic strategies. METHODS: A proteomic screen was performed to reveal altered protein kinase phosphorylation in oxaliplatin-resistant (OR) CRC tumour spheroids. The function of CHK2 was characterised using several biochemical techniques and evident using in vitro cell and in vivo tumour models. RESULTS: We revealed that the level of phospho-CHK2(Thr68) was elevated in OR CRC cells and in ~30% of tumour samples from patients with OR CRC. We demonstrated that oxaliplatin activated several phosphatidylinositol 3-kinase-related kinases (PIKKs) and CHK2 downstream effectors and enhanced CHK2/PARP1 interaction to facilitate DNA repair. A phosphorylation mimicking CHK2 mutant, CHK2T68D, but not a kinase-dead CHK2 mutant, CHK2D347A, promoted DNA repair, the CHK2/PARP1 interaction, and cell growth in the presence of oxaliplatin. Finally, we showed that a CHK2 inhibitor, BML-277, reduced protein poly(ADP-ribosyl)ation (PARylation), FANCD2 monoubiquitination, homologous recombination and OR CRC cell growth in vitro and in vivo. CONCLUSION: Our findings suggest that CHK2 activity is critical for modulating oxaliplatin response and that CHK2 is a potential therapeutic target for OR CRC.

AUY922 induces retinal toxicity through attenuating TRPM1.

BACKGROUND: Ocular adverse events are common dose-limiting toxicities in cancer patients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed. METHODS: The impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)­based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches. RESULTS: Here, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922. CONCLUSIONS: Our study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity.

Lymphatic vessel remodeling and invasion in pancreatic cancer progression.

BACKGROUND: The lymphatic system is involved in metastasis in pancreatic cancer progression. In cancer staging, lymphatic spread has been used to assess the invasiveness of tumor cells. However, from the endothelium's perspective, the analysis downplays the peri-lesional activities of lymphatic vessels. This unintended bias is largely due to the lack of 3-dimensional (3-D) tissue information to depict the lesion microstructure and vasculature in a global and integrated fashion. METHODS: We targeted the pancreas as the model organ to investigate lymphatic vessel remodeling in cancer lesion progression. Transparent pancreases were prepared by tissue clearing to facilitate deep-tissue, tile-scanning microscopy for 3-D lymphatic network imaging. FINDINGS: In human pancreatic ductal adenocarcinoma, we identify the close association between the pancreatic intraepithelial neoplasia (PanIN) lesions and the lymphatic network. In mouse models of PanIN (elastase-CreER;LSL-KrasG12D and elastase-CreER;LSL-KrasG12D;p53+/-), the 3-D image data reveal the peri-lesional lymphangiogenesis, endothelial invagination, formation of the bridge/valve-like luminal tubules, vasodilation, and luminal invasion. In the orthotopic mouse model of pancreatic cancer, we identify the localized, graft-induced lymphangiogenesis and the peri- and intra-tumoral lymphatic vessel invasion. INTERPRETATION: The integrated view of duct lesions and vascular remodeling suggests an active role, rather than a passive target, of lymphatic vessels in the metastasis of pancreatic cancer. Our 3-D image data provide insights into the pancreatic cancer microenvironment and establish the technical and morphological foundation for systematic detection and 3-D analysis of lymphatic vessel invasion. FUND: Taiwan Academia Sinica (AS-107-TP-L15 and AS-105-TP-B15), Ministry of Science and Technology (MOST 106-2321-B-001-048, 106-0210-01-15-02, 106-2321-B-002-034, and 106-2314-B-007-004-MY2), and Taiwan National Health Research Institutes (NHRI EX107-10524EI).

The Potential of Targeting P53 and HSP90 Overcoming Acquired MAPKi-Resistant Melanoma.

OPINION STATEMENT: Melanoma is the deadliest form of skin cancer worldwide. The rising melanoma incidence and mortality, along with its high propensity for metastasis highlights the urgency to identify more effective therapeutic targets. Approximately, one half of advanced melanoma bears a mutation in the BRAF gene that makes BRAF as an important therapeutic target. Significant clinical benefit is associated with BRAF and MEK inhibitors (MAPKi) on targeting patients with BRAF V600 mutations. However, the frequent and rapid development of acquired resistance still is the major challenge facing the melanoma. Several mechanisms by which melanoma passes the inhibitory effects of MAPKi have been characterized and clinically translated, but additional alternations of genetic and epigenetic regulators outside of MAPK and/or AKT networks occurs in a quarter of patients with acquired MAPKi resistance. These studies implicate that targeting signaling networks external MAPK or AKT pathways is critical. In this review, we will focus on two approaches that are under evaluating for targeting melanoma: (1) against genome instability by p53 network restoration and (2) disrupt cancer proteome by chaperone inhibition.

Elevation of ß-galactoside α2,6-sialyltransferase 1 in a fructoseresponsive manner promotes pancreatic cancer metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of pancreatic cancer with clinical characteristics of local invasion and early metastasis. Recent cohort studies indicate high fructose intake is associated with an increase in pancreatic cancer risk. However, the mechanisms by which fructose promotes pancreatic tumorigenesis remain unclear. Herein, Kras+/LSLG12D mice were crossed with Elas-CreER transgenic mice to determine whether fructose intake directly contributes to tumor formation. Orthotopic tumor-xenograft experiments were performed to determine whether fructose substitution enhances the metastatic potential of PDAC cells. The mechanisms underlying the effects of fructose were explored by RNAseq analysis in combination with high-performance anion exchange chromatography. Dietary fructose was initially found to promote the development of aggressive pancreatic cancer in mice conditionally expressing KrasG12D in the adult pancreas. We further revealed that fructose substitution enhanced the metastatic potential of human PDAC cell via selective outgrowth of aggressive ABCG2-positive subpopulations and elevating N-acetylmannosamine levels that upregulated ß-galactoside α2,6-sialyltransferase 1 (ST6Gal1), thereby promoting distant metastasis. Finally, we observed that PDAC patients expressing higher levels of ST6Gal1 and GLUT5 presented poorer prognosis compared to other groups. In conclusion, our findings have elucidated a crucial role of ST6Gal1 in regulating the invasiveness of PDACs in a fructose-responsive manner.

Differentiation of pancreatic acinar cells to hepatocytes requires an intermediate cell type.

BACKGROUND & AIMS: The appearance of hepatic foci in pancreas has been well-documented in animal experiments and in patients with pancreatic cancer. We previously demonstrated that transdifferentiation of pancreatic exocrine cells to hepatocytes required members of the CCAAT enhancer binding protein family. Although the molecular basis of hepatic transdifferentiation is understood, the early cellular events remain to be defined. METHODS: Dexamethasone and oncostatin M were used to induce transdifferentiation of primary cultures of mouse acinar cells and exocrine cell lines into hepatocytes. Fluorescent-activated cell sorting was used to identify intermediate cell types and side-population characteristics. Cre-loxP-based lineage tracing was used to investigate whether acinar cells contribute directly to hepatocytes via intermediates that express adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2). RESULTS: Lineage tracing studies showed that hepatocytes were derived directly from pancreatic cells via ABCG2-expressing intermediates. Exposure of cells to insulin increased Akt phosphorylation, ABCG2 expression, and hepatic transdifferentiation. Inhibition of the phosphoinositide 3-kinase pathway, through addition of LY294002 or overexpression of a dominant-negative form of Akt, was sufficient to prevent transdifferentiation. When ABCG2-expressing cells were incubated with glucagon-like-peptide 1 or epidermal growth factor, the intermediate cells could differentiate into insulin-producing beta-like cells. CONCLUSIONS: The phosphoinositide 3-kinase pathway is important in the transdifferentiation of acinar cells to hepatocytes and those hepatocytes arise from acinar cells via ABCG2-expressing intermediates. Furthermore, ABCG2-expressing cells are multipotent and able to differentiate into hepatocytes and insulin-producing beta cells.