Leukocyte cell-derived chemotaxin 2 regulates epithelial-mesenchymal transition and cancer stemness in hepatocellular carcinoma.

Leukocyte cell-derived chemotaxin 2 (LECT2) acts as a tumor suppressor in hepatocellular carcinoma (HCC). However, the antineoplastic mechanism of LECT2, especially its influence on hepatic cancer stem cells (CSCs), remains largely unknown. In The Cancer Genome Atlas cohort, LECT2 mRNA expression was shown to be associated with stage, grade, recurrence, and overall survival in human HCC patients, and LECT2 expression was downregulated in hepatoma tissues compared with the adjacent nontumoral liver. Here, we show by immunofluorescence and immunoblot analyses that LECT2 was expressed at lower levels in tumors and in poorly differentiated HCC cell lines. Using functional assays, we also found LECT2 was capable of suppressing oncogenic behaviors such as cell proliferation, anchorage-independent growth, migration, invasiveness, and epithelial-mesenchymal transition in hepatoma cells. Moreover, we show exogenous LECT2 treatment inhibited CSC functions such as tumor sphere formation and drug efflux. Simultaneously, hepatic CSC marker expression was also downregulated, including expression of CD133 and CD44. This was supported by infection with adenovirus encoding LECT2 (Ad-LECT2) in HCC cells. Furthermore, in animal experiments, Ad-LECT2 gene therapy showed potent efficacy in treating HCC. We demonstrate LECT2 overexpression significantly promoted cell apoptosis and reduced neovascularization/CSC expansion in rat hepatoma tissues. Mechanistically, we showed using immunoblot and immunofluorescence analyses that LECT2 inhibited ß-catenin signaling via the suppression of the hepatocyte growth factor/c-MET axis to diminish CSC properties in HCC cells. In summary, we reveal novel functions of LECT2 in the suppression of hepatic CSCs, suggesting a potential alternative strategy for HCC therapy.

Cisplatin-Induced Giant Cells Formation Is Involved in Chemoresistance of Melanoma Cells.

Melanoma is notoriously resistant to current cancer therapy. However, the chemoresistance mechanism of melanoma remains unclear. The present study unveiled that chemotherapy drug cisplatin induced the formation of giant cells, which exhibited enlargement in cell diameter and nucleus in mice and human melanoma cells. Giant cells were positive with melanoma maker S100 and cancer stem cell markers including ABCB5 and CD133 in vitro and in vivo. Moreover, giant cells retained the mitotic ability with expression of proliferation marker Ki-67 and exhibited multiple drug resistance to doxorubicin and actinomycin D. The mitochondria genesis/activities and cellular ATP level were significantly elevated in giant cells, implicating the demand for energy supply. Application of metabolic blockers such as sodium azide or 2-deoxy glucose abolished the cisplatin-induced giant cells formation and expression of cancer stemness markers. The present study unveils a novel chemoresistance mechanism of melanoma cells via size alteration and the anti-neoplastic strategy by targeting giant cells.

Nanoscale characterization illustrates the cisplatin-mediated biomechanical changes of B16-F10 melanoma cells.

Cells reorganize their membrane biomechanical dynamics in response to environmental stimuli or inhibitors associated with their physiological/pathological processes, and disease therapeutics. To validate the biophysical dynamics during cell exposure to anti-cancer drugs, we investigate the nanoscale biological characterization in melanoma cells undergoing cisplatin treatment. Using atomic force microscopy, we demonstrate that the cellular morphology and membrane ultrastructure are altered after exposure to cisplatin. In contrast to their normal spindle-like shape, cisplatin causes cell deformation rendering cells flat and enlarged, which increases the cell area by 3-4 fold. Additionally, cisplatin decreases the topography height values for both the cytoplasmic and nuclear regions (by 40-80% and 60%, respectively). Furthermore, cisplatin increases the cytoplasmic root mean square roughness by 110-240% in correlation with the drug concentration and attenuates the nuclear RMS by 60%. Moreover, the cellular adhesion force was enhanced, while the Young's modulus elasticity was attenuated by ∼2 and ∼2.3 fold, respectively. F-actin phalloidin staining revealed that cisplatin enlarges the cell size through enhanced stress fiber formation and promotes cytoskeletal reorganization. Immunoblot analyses further revealed that the activities of focal adhesion proteins, such as FAK and c-Src, are upregulated by cisplatin through phosphorylation at tyrosine 397 and 530, respectively. Collectively, these results show that cisplatin-treated melanoma cells not only exhibit the upregulation of FAK-mediated signaling to enhance the cytoskeleton mechanical stretch, but also promote the cytoskeletal rearrangement resulting in 43% decrease in the cell modulus. These mechanisms thus promote the malignancy and invasiveness of the melanoma cells.

Ultraviolet C irradiation induces different expression of cyclooxygenase 2 in NIH 3T3 cells and A431 cells: the roles of COX-2 are different in various cell lines.

Ultraviolet C (UVC) is a DNA damage inducer, and 20 J/m(2) of UVC irradiation caused cell growth inhibition and induced cell death after exposure for 24-36 h. The growth of NIH 3T3 cells was significantly suppressed at 24 h after UVC irradiation whereas the proliferation of A431 cells was inhibited until 36 h after UVC irradiation. UVC irradiation increased COX-2 expression and such up-regulation reached a maximum during 3-6 h in NIH 3T3 cells. In contrast, UVC-induced COX-2 reached a maximum after 24-36 h in A431 cells. Measuring prostaglandin E2 (PGE2) level showed a biphasic profile that PGE2 release was rapidly elevated in 1-12 h after UVC irradiation and increased again at 24 h in both cell lines. Treatment with the selective COX-2 inhibitor, SC-791, during maximum expression of COX-2 induction, attenuated the UVC induced-growth inhibition in NIH 3T3 cells. In contrast, SC-791 treatment after UVC irradiation enhanced death of A431 cells. These data showed that the patterns of UVC-induced PGE2 secretion from NIH 3T3 cells and A431 cells were similar despite the differential profile in UVC-induced COX-2 up-regulation. Besides, COX-2 might play different roles in cellular response to UVC irradiation in various cell lines.

Somatic mutations of PREX2 gene in patients with hepatocellular carcinoma.

Characterized with a high recurrence rate and low detection rate, prevention is the best approach to reduce mortality in hepatocellular carcinoma (HCC). The overexpression of Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 2 (PREX2) is observed in various tumors, including HCC; and the frequent PREX2 mutations in melanoma are associated with invasiveness. We sought to identify somatic mutations and the functional changes in mutational signatures of PREX2. Genomic DNA sequencing was performed in 68 HCC samples with three types of hepatitis viral infection status: HBs Ag-positive, anti-HCV Ab-positive, and negative for any hepatitis B or C markers. Stabilities and interactions of proteins as well as cell proliferation and migration were evaluated. Fourteen non-silent point mutations in PREX2 were detected, with 16 of 68 HCC patients harboring at least one non-silent mutation. All mutant forms of PREX2, except for K400f, had an extended half-life compared with wild-type PREX2. Moreover, only the half-life of S1113R was twice that of the wild-type. PREX2 mutant-S1113R also promoted migration and activated the AKT pathway as well as impaired HectH9-mediated ubiquitination. Our study identified a gain-of-function mutation of PREX2 - S1113R in HCC. Such mutation enhanced PREX2 protein stability, promoted cell proliferation, and was associated with aggressiveness of HCC.

Epithelial-mesenchymal transition (EMT) beyond EGFR mutations per se is a common mechanism for acquired resistance to EGFR TKI.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is a major advance in treating NSCLC with EGFR-activating mutations. However, acquired resistance, due partially to secondary mutations limits their use. Here we report that NSCLC cells with acquired resistance to gefitinib or osimertinib (AZD9291) exhibit EMT features, with a decrease in E-cadherin, and increases in vimentin and stemness, without possessing any EGFR secondary mutations. Knockdown of E-cadherin in parental cells increased gefitinib resistance and stemness, while knockdown of vimentin in resistant cells resulted in opposite effects. Src activation and Hakai upregulation were found in gefitinib-resistant cells. Knockdown of Hakai elevated E-cadherin expression, attenuated stemness, and resensitized the cells to gefitinib. Clinical cancer specimens with acquired gefitinib resistance also showed a decrease in E-cadherin and an increase in Hakai expression. The dual HDAC and HMGR inhibitor JMF3086 inhibited the Src/Hakai and Hakai/E-cadherin interaction to reverse E-cadherin expression, and attenuated vimentin and stemness to restore gefitinib sensitivity. The EMT features of AZD9291-resistant H1975 cells were related to the upregulation of Zeb1. Both gefitinib and AZD9291 sensitivity was restored by JMF3086 through reversing EMT. Our study not only revealed a common mechanism of EMT in both gefitinib and AZD9291 resistance beyond EGFR mutations per se, but also provides a new strategy to overcome it.

Involvement of nucleophosmin/B23 in the cellular response to curcumin.

Nucleophosmin (NPM/B23) is a nucleolar phosphoprotein involved in cellular response to many different stimuli. Herein, we studied the molecular mechanism of NPM/B23 induction by curcumin, a natural AP-1 inhibitor with antitumor properties. Exposure to 5-30 µM curcumin significantly and dose-dependently increased the level of NPM/B23 in non-transformed NIH 3T3 cells but not HeLa cells and F9 cells. Besides, the transformed F9 and HeLa cells are more sensitive to curcumin-induced cell death and growth inhibition than NIH 3T3 cells. Overexpression of c-Jun, but not c-Fos, decreased ∼40% of NPM/B23 and enhanced the sensitivity of NIH 3T3 cells to 30 µM curcumin. Furthermore, down-regulation of NPM/B23 by transfection with NPM/B23 antisense plasmid enhanced the sensitivity to curcumin-induced cell death and growth inhibition. These results indicated that NPM/B23 expression regulates cellular sensitivity to curcumin. Besides, NPM/B23 knockdown may facilitate as a novel strategy to promote the sensitivity of cancer cells to curcumin.

Systemic pro-opiomelanocortin expression induces melanogenic differentiation and inhibits tumor angiogenesis in established mouse melanoma.

Malignant melanoma is one of the leading causes of cancer mortality worldwide, underlining the need for effective novel therapies. In this study, the therapeutic efficacy and mechanism of systemic pro-opiomelanocortin (POMC) therapy were evaluated in mice bearing established melanoma. Injection of adenovirus encoding POMC (Ad-POMC) led to hepatic POMC overexpression and elevated adrenocorticotropin (ACTH) levels in the circulation. Systemic POMC therapy significantly attenuated the growth of established melanoma and prolonged the survival of tumor-bearing mice. Histological analysis revealed that systemic POMC therapy induced melanogenic differentiation while reducing melanoma growth. In addition, POMC therapy also elicited a significant reduction in the neovascular network of melanoma. Last, we demonstrated that POMC-derived peptides, including ACTH, α-melanocyte-stimulating hormone (α-MSH), and ß-MSH, are involved in POMC-mediated melanogenic differentiation and angiogenesis inhibition. In summary, systemic POMC therapy suppresses melanoma growth via induction of melanogenic differentiation and angiogenesis blockade, thereby demonstrating its potential as a novel treatment modality for melanoma.