In Vitro Treatment of Melanoma Brain Metastasis by Simultaneously Targeting the MAPK and PI3K Signaling Pathways

Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAF^V600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway [1]. In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN [2]. Here, we treated two melanoma brain metastasis cell lines, H1_DL2, harboring a BRAF^V600E mutation and PTEN loss, and H3, harboring WT (wild-type) BRAF and PTEN loss, with the MAPK (BRAF) inhibitor vemurafenib and the PI3K pathway associated mTOR inhibitor temsirolimus. Combined use of the drugs inhibited tumor cell growth and proliferation in vitro in H1_DL2 cells, compared to single drug treatment. Treatment was less effective in the H3 cells. Furthermore, a strong inhibitory effect on the viability of H1_DL2 cells, when grown as 3D multicellular spheroids, was seen. The treatment inhibited the expression of pERK1/2 and reduced the expression of pAKT and p-mTOR in H1_DL2 cells, confirming that the MAPK and PI3K pathways were inhibited after drug treatment. Microarray experiments followed by principal component analysis (PCA) mapping showed distinct gene clustering after treatment, and cell cycle checkpoint regulators were affected. Global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment. In conclusion, we demonstrate for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro. The presented results highlight the potential of combined treatment to overcome treatment resistance that may develop after vemurafenib treatment of melanomas.     

PI3K/PTEN/AKT Signaling Pathways in Germ Cell Development and Their Involvement in Germ Cell Tumors and Ovarian Dysfunctions

Several studies indicate that the PI3K/PTEN/AKT signaling pathways are critical regulators of ovarian function including the formation of the germ cell precursors, termed primordial germ cells, and the follicular pool maintenance. This article reviews the current state of knowledge of the functional role of the PI3K/PTEN/AKT pathways during primordial germ cell development and the dynamics of the ovarian primordial follicle reserve and how dysregulation of these signaling pathways may contribute to the development of some types of germ cell tumors and ovarian dysfunctions.     

Combined Phosphatase and Tensin Homolog (PTEN) Loss and Fatty Acid Synthase (FAS) Overexpression Worsens the Prognosis of Chinese Patients with Hepatocellular Carcinoma

We aimed to investigate the expression pattern of phosphatase and tensin homolog (PTEN), to evaluate the relationship between PTEN expression and clinicopathological characteristics, including fatty acid synthase (FAS) expression, and to determine the correlations of PTEN and FAS expression with survival in Chinese patients with hepatocellular carcinoma (HCC). The expression patterns of PTEN and FAS were determined using tissue microarrays and immunohistochemistry. The expression of PTEN was compared with the clinicopathological characteristics of HCC, including FAS expression. Receiver operator characteristic curves were used to calculate the clinical sensitivity and specificity of PTEN expression. Kaplan-Meier survival curves were constructed to evaluate the correlations of PTEN loss and FAS overexpression with overall survival. We found that the loss of PTEN expression occurred predominantly in the cytoplasm, while FAS was mainly localized to the cytoplasm. Cytoplasmic and total PTEN expression levels were significantly decreased in HCC compared with adjacent non-neoplastic tissue (both, p < 0.0001). Decreased cytoplasmic and total PTEN expression showed significant clinical sensitivity and specificity for HCC (both, p < 0.0001). Downregulation of PTEN in HCC relative to non-neoplastic tissue was significantly correlated with histological grade (p = 0.043 for histological grades I-II versus grade III). Loss of total PTEN was significantly correlated with FAS overexpression (p = 0.014). Loss of PTEN was also associated with poor prognosis of patients with poorly differentiated HCC (p = 0.049). Moreover, loss of PTEN combined with FAS overexpression was associated with significantly worse prognosis compared with other HCC cases (p = 0.011). Our data indicate that PTEN may serve as a potential diagnostic and prognostic marker of HCC. Upregulating PTEN expression and inhibiting FAS expression may offer a novel therapeutic approach for HCC.     

The miRNA-21-5p Payload in Exosomes from M2 Macrophages Drives Tumor Cell Aggression via PTEN/Akt Signaling in Renal Cell Carcinoma

M2 macrophages in the tumor microenvironment are important drivers of cancer metastasis. Exosomes play a critical role in the crosstalk between different cells by delivering microRNAs or other cargos. Whether exosomes derived from pro-tumorigenic M2 macrophages (M2-Exos) could modulate the metastatic behavior of renal cell carcinoma (RCC) is unclear. This study found that M2-Exos promotes migration and invasion in RCC cells. Inhibiting miR-21-5p in M2-Exos significantly reversed their pro-metastatic effects on RCC cells in vitro and in the avian embryo chorioallantoic membrane in vivo tumor model. We further found that the pro-metastatic mechanism of miR-21-5p in M2-Exos is by targeting PTEN-3′UTR to regulate PTEN/Akt signaling. Taken together, our results demonstrate that M2-Exos carries miR-21-5p promote metastatic features of RCC cells through PTEN/Akt signaling. Reversing this could serve as a novel approach to control RCC metastasis.     

BPA Decreases PDCD4 in Bovine Granulosa Cells Independently of miR-21 Inhibition

microRNAs (miRNAs) are susceptible to environmental factors that might affect cellular function and impose negative effects on female reproduction. miR-21 is the most abundant miRNA in bovine granulosa cells and is widely reported as affected by Bisphenol A (BPA) exposure, yet the cause and consequences are not entirely elucidated. BPA is a synthetic endocrine disruptor associated with poor fertility. miR-21 function in bovine granulosa cells is investigated utilizing locked nucleic acid (LNA) oligonucleotides to suppress miR-21. Before measuring apoptosis and quantifying miR-21 apoptotic targets PDCD4 and PTEN, transfection was optimized and validated. BPA was introduced to see how it affects miR-21 regulation and which BPA-mediated effects are influenced by miR-21. miR-21 knockdown and specificity against additional miRNAs were confirmed. miR-21 was found to have antiapoptotic effects, which could be explained by its effect on the proapoptotic target PDCD4, but not PTEN. Previous findings of miR-21 overexpression were validated using BPA treatments, and the temporal influence of BPA on miR-21 levels was addressed. Finally, BPA effects on upstream regulators, such as VMP1 and STAT3, explain the BPA-dependent upregulation of miR-21 expression. Overall, this research enhances our understanding of miR-21 function in granulosa cells and the mechanisms of BPA-induced reproductive impairment.     

Inhibitory Effects of Ursolic Acid on the Stemness and Progression of Human Breast Cancer Cells by Modulating Argonaute-2

The stemness and metastasis of cancer cells are crucial features in determining cancer progression. Argonaute-2 (AGO2) overexpression was reported to be associated with microRNA (miRNA) biogenesis, supporting the self-renewal and differentiation characteristics of cancer stem cells (CSCs). Ursolic acid (UA), a triterpene compound, has multiple biological functions, including anticancer activity. In this study, we find that UA inhibits the proliferation of MDA-MB-231 and MCF-7 breast cancer cell lines using the CCK-8 assay. UA induced a significant decrease in the fraction of CSC in which it was examined by changes in the expression of stemness biomarkers, including the Nanog and Oct4 genes. UA altered invasion and migration capacities by significant decreases in the levels of epithelial-to-mesenchymal transition (EMT) proteins of slug and vimentin. Furthermore, the co-reduction in oncogenic miRNA levels (miR-9 and miR-221) was a result of the down-modulation in AGO2 in breast cancer cells in vitro. Mechanically, UA increases PTEN expression to inactivate the FAK/PI3K/Akt/mTOR signaling pathway and the decreased level of c-Myc in quantitative RT-PCR and Western blot imaging analyses. Our current understanding of the anticancer potential of UA in interrupting between EMT programming and the state of CSC suggests that UA can contribute to improvements in the clinical practice of breast cancer.     

Exploring Prostate Cancer Genome Reveals Simultaneous Losses of PTEN,FAS and PAPSS2 in Patients with PSA Recurrence after Radical Prostatectomy

The multifocal nature of prostate cancer (PCa) creates a challenge to patients’ outcome prediction and their clinical management. An approach that scrutinizes every cancer focus is needed in order to generate a comprehensive evaluation of the disease, and by correlating to patients’ clinico-pathological information, specific prognostic biomarker can be identified. Our study utilized the Affymetrix SNP 6.0 Genome-wide assay to investigate forty-three fresh frozen PCa tissue foci from twenty-three patients. With a long clinical follow-up period that ranged from 2.0–9.7 (mean 5.4) years, copy number variation (CNV) data was evaluated for association with patients’ PSA status during follow-up. From our results, the loss of unique genes on 10q23.31 and 10q23.2–10q23.31 were identified to be significantly associated to PSA recurrence (p < 0.05). The implication of PTEN and FAS loss (10q23.31) support previous reports due to their critical roles in prostate carcinogenesis. Furthermore, we hypothesize that the PAPSS2 gene (10q23.2–10q23.31) may be functionally relevant in post-operative PSA recurrence because of its reported role in androgen biosynthesis. It is suggestive that the loss of the susceptible region on chromosome 10q, which implicates PTEN, FAS and PAPSS2 may serve as genetic predictors of PSA recurrence after radical prostatectomy.     

PTEN再表达对细胞内抗氧化蛋白表达和DNA氧化损伤的影响

目的研究10号染色体缺失磷酸酶及张力蛋白同源体(Phosphatase and tensin homolog deleted on chromosome ten,PTEN)再表达对细胞内抗氧化蛋白Prdx1,2,5,6和Cu/Zn SOD的表达、活性氧(ROS)和DNA氧化损伤水平及细胞抗氧化能力的影响。方法采用Western blot法比较Pten-/-MEFs细胞(对照细胞)与PTEN再表达的Pten-/-MEFs细胞内抗氧化蛋白Prdx1,2,5,6和Cu/Zn SOD的表达差异;采用化学/荧光发光法分析对照细胞与PTEN再表达细胞内基础ROS水平差异;采用中性单细胞凝胶电泳比较不同浓度(0、0.01、0.05、0.10mmol/L)H2O2处理后,对照细胞与PTEN再表达细胞内DNA双链断裂(DSBs)的变化。结果与对照细胞相比,PTEN再表达细胞中Prdx1,2,5,6和Cu/Zn SOD蛋白的表达水平增高,细胞内基础ROS水平降低,DSBs减少,细胞抵抗外源性H2O2的能力增强。结论 PTEN可能通过对Prdx1,2,5,6和Cu/Zn SOD等抗氧化蛋白的表达调控,增强细胞抗氧化能力,清除细胞内过量的ROS,从而保护细胞免受氧化压力导致的DNA氧化损伤,维持基因组稳定性。     

miRNA-21对肺癌A549细胞抑癌基因PTEN表达的调控

目的探讨miRNA-21对肺癌A549细胞抑癌基因PTEN表达的调控作用。方法人工合成miRNA-21序列,插入到质粒pGenesil-1中,构建重组真核表达质粒,并转染至肺癌A549细胞,采用RT-PCR和Western blot分别检测细胞PTEN基因mRNA转录水平和蛋白的表达水平。结果经酶切和测序鉴定,表明重组真核表达质粒构建正确。转染重组真核表达质粒的A549细胞PTEN基因mRNA转录水平与未转染组相比,差异无统计学意义,蛋白表达水平较未转染组明显降低。结论miRNA-21可能主要在翻译水平调控PTEN基因的表达。