Shih Sheng Jiang

Caffeic Acid Phenethyl Ester Causes p21Cip1 Induction, Akt Signaling Reduction, and Growth Inhibition in PC-3 Human Prostate Cancer Cells

Caffeic acid phenethyl ester (CAPE) treatment suppressed proliferation, colony formation, and cell cycle progression in PC-3 human prostate cancer cells. CAPE decreased protein expression of cyclin D1, cyclin E, SKP2, c-Myc, Akt1, Akt2, Akt3, total Akt, mTOR, Bcl-2, Rb, as well as phosphorylation of Rb, ERK1/2, Akt, mTOR, GSK3α, GSK3β, PDK1; but increased protein expression of KLF6 and p21Cip1. Microarray analysis indicated that pathways involved in cellular movement, cell death, proliferation, and cell cycle were affected by CAPE. Co-treatment of CAPE with chemotherapeutic drugs vinblastine, paclitaxol, and estramustine indicated synergistic suppression effect. CAPE administration may serve as a potential adjuvant therapy for prostate cancer.

Downregulated miR329 and miR410 promote the proliferation and invasion of oral squamous cell carcinoma by targeting Wnt-7b.

microRNA (miRNA) dysregulation contributes widely to human cancer but has not been fully assessed in oral cancers. In this study, we conducted a global microarray analysis of miRNA expression in 40 pairs of betel quid-associated oral squamous cell carcinoma (OSCC) specimens and their matched nontumorous epithelial counterparts. Eighty-four miRNAs were differentially expressed in the OSCC specimens compared with the matched tissue. Among these downregulated miRNAs, 19 miRNAs were found and mapped to the chromosome 14q32.2 miRNA cluster region, which resides within a parentally imprinted region designated as Dlk-Dio3 and known to be important in development and growth. Bioinformatic analysis predicted two miRNAs from the cluster region, miR329 and miR410, which could potentially target Wnt-7b, an activator of the Wnt-β-catenin pathway, thereby attenuating the Wnt-β-catenin signaling pathway in OSCC. Stable ectopic expression of Wnt-7b in OSCC cells overexpressing miR329 or miR410 restored proliferation and invasion capabilities abolished by these miRNA. Combining a demethylation agent and a histone deacetylase inhibitor was sufficient to reexpress miR329, miR410, and Meg3, consistent with epigenetic regulation of these miRNA in human OSCC. Specifically, arecoline, a major betel nut alkaloid, reduced miR329, miR410, and Meg3 gene expression. Overall, our results provide novel molecular insights into how betel quid contributes to oral carcinogenesis through epigenetic silencing of tumor-suppressor miRNA that targets Wnt-β-catenin signaling.

Increased Cdc7 expression is a marker of oral squamous cell carcinoma and overexpression of Cdc7 contributes to the resistance to DNA-damaging agents

Cdc7-Dbf4 kinase (Dbf4-dependent kinase, DDK) is an essential factor of DNA replication and DNA damage response (DDR), which is associated with tumorigenesis. However, Cdc7 expression has never been associated to the outcome of oral squamous cell carcinoma (OSCC) patients, and the mechanism underlying cancer cell survival mediated by Cdc7 remains unclear. The Cdc7 protein expression of 105 OSCC tumor and 30 benign tissues was examined by immunohistochemistry assay. Overall survival rates of 80 OSCC patients were measured using Kaplan-Meier estimates and the log-rank tests. Cdc7 overexpression by adenovirus system was used to scrutinize the underlying mechanism contributed to cancer cell survival upon DDR. In silico analysis showed that increased Cdc7 is a common feature of cancer. Cdc7 overexpression was found in 96 of 105 (91.4%) studied cases of OSCC patients. Patients with higher Cdc7 expression, either categorized into two groups: Cdc7 high expression (2+ to 3+) versus Cdc7 low expression (0 to 1+) [hazard ratios (HR)=2.6; 95% confidence interval (CI)=1.28-5.43; P=0.0087] or four groups (0 to 3+) [HR=1.71; 95% CI=1.20-2.44; P=0.0032], exhibited a poorer outcome. Multivariate analysis showed that Cdc7 is an independent marker for survival prediction. Overexpressed Cdc7 inhibits genotoxin-induced apoptosis to increase the survival of cancer cells. In summary, Cdc7 expression, which is universally upregulated in cancer, is an independent prognostic marker of OSCC. Cdc7 inhibits genotoxin-induced apoptosis and increases survival in cancer cells upon DDR, suggesting that high expression of Cdc7 enhances the resistance to chemotherapy.

miR-520h is crucial for DAPK2 regulation and breast cancer progression

MicroRNAs (miRNAs) are small RNAs that suppress gene expression by their interaction with 3’untranslated region of specific target mRNAs. Although the dysregulation of miRNAs has been identified in human cancer, only a few of these miRNAs have been functionally documented in breast cancer. Thus, defining the important miRNA and functional target involved in chemoresistance is an urgent need for human breast cancer treatment. In this study, we, for the first time, identified a key role of miRNA 520h (miR-520h) in drug resistance. Through protecting cells from paclitaxel-induced apoptosis, expression of miR-520h promoted the drug resistance of human breast cancer cells. Bioinformatics prediction, compensatory mutation and functional validation further confirmed the essential role of miR-520h-suppressed Death-associated protein kinase 2 (DAPK2) expression, as restoring DAPK2 abolished miR-520h-promoted drug resistance, and knockdown of DAPK2 mitigated cell death caused by the depletion of miR-520h. Furthermore, we observed that higher level of miR-520h is associated with poor prognosis and lymph node metastasis in human breast cancer patients. These results show that miR-520h is not only an independent prognostic factor, but is also a potential functional target for future applications in cancer therapeutics.

Lysine demethylase 2A promotes stemness and angiogenesis of breast cancer by upregulating Jagged1

Alterations of histone methylation dynamically regulated by methyltransferases and demethylases are frequently found in human cancers. Here, we showed that expression of lysine demethylase 2A (KDM2A) is markedly increased in human breast cancer and its overexpression is associated with tumor progression and poor prognosis. Knockdown of KDM2A in breast cancer cells reduced proliferation but not viability. Gene set enrichment analysis revealed that inhibition of KDM2A down-regulates angiogenic genes with concurrent reduction of Jagged1 (JAG1), NOTCH1 and HEY1 in the NOTCH signaling. Chromatin immunoprecipitation- quantitative polymerase chain reaction (ChIP-qPCR) demonstrated the binding of KDM2A to the JAG1 promoter and the increase of methylation of Lys-36 of histone H3 (H3K36) in KDM2A-depleted MDA-MB-231 cells. Tumorsphere formation was significantly reduced in KDM2A-depleted cells which could be reversed by ectopic expression of JAG1. A selective KDM2A inhibitor daminozide also decreased the number of tumorsphere and the number of CD24−/CD44hi cells. In addition, daminozide acted synergistically with cisplatin in cell killing. We identified SOX2 as a direct transcriptional target of KDM2A to promote cancer stemness. Depletion of KDM2A in MDA-MB-231 cells attenuated NOTCH activation and tube formation in co-cultured endothelial cells. Two pro-angiogenic factors JAG1 and PDGFA are key mediators for KDM2A to enhance angiogenesis. Finally, inhibition of KDM2A significantly decreased tumor growth and angiogenesis in orthotopic animal experiments. Collectively, we conclude that KDM2A functions as an oncogene in breast cancer by upregulating JAG1 to promote stemness, chemoresistance and angiogenesis.

AKT3 promotes prostate cancer proliferation cells through regulation of Akt, B-Raf & TSC1/TSC2

The qRT-PCR analysis of 139 clinical samples and analysis of 150 on-line database clinical samples indicated that AKT3 mRNA expression level was elevated in primary prostate tumors. Immunohistochemical staining of 65 clinical samples revealed that AKT3 protein expression was higher in prostate tumors of stage I, II, III as compared to nearby normal tissues. Plasmid overexpression of AKT3 promoted cell proliferation of LNCaP, PC-3, DU-145, and CA-HPV-10 human prostate cancer (PCa) cells, while knockdown of AKT3 by siRNA reduced cell proliferation. Overexpression of AKT3 increased the protein expression of total AKT, phospho-AKT S473, phospho-AKT T308, B-Raf, c-Myc, Skp2, cyclin E, GSK3β, phospho-GSK3β S9, phospho-mTOR S2448, and phospho-p70S6K T421/S424, but decreased TSC1 (tuberous sclerosis 1) and TSC2 (tuberous Sclerosis Complex 2) proteins in PC-3 PCa cells. Overexpression of AKT3 also increased protein abundance of phospho-AKT S473, phospho-AKT T308, and B-Raf but decreased expression of TSC1 and TSC2 proteins in LNCaP, DU-145, and CA-HPV-10 PCa cells. Oncomine datasets analysis suggested that AKT3 mRNA level was positively correlated to BRAF. Knockdown of AKT3 in DU-145 cells with siRNA increased the sensitivity of DU-145 cells to B-Raf inhibitor treatment. Knockdown of TSC1 or TSC2 promoted the proliferation of PCa cells. Our observations implied that AKT3 may be a potential therapeutic target for PCa treatment.

IL-8 induces miR-424-5p expression and modulates SOCS2/ STAT5 signaling pathway in oral squamous cell carcinoma

Suppressor of cytokine signaling (SOCS) proteins are negative feedback regulators of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Dysregulation of SOCS protein expression in cancers can be one of the mechanisms that maintain STAT activation, but this mechanism is still poorly understood in oral squamous cell carcinoma (OSCC). Here, we report that SOCS2 protein is significantly downregulated in OSCC patients and its levels are inversely correlated with miR‐424‐5p expression. We identified the SOCS2 protein, which modulates STAT5 activity, as a direct target of miR‐424‐5p. The miR‐424‐5p‐induced STAT5 phosphorylation, matrix metalloproteinases (MMPs) expression, and cell migration and invasion were blocked by SOCS2 restoration, suggesting that miR‐424‐5p exhibits its oncogenic activity through negatively regulating SOCS2 levels. Furthermore, miR‐424‐5p expression could be induced by the cytokine IL‐8 primarily through enhancing STAT5 transcriptional activity rather than NF‐κB signaling. Antagomir‐mediated inactivation of miR‐424‐5p prevented the IL‐8‐induced cell migration and invasion, indicating that miR‐424‐5p is required for IL‐8‐induced cellular invasiveness. Taken together, these data indicate that STAT5‐dependent expression of miR‐424‐5p plays an important role in mediating IL‐8/STAT5/SOCS2 feedback loop, and scavenging miR‐424‐5p function using antagomir may have therapeutic potential for the treatment of OSCC.

Epigenetic switch between SOX2 and SOX9 regulates cancer cell plasticity

Cell differentiation within stem cell lineages can check proliferative potential, but nodal pathways that can limit tumor growth are obscure. Here, we report that lung cancer cell populations generate phenotypic and oncogenic plasticity via a switch between differentiation programs controlled by SOX2 and SOX9, thus altering proliferative and invasive capabilities. In lung cancer cells, SOX2 bound the EPCAM promoter to induce EpCAM-p21Cip1-cyclin A2 signaling, encouraging cell proliferation as well as barrier properties. In contrast, SOX9 bound the SLUG promoter to induce SLUG-mediated cell invasion with a spindle-like phenotype. Pharmacologic inhibition of HDAC elevated a SOX9-positive cell population from SOX2-positive cells, whereas ectopic expression of SOX2 inhibited SOX9 with increased H3K9me2 levels on the SOX9 promoter. In clinical specimens, the expression of SOX2 and SOX9 correlated negatively and positively with lung tumor grade, respectively. Our findings identify SOX2 and SOX9 as nodal epigenetic regulators in determining cancer cell plasticity and metastatic progression. Cancer Res; 76(23); 7036-48. ©2016 AACR.

Molecular profiling of prostatic acinar morphogenesis identifies PDCD4 and KLF6 as tissue architecture-specific prognostic markers in prostate cancer.

Histopathological classification of human prostate cancer (PCA) relies on the morphological assessment of tissue specimens but has limited prognostic value. To address this deficiency, we performed comparative transcriptome analysis of human prostatic acini generated in a three-dimensional basement membrane that recapitulates the differentiated morphological characteristics and gene expression profile of a human prostate glandular epithelial tissue. We then applied an acinar morphogenesis-specific gene profile to two independent cohorts of patients with PCA (total n = 79) and found that those with tumors expressing this profile, which we designated acini-like tumors, had a significantly lower risk of postoperative relapse compared with those tumors with a lower correlation (hazard ratio, 0.078; log-rank test P = 0.009). Multivariate analyses showed superior prognostic prediction performance using this classification system compared with clinical criteria and Gleason scores. We prioritized the genes in this profile and identified programmed cell death protein 4 (PDCD4) and Kruppel-like factor 6 (KLF6) as critical regulators and surrogate markers of prostatic tissue architectures, which form a gene signature that robustly predicts clinical prognosis with a remarkable accuracy in several large series of PCA tumors (total n = 161; concordance index, 0.913 to 0.951). Thus, by exploiting the genomic program associated with prostate glandular differentiation, we identified acini-like PCA and related molecular markers that significantly enhance prognostic prediction of human PCA.

Difference in protein expression profile and chemotherapy drugs response of different progression stages of LNCaP sublines and other human prostate cancer cells

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months after treatment with a median overall survival time of 1-2 years after relapse. LNCaP is a commonly used cell line established from a human lymph node metastatic lesion of prostatic adenocarcinoma. We previously established two relapsed androgen receptor (AR)-rich androgen-independent LNCaP sublines 104-R1 (androgen depleted for 12 months) and 104-R2 cells (androgen depleted for 24 months) from AR-positive androgen-dependent LNCaP 104-S cells. LNCaP 104-R1 and 104-R2 mimics the AR-positive hormone-refractory relapsed tumors in patients receiving androgen ablation therapy. Androgen treatment stimulates proliferation of 104-S cells, but causes growth inhibition and G1 cell cycle arrest in 104-R1 and 104-R2 cells. We investigated the protein expression profile difference between LNCaP 104-S vs. LNCaP 104-R1, 104-R2, PC-3, and DU-145 cells as well as examined the sensitivity of these prostate cancer cells to different chemotherapy drugs and small molecule inhibitors. Compared to 104-S cells, 104-R1 and 104-R2 cells express higher protein levels of AR, PSA, c-Myc, Skp2, BCL-2, P53, p-MDM2 S166, Rb, and p-Rb S807/811. The 104-R1 and 104-R2 cells express higher ratio of p-Akt S473/Akt, p-EGFR/EGFR, and p-Src/Src, but lower ratio of p-ERK/ERK than 104-S cells. PC-3 and DU-145 cells express higher c-Myc, Skp2, Akt, Akt1, and phospho-EGFR but less phospho-Akt and phospho-ERK. Overexpression of Skp2 increased resistance of LNCaP cells to chemotherapy drugs. Paclitaxel, androgen, and inhibitors for PI3K/Akt, EGFR, Src, or Bcl-2 seem to be potential choices for treatment of advanced prostate cancers. Our study provides rationale for targeting Akt, EGFR, Src, Bcl-2, and AR signaling as a treatment for AR-positive relapsed prostate tumors after hormone therapy.