Chih Yang Wang

Meta-Analysis of Public Microarray Datasets Reveals Voltage-Gated Calcium Gene Signatures in Clinical Cancer Patients

Voltage-gated calcium channels (VGCCs) are well documented to play roles in cell proliferation, migration, and apoptosis; however, whether VGCCs regulate the onset and progression of cancer is still under investigation. The VGCC family consists of five members, which are L-type, N-type, T-type, R-type and P/Q type. To date, no holistic approach has been used to screen VGCC family genes in different types of cancer. We analyzed the transcript expression of VGCCs in clinical cancer tissue samples by accessing ONCOMINE (www.oncomine.org), a web-based microarray database, to perform a systematic analysis. Every member of the VGCCs was examined across 21 different types of cancer by comparing mRNA expression in cancer to that in normal tissue. A previous study showed that altered expression of mRNA in cancer tissue may play an oncogenic role and promote tumor development; therefore, in the present findings, we focus only on the overexpression of VGCCs in different types of cancer. This bioinformatics analysis revealed that different subtypes of VGCCs (CACNA1C, CACNA1D, CACNA1B, CACNA1G, and CACNA1I) are implicated in the development and progression of diverse types of cancer and show dramatic up-regulation in breast cancer. CACNA1F only showed high expression in testis cancer, whereas CACNA1A, CACNA1C, and CACNA1D were highly expressed in most types of cancer. The current analysis revealed that specific VGCCs likely play essential roles in specific types of cancer. Collectively, we identified several VGCC targets and classified them according to different cancer subtypes for prospective studies on the underlying carcinogenic mechanisms. The present findings suggest that VGCCs are possible targets for prospective investigation in cancer treatment.

RasGRP1 opposes proliferative EGFR-SOS1-Ras signals and restricts intestinal epithelial cell growth

The character of EGFR signals can influence cell fate but mechanistic insights into intestinal EGFR-Ras signalling are limited. Here we show that two distinct Ras nucleotide exchange factors, RasGRP1 and SOS1, lie downstream of EGFR but act in functional opposition. RasGRP1 is expressed in intestinal crypts where it restricts epithelial growth. High RasGRP1 expression in colorectal cancer (CRC) patient samples correlates with a better clinical outcome. Biochemically, we find that RasGRP1 creates a negative feedback loop that limits proliferative EGFR–SOS1–Ras signals in CRC cells. Genetic Rasgrp1 depletion from mice with either an activating mutation in KRas or with aberrant Wnt signalling due to a mutation in Apc resulted in both cases in exacerbated Ras–ERK signalling and cell proliferation. The unexpected opposing cell biological effects of EGFR–RasGRP1 and EGFR–SOS1 signals in the same cell shed light on the intricacy of EGFR-Ras signalling in normal epithelium and carcinoma.

Systematic Analysis of Gene Expression Alterations and Clinical Outcomes for Long-Chain Acyl-Coenzyme A Synthetase Family in Cancer.

Dysregulated lipid metabolism contributes to cancer progression. Our previous study indicates that long-chain fatty acyl-Co A synthetase (ACSL) 3 is essential for lipid upregulation induced by endoplasmic reticulum stress. In this report, we aimed to identify the role of ACSL family in cancer with systematic analysis and in vitro experiment. We explored the ACSL expression using Oncomine database to determine the gene alteration during carcinogenesis and identified the association between ACSL expression and the survival of cancer patient using PrognoScan database. ACSL1 may play a potential oncogenic role in colorectal and breast cancer and play a potential tumor suppressor role in lung cancer. Co-expression analysis revealed that ACSL1 was coexpressed with MYBPH, PTPRE, PFKFB3, SOCS3 in colon cancer and with LRRFIP1, TSC22D1 in lung cancer. In accordance with PrognoScan analysis, downregulation of ACSL1 in colon and breast cancer cell line inhibited proliferation, migration, and anchorage-independent growth. In contrast, increase of oncogenic property was observed in lung cancer cell line by attenuating ACSL1. High ACSL3 expression predicted a better prognosis in ovarian cancer; in contrast, high ACSL3 predicted a worse prognosis in melanoma. ACSL3 was coexpressed with SNUPN, TRIP13, and SEMA5A in melanoma. High expression of ACSL4 predicted a worse prognosis in colorectal cancer, but predicted better prognosis in breast, brain and lung cancer. ACSL4 was coexpressed with SERPIN2, HNRNPCL1, ITIH2, PROCR, LRRFIP1. High expression of ACSL5 predicted good prognosis in breast, ovarian, and lung cancers. ACSL5 was coexpressed with TMEM140, TAPBPL, BIRC3, PTPRE, and SERPINB1. Low ACSL6 predicted a worse prognosis in acute myeloid leukemia. ACSL6 was coexpressed with SOX6 and DARC. Altogether, different members of ACSLs are implicated in diverse types of cancer development. ACSL-coexpressed molecules may be used to further investigate the role of ACSL family in individual type of cancers.

Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer

CD90 is used as a marker for cancer stem cell in liver cancer. We aimed to study the mechanism by which CD90 promoted liver cancer progression and identify the new therapeutic targets on CD90 signal pathway. Ectopic expression of CD90 in liver cancer cell lines enhanced anchorage-independent growth and tumor progression. Furthermore, CD90 promoted sphere formation in vitro and upregulated the expression of the cancer stem cell marker CD133. The CD133 expression was higher in CD45-CD90+ cells in liver cancer specimen. The natural carcinogenic molecules TGF-β-1, HGF, and hepatitis B surface antigen increased the expression of CD90 and CD133. Inhibition of CD90 by either shRNA or antibody attenuated the induction of CD133 and anchorage-independent growth. Lentiviral delivery of CD133 shRNA abolished the tumorigenicity induced by CD90. Ectopic expression of CD90 induced mTOR phosphorylation and AMPK dephosphorylation. Mutation of integrin binding-RLD domain in CD90 attenuated the induction of CD133 and anchorage-independent growth. Similar results were observed after silencing β3 integrin. Signaling analyses revealed that AMPK/mTOR and β3 integrin were required for the induction of CD133 and tumor formation by CD90. Importantly, the energy restriction mimetic agent OSU-CG5 reduced the CD90 population in fresh liver tumor sample and repressed the tumor growth. In contrast, sorafenib did not decrease the CD90+ population. In conclusion, the signal axis of CD90-integrin-mTOR/AMPK-CD133 is critical for promoting liver carcinogenesis. Molecules inhibiting the signal axis, including OSU-CG5 and other inhibitors, may serve as potential novel cancer therapeutic targets in liver cancer.

A novel cancer therapeutic using thrombospondin 1 in dendritic cells

Induction of thrombospondin 1 (TSP-1) is generally assumed to suppress tumor growth through inhibiting angiogenesis; however, it is less clear how TSP-1 in dendritic cells (DCs) influences tumor progression. We investigated tumor growth and immune mechanism by downregulation of TSP-1 in dendritic cells. Administration of TSP-1 small hairpin RNA (shRNA) through the skin produced anticancer therapeutic effects. Tumor-infiltrating CD4(+) and CD8(+) T cells were increased after the administration of TSP-1 shRNA. The expression of interleukin-12 and interferon-γ in the lymph nodes was enhanced by injection of TSP-1 shRNA. Lymphocytes from the mice injected with TSP-1 shRNA selectively killed the tumor cells, and the cytotoxicity of lymphocytes was abolished by depletion of CD8(+) T cells. Injection of CD11c(+) TSP-1-knockout (TSP-1-KO) bone marrow-derived DCs (BMDCs) delayed tumor growth in tumor-bearing mice. Similarly, antitumor activity induced by TSP-1-KO BMDCs was abrogated by depletion of CD8(+) T cells. In contrast, the administration of shRNAs targeting TSP-2, another TSP family member, did not extend the survival of tumor-bearing mice. Finally, TSP-1 shRNA functioned as an immunotherapeutic adjuvant to augment the therapeutic efficacy of Neu DNA vaccination. Collectively, the downregulation of TSP-1 in DCs produces an effective antitumor response that is opposite to the protumor effects by silencing of TSP-1 within tumor cells.

Voltage-gated calcium channels: Novel targets for cancer therapy

Voltage-gated calcium channels (VGCCs) comprise five subtypes: The L-type; R-type; N-type; P/Q-type; and T-type, which are encoded by α1 subunit genes. Calcium ion channels also have confirmed roles in cellular functions, including mitogenesis, proliferation, differentiation, apoptosis and metastasis. An association between VGCCs, a reduction in proliferation and an increase in apoptosis in prostate cancer cells has also been reported. Therefore, in the present study, the online clinical database Oncomine was used to identify the alterations in the mRNA expression level of VGCCs in 19 cancer subtypes. Overall, VGCC family genes exhibited under-expression in numerous types of cancer, including brain, breast, kidney and lung cancers. Notably, the majority of VGCC family members (CACNA1C, CACNA1D, CACNA1A, CACNA1B, CACNA1E, CACNA1H and CACNA1I) exhibited low expression in brain tumors, with mRNA expression levels in the top 1–9% of downregulated gene rankings. A total of 5 VGCC family members (CACNA1A, CACNA1B, CACNA1E, CACNA1G and CACNA1I) were under-expressed in breast cancer, with a gene ranking in the top 1–10% of the low-expressed genes compared with normal tissue. In kidney and lung cancers, CACNA1S, CACNA1C, CACNA1D, CACNA1A and CACNA1H exhibited low expression, with gene rankings in the top 1–8% of downregulated genes. In conclusion, the present findings may contribute to the development of new cancer treatment approaches by identifying target genes involved in specific types of cancer.

MST3 promotes proliferation and tumorigenicity through the VAV2/Rac1 signal axis in breast cancer

MST3 (mammalian STE20-like kinase 3) belongs to the Ste20 serine/threonine protein kinase family. The role of MST3 in tumor growth is less studied; therefore, we investigates the function of MST3 in breast cancer. Here, we demonstrate that MST3 is overexpressed in human breast tumors. Online Kaplan-Meier plotter analysis reveals that overexpression of MST3 predicts poor prognosis in breast cancer patients. Knockdown of MST3 with shRNA inhibits proliferation and anchorage-independent growth in vitro. Downregulation of MST3 in triple-negative MDA-MB-231 and MDA-MB-468 breast cancer cells decreases tumor formation in NOD/SCID mice. MST3 interacts with VAV2, but not VAV3, as demonstrated by co-immunoprecipitation and confocal microscopy. By domain mapping of MST3, we determine that the proline-rich region of MST3 (353KDIPKRP359) interacts with the SH3 domain of VAV2. Mutation of the two proline residues in this domain significantly attenuates the interaction between MST3 and VAV2. Overexpression of wild-type MST3 (WT-MST3), but not proline-rich-deleted MST3 (ΔP-MST3), enhances the proliferation rate and anchorage-independent growth of MDA-MB-468 cells. Overexpression of MST3 increases VAV2 phosphorylation and GTP-Rac1, whereas downregulation of MST3 or delivery of ΔP-MST3 results in a reduction of VAV2 and Rac1 activation. Knockdown of MST3 inhibits cyclin D1 protein expression. The Rac1 inhibitor EHop-016 attenuates cell proliferation induced by WT-MST3. Finally, Knockdown of MST3 or Rac1 inhibitor decreases cyclin D protein expression, which is important for tumor growth. These results indicate that MST3 interacts with VAV2 to activate Rac1 and promote the tumorigenicity of breast cancer.

Argininosuccinate lyase is a potential therapeutic target in breast cancer.

Arginine is a non-essential amino acid that modulates nitric oxide production and cancer homeostasis. In our previous study, we observed that blocking argininosuccinate lyase (ASL) attenuates tumor progression in liver cancer. However, the role of ASL in human breast cancer has been studied to a lesser degree. In the present study, we investigated the effect of targeting ASL in breast cancer. We found that ASL was induced by ER stress and was significantly upregulated in breast cancer tissues compared to that in the corresponding normal tissues. Downregulation of ASL inhibited the growth of breast cancer in vitro and in vivo. The level of cell cycle-related gene, cyclin A2, was reduced and was accompanied by a delay in G2/M transition. ASL shRNA-induced cell inhibition was rescued by exogenous cyclin A2. Furthermore, autophagy was observed in the cells expressing ASL shRNA, and inhibition of autophagy reduced cell growth, indicating that autophagy played a cell survival role in the ASL knockdown cells. Moreover, inhibition of ASL reduced NO content. Introduction of the NO donor partially restored the growth inhibition by ASL shRNA. Thus, the mechanism induced by ASL shRNA which occurred in human breast cancer may be attributed to a decrease in cyclin A2 and NO.

Differential Expression Pattern of THBS1 and THBS2 in Lung Cancer: Clinical Outcome and a Systematic-Analysis of Microarray Databases

Thrombospondin 1 and thrombospondin 2 (THBS1 and THBS2) share similar multifunctional domains, and are known to be antiangiogenic. However, the expression pattern of THBS1 and THBS2 is different, and the specific role of THBS2 in different subtypes of lung cancer remains largely unclear. To evaluate the significance of THBS1 and THBS2 in the development of lung cancer, the present study performed a microarray-based systematic-analysis to determine the transcript levels of thrombospondins and their relation to the prognosis in lung cancer. THBS1 was in general underexpressed in lung cancer; in contrast, mRNA levels of THBS2 were markedly overexpressed in a number of datasets of non-small cell lung carcinoma (NSCLC), including lung adenocarcinoma (AC) and squamous cell carcinoma. Similar expression pattern of THBS1 and THBS2 was verified in pulmonary AC cell lines with real-time PCR analysis. The survival of lung AC patients with high THBS2 mRNA expression levels was poorer than patients with low levels of expression of THBS2. In a microarray-based analysis, genes coexpressed with THBS1 or THBS2 were determined. Pulmonary AC patients with a high expression level of sevenTSHB1-coexpressed genes (CCL5, CDH11, FYB, GZMK, LA-DQA1, PDE4DIP, and SELL) had better survival rates than those with a low expression level. Patients with a high expression of seven TSHB2-coexpressed genes (CHI3L1, COL5A2, COL11A1, FAP, MXRA5, THY1, and VCAN) had poor survival rates. Downregulation of VCAN and THBS2 with shRNA inhibited the cell proliferation in the A549 cell line. In summary, THBS1 functions as a tumor suppressor in lung adenocarcinoma. However, THBS2 may play a double-edged role in the progression of lung AC, i.e. anti-angiogenic and oncogenic function. Further study on the mechanism underlying the activity of THBS2 is warranted to have further implications for cancer diagnosis and treatment of pulmonary AC.

Cyclin D1 overexpression correlates with poor tumor differentiation and prognosis in gastric cancer

Overexpression of cyclin D is associated with the molecular tumorigenesis of gastric cancer. The purpose of the present study was to investigate the expression of cyclin D in human gastric cancer and to determine the potential correlations between cyclin D expression and clinicopathological characteristics of specific histological types, as well as its prognostic significance. In the present study, the expression of the cyclin D1 (CCND1), cyclin D2 (CCND2) and cyclin D3 (CCND3) genes in gastric cancer patients was explored using the Oncomine database, and their correlation with overall survival (OS) and progression-free survival (PFS) was evaluated using Kaplan-Meier analysis. The prognostic significance of CCND1 protein expression was evaluated by western blot analysis of 32 matched specimens of gastric adenocarcinomas and normal tissues obtained from patients treated at the National Cheng Kung University Hospital (Tainan, Taiwan). Analysis of the Oncomine cancer microarray database revealed that CCND1 gene expression was significantly increased in gastric intestinal-type adenocarcinoma, while CCND2 was significantly increased in diffuse gastric adenocarcinoma, gastric intestinal-type adenocarcinoma and gastric mixed adenocarcinoma. Kaplan-Meier analysis indicated that overexpression of CCND1 was associated with reduced OS and PFS. In addition, overexpression of CCND1 and downregulation of CCND2 were significantly correlated with receptor tyrosine-protein kinase erb-2-negative tumors and poor differentiation. The ratio of relative CCND1 expression (expressed as the CCND1/β-actin ratio) in tumor tissues compared with that in normal tissues was correlated with poor differentiation (P=0.0018). In summary, CCND1 overexpression is associated with shorter survival in patients with gastric cancer and with poorly differentiated tumors.