Jirong Zhang

VILIP-1 Downregulation in Non-Small Cell Lung Carcinomas: Mechanisms and Prediction of Survival

VILIP-1, a member of the neuronal Ca++ sensor protein family, acts as a tumor suppressor gene in an experimental animal model by inhibiting cell proliferation, adhesion and invasiveness of squamous cell carcinoma cells. Western Blot analysis of human tumor cells showed that VILIP-1 expression was undetectable in several types of human tumor cells, including 11 out of 12 non-small cell lung carcinoma (NSCLC) cell lines. The down-regulation of VILIP-1 was due to loss of VILIP-1 mRNA transcripts. Rearrangements, large gene deletions or mutations were not found. Hypermethylation of the VILIP-1 promoter played an important role in gene silencing. In most VILIP-1-silent cells the VILIP-1 promoter was methylated. In vitro methylation of the VILIP-1 promoter reduced its activity in a promoter-reporter assay. Transcriptional activity of endogenous VILIP-1 promoter was recovered by treatment with 5′-aza-2′-deoxycytidine (5′-Aza-dC). Trichostatin A (TSA), a histone deacetylase inhibitor, potently induced VILIP-1 expression, indicating that histone deacetylation is an additional mechanism of VILIP-1 silencing. TSA increased histone H3 and H4 acetylation in the region of the VILIP-1 promoter. Furthermore, statistical analysis of expression and promoter methylation (n = 150 primary NSCLC samples) showed a significant relationship between promoter methylation and protein expression downregulation as well as between survival and decreased or absent VILIP-1 expression in lung cancer tissues (p<0.0001). VILIP-1 expression is silenced by promoter hypermethylation and histone deacetylation in aggressive NSCLC cell lines and primary tumors and its clinical evaluation could have a role as a predictor of short-term survival in lung cancer patients.

VILIP-1 expression in vivo results in decreased mouse skin keratinocyte proliferation and tumor development

VILIP-1, a member of the neuronal Ca2+ sensor protein family, is able to act as a tumor suppressor in carcinoma cells by inhibiting cell proliferation and migration. In order to study the role of VILIP-1 in skin carcinogenesis we generated transgenic mice overexpressing VILIP-1 in epidermis under the control of the bovine keratin K5 promoter (K5-VILIP-1). We studied the susceptibility of FVB wild type and VILIP-1 transgenic mice to chemically mediated carcinogenesis. After 30 weeks of treatment with a two-stage carcinogenesis protocol, all animals showed numerous skin tumors. Nevertheless, K5-VILIP-1 mice showed decreased squamous cell carcinoma (SCC) multiplicity of ∼49% (p<0.02) with respect to the corresponding SCC multiplicity observed in wild type (WT) mice. In addition, the relative percentage of low-grade cutaneous SCCs grade I (defined by the differentiation pattern according to the Broders grading scale) increased approximately 50% in the K5-VILIP1 mice when compared with SCCs in WT mice. Similar tendency was observed using a complete carcinogenesis protocol for skin carcinogenesis using benzo(a)pyrene (B(a)P). Further studies of tumors and primary epidermal keratinocyte cultures showed that matrix metalloproteinase 9 (MMP-9) levels and cell proliferation decreased in K5-VILIP-1 mice when compared with their wild counterparts. In addition tissue inhibitor of metalloproteinase 1 (TIMP-1) expression was higher in K5-VILIP-1 keratinocytes. These results show that VILIP-1 overexpression decreases the susceptibility to skin carcinogenesis in experimental mouse cancer models, thus supporting its role as a tumor suppressor gene.

Enhanced aggressiveness of benzopyrene-induced squamous carcinomas in transgenic mice overexpressing the proprotein convertase PACE4 (PCSK6)

PACE4 (PCSK6) is a proprotein convertase (PC) capable of processing numerous substrates involved in tumor growth, invasion, and metastasis. Because of the human relevancy of the tobacco‐associated carcinogen benzo[a]pyrene (B(a)P) we investigated whether transgenic mice in which this PC is targeted to the epidermis (K5‐PACE4) may be more susceptible to B(a)P complete carcinogenesis than wild type (WT) mice. In an in vitro experiment, using cell lines derived from skin tumors obtained after B(a)P treatment, we observed that PACE4 overexpression and activity accounts for an increased proliferation rate, exaggerated sensitivity to the PC inhibitor CMK, and interference with IGF‐1R autophosphorylation. Squamous cell carcinomas, obtained from K5‐PACE4 mice subjected to complete chemical carcinogenesis, were characterized by a 50% increase in cell proliferation, when compared with similar tumors from WT mice. In addition, tumors from K5‐PACE4 mice showed deeper invasion into the underlying dermis. Thus, mice overexpressing PACE4 exhibited tumors of increased growth rate and invasive potential when exposed to the human carcinogen B(a)P, further supporting the significance of PCs in tumor growth and progression.

Promoter Regulation of the Visinin-like Subfamily of Neuronal Calcium Sensor Proteins by Nuclear Respiratory Factor-1

VILIP-1 (gene name VSNL1), a member of the neuronal Ca2+ sensor protein family, acts as a tumor suppressor gene by inhibiting cell proliferation, adhesion, and invasiveness. VILIP-1 expression is down-regulated in several types of human cancer. In human non-small cell lung cancer, we found that down-regulation was due to epigenetic changes. Consequently, in this study we analyzed the VSNL1 promoter and its regulation. Serial truncation of the proximal 2-kb VSNL1 promoter (VP-1998) from its 5′ terminus disclosed that the last 3′ terminal 100-bp promoter fragment maintained similar promoter activity as compared with VP-1998 and therefore was referred to as VSNL1 minimal promoter. When the 5′ terminal 50 bp were deleted from the minimal promoter, the activity was dramatically decreased, suggesting that the deleted 50 bp contained a potential cis-acting element crucial for promoter activity. Deletion and site-directed mutagenesis combined with in silico transcription factor binding analysis of VSNL1 promoter identified nuclear respiratory factor (NRF)-1/α-PAL as a major player in regulating VSNL1 minimal promoter activity. The function of NRF-1 was further confirmed using dominant-negative NRF-1 overexpression and NRF-1 small interfering RNA knockdown. Electrophoretic mobility shift assay and chromatin immunoprecipitation provided evidence for direct NRF-1 binding to the VSNL1 promoter. Methylation of the NRF-1-binding site was found to be able to regulate VSNL1 promoter activity. Our results further indicated that NRF-1 could be a regulatory factor for gene expression of the other visinin-like subfamily members including HPCAL4, HPCAL1, HPCA, and NCALD.

Regulation of HIF-1 alpha by the proprotein convertases furin and PC7 in human squamous carcinoma cells.

Proprotein convertases (PC), a family of serine proteases, process cancer‐related substrates such as growth factors, growth factor receptors, cell adhesion molecules, metalloproteinases, etc. HIF‐1α is a major transcription factor involved in tumorigenesis by sensing intratumoral hypoxia. Furin (PCSK3) is one of the numerous target genes regulated by HIF‐1α transactivation and its distribution into endosomal compartments and onto the cell surface can be triggered by hypoxia via HIF‐1α. siRNAs to knockdown PCs were transfected into cells alone or in combination with different drug treatments. Protein and RNA expression levels were analyzed by Western blotting or RT‐PCR, respectively. PC7 (PCSK7) and furin siRNAs upregulated HIF‐1α protein under normoxic condition to a level similar to that obtained by cobalt chloride treatment, eventually leading to activation of VEGF‐A synthesis in two human head and neck squamous cell carcinoma cell lines. The unchanged levels of HIF‐1α mRNA expression under siRNA treatment and the additive HIF‐1α induction of PC siRNAs and either cobalt chloride or the 26S ribosome inhibitor, MG‐132, suggested a post‐transcriptional PC‐mediated regulation. Furthermore, cycloheximide chase showed that PC7/furin siRNA regulation occurred at the level of HIF‐1α translation. A specific IGF‐1R signaling inhibitor was able to attenuate the PC siRNA induction of HIF‐1α, suggesting the involvement of the IGF‐1R pathway. Thus, the data show that PCs regulate HIF‐1α. Furin and PC7 siRNAs induced HIF‐1α protein by increasing its translation, resulting in upregulation of VEGF‐A. This finding may provide insight into intricate PC functions that seem to be independent from their substrate‐processing activity.

Targeting proprotein convertases in furin‐rich lung cancer cells results in decreased in vitro and in vivo growth

Proprotein convertases (PCs) are serine proteases with an active role in the post‐translational processing of numerous inactive proteins to active proteins including many substrates of paramount importance in cancer development and progression. Furin (PCSKC3), a well‐studied member of this family, is overexpressed in numerous human and experimental malignancies. In the present communication, we treated two furin‐overexpressing non‐small cell carcinoma (NSCLC) cell lines (Calu‐6 and HOP‐62) with the PC inhibitor CMK (Decanoyl‐Arg‐Val‐Lys‐Arg‐chloromethylketone). This resulted in a diminished IGF‐1R processing and a simultaneous decrease in cell proliferation of two NSCLC lines. Similarly, growth of subcutaneous xenografts of both cell lines, were partially inhibited by an in vivo treatment with the same drug. These observations point to a potential role of PC inhibitors in cancer therapy.