When a normal copy of chromosome 6 (chr. 6) was transferred into the human melanoma cell line C8161, metastasis was suppressed and expression of KISS1 and VDUP1 increased. KISS1, a known metastasis suppressor gene (MSG), and VDUP1, a putative MSG, are located on chr.1. This observation led us to hypothesize that a third gene, located on chr.6, may regulate the transcription of these two genes.  DRIP130 is one of the 13 components of the Vitamin D Receptor Interacting Protein (DRIP) complex, which regulates transcription in cells via interaction with Vitamin D-VDR complexes. The gene encoding DRIP130 maps to ~6q22-q24, which is in a region frequently reported to be deleted in malignant melanoma. Experiments performed in our lab implicate the involvement of a gene in this region with melanoma metastasis. RPCI-5-914N13, a phage-derived artificial chromosome (PAC) with an insert containing the DRIP130 gene sequence, has been isolated. The PAC insert has been retrofitted into the pPAC4 vector and is being transfected into the highly metastatic human melanoma cell lines MelJuSo and C8161. Transfected cells will be used in future studies to determine the effect of Vitamin D3 on expression of KISS1 and VDUP1. Subsequent studies will be performed to evaluate if Vitamin D3 treatment modulates metastatic potential in nude mice.
 

The gene that encodes connexin 43 (CX43), a gap junctional protein, is located on chromosome 6 in a region (6q16.3-q23) frequently deleted in malignant melanomas. Changes in gap junctional activity have been implicated in tumor progression.  Thus, we hypothesize that CX43 may play a role in the regulation of human melanoma metastasis.  More specifically, we proposed that increased CX43 expression may decrease metastatic potential in melanoma cells. In a panel of highly metastatic human melanoma cell lines RT-PCR revealed that these cell lines expressed little or no CX43 mRNA. To test our hypothesis, CX43 (i.e., CX43pcDNA3.1/hygromycin) is being transfected into two human metastatic melanoma cell lines, MelJuSo and M24met. Frequently, CX43 expression is inversely correlated with osteopontin (OPN) levels, a secreted integrin-binding protein. Other investigators have reported that OPN is overexpressed in tumors when compared to normal tissue and is prominent at sites of tumor invasion and necrosis. Furthermore, increased OPN levels have been found in the blood of patients with metastatic cancers, such as colon, breast, prostate, lung, and stomach. These findings led us to assay several melanoma cell lines for OPN expression. RT-PCR demonstrated that nine human metastatic melanoma cell lines expressed varying levels of OPN. CX43 transfected melanoma cells will be evaluated for gap junctional activity and metastatic ability in nude mice in future experiments.

Cancer progression results from deregulation of normal gene expression in cells.  For cancers to occur, the expression of oncogenes may be increased, and/or the expression tumor suppressor and metastasis suppressor genes may be decreased.  Therefore, transcriptional regulators likely play a key role in cancer progression and metastasis. Histone deacetylase 2 (HDAC2) and CBP/p300-interacting transactivator (p300) are known to act in transcriptional regulation. These two genes are located on the long arm of chromosome 6, a region that is often mutated or deleted in melanoma cells and previously shown by our laboratory to harbor a metastasis-suppressor locus. The hypothesis being tested is that HDAC2 and p300 function coordinately as transcriptional regulators in pathways leading to metastasis suppression.  Therefore, it is expected that more metastatic cells may lack expression of HDAC2 and p300, while cells from earlier stages of melanoma would express higher levels of one or more of these genes resulting in inhibition of metastasis.  To test this theory, melanoma cell lines representing various stages of tumor progression were cultured and total RNA was isolated from them.  Expression of the HDAC2 and p300 genes was determined for each cell line by reverse transcription-polymerase chain reaction (RT-PCR). Analysis of these results will provide the basis for future directions of this project.

Previous studies show that introduction of a human chromosome 6 (chr6) suppresses metastasis in a human malignant melanoma model. Additional experiments employing cells containing an added chr6 lacking 6q16.3-23.3 (6qdel), found that these hybrids were metastatic.  Taken together, these experiments provide evidence that a metastasis suppressor locus resides in this region.  Search for genes involved in metastasis suppression was evaluated by comparing gene expression in non-metastatic chr6-containing cells to metastatic cell lines.  Gene expression of two putative metastasis-suppressor genes that localize to 6qdel were measured in various metastatic and non-metastatic hybrid cell lines originating from the parent C8161 melanoma cell line. These two genes, Connexin 43 (CX43) and Vitamin D receptor interacting protein 130 (DRIP130), were analyzed using RT-PCR. Results indicate that multiple gene interactions are involved in regulating metastasis suppression. Differential gene expression between metastatic and non-metastatic cells was assessed by microarray experiments to identify additional genes involved in melanoma progression.  In two independent experiments, thioredoxin interacting protein (TXNIP) was found to be upregulated in non-metastatic cells.  Thus further studies will be initiated to investigate whether the TXNIP gene acts to inhibit tumor progression by its interference in the metastatic cascade.