Lewis J. Stafford

Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation

Cyclin D1 is a proto-oncogene that is overexpressed in many cancers including breast and prostate. It plays a role in cell proliferation through activation of cyclin-dependent kinases. Curcumin, a diferuloylmethane, is a chemopreventive agent known to inhibit the proliferation of several breast and prostate cancer cell lines. It is possible that the effect of curcumin is mediated through the regulation of cyclin D1. In the present report we show that inhibition of the proliferation of various prostate, breast and squamous cell carcinoma cell lines by curcumin correlated with the down-regulation of the expression of cyclin D1 protein. In comparison, the down-regulation by curcumin of cyclin D2 and cyclin D3 was found only in selective cell lines. The suppression of cyclin D1 by curcumin led to inhibition of CDK4-mediated phosphorylation of retinoblastoma protein. We found that curcumin-induced down-regulation of cyclin D1 was inhibited by lactacystin, an inhibitor of 26S proteosome, suggesting that curcumin represses cyclin D1 expression by promoting proteolysis. We found that curcumin also down-regulated mRNA expression, thus suggesting transcriptional regulation. Curcumin also inhibited the activity of the cyclin D1 promoter-dependent reporter gene expression. Overall our results suggest that curcumin down-regulates cyclin D1 expression through activation of both transcriptional and post-transcriptional mechanisms, and this may contribute to the antiproliferative effects of curcumin against various cell types.

Metastasis suppressors genes in cancer.

The major problem for cancer patients is metastasis of the cancer from the primary tumor to secondary sites. Metastasis is the process by which tumor cells disseminate from the primary tumor, migrate through the basement membrane, survive in the circulatory system, invade into a secondary site, and start to proliferate. In the past, research had concentrated on the biology, taking more of a global view instead of a molecular view. More recently, the focus has been determining the molecular underpinnings, looking at genes that induce or inhibit metastasis. Metastasis suppressors, by definition, inhibit metastasis at any step of the metastatic cascade without blocking primary tumor growth. The expanding list of metastasis suppressors exist with every cellular compartment and have been shown to work by regulating signaling pathways that inhibit proliferation, cell migration and growth at the secondary site. Still, the biochemical basis of their inhibition is not completely known. Here we review the known metastasis suppressors and summarize the suspected mechanisms by which they inhibit metastasis.

Breast cancer metastasis suppressor 1 coordinately regulates metastasis-associated microRNA expression

Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple tumor types without blocking tumorigenesis. BRMS1 forms complexes with SIN3, histone deacetylases and selected transcription factors that modify metastasis‐associated gene expression (e.g., EGFR, OPN, PI4P5K1A, PLAU). microRNA (miRNA) are a recently discovered class of regulatory, noncoding RNA, some of which are involved in neoplastic progression. Based on these data, we hypothesized that BRMS1 may also exert some of its antimetastatic effects by regulating miRNA expression. MicroRNA arrays were done comparing small RNAs that were purified from metastatic MDA‐MB‐231 and MDA‐MB‐435 and their nonmetastatic BRMS1‐transfected counterparts. miRNA expression changed by BRMS1 were validated using SYBR Green RT‐PCR. BRMS1 decreased metastasis‐promoting (miR‐10b, ‐373 and ‐520c) miRNA, with corresponding reduction of their downstream targets (e.g., RhoC which is downstream of miR‐10b). Concurrently, BRMS1 increased expression of metastasis suppressing miRNA (miR‐146a, ‐146b and ‐335). Collectively, these data show that BRMS1 coordinately regulates expression of multiple metastasis‐associated miRNA and suggests that recruitment of BRMS1‐containing SIN3:HDAC complexes to, as yet undefined, miRNA promoters might be involved in the regulation of cancer metastasis.

Regulation of the p21-activated kinase (PAK) by a human Gβ-like WD-repeat protein, hPIP1

The family of p21-activated protein kinases (PAKs) is composed of serine–threonine kinases whose activity is regulated by the small guanosine triphosphatases (GTPases) Rac and Cdc42. In mammalian cells, PAKs have been implicated in the regulation of mitogen-activated protein cascades, cellular morphological and cytoskeletal changes, neurite outgrowth, and cell apoptosis. Although the ability of Cdc42 and Rac GTPases to activate PAK is well established, relatively little is known about the negative regulation of PAK or the identity of PAK cellular targets. Here, we describe the identification and characterization of a human PAK-interacting protein, hPIP1. hPIP1 contains G protein β-like WD repeats and shares sequence homology with the essential fission yeast PAK regulator, Skb15, as well as the essential budding yeast protein, MAK11. Interaction of hPIP1 with PAK1 inhibits the Cdc42/Rac-stimulated kinase activity through the N-terminal regulatory domains of PAK1. Cotransfection of hPIP1 in mammalian cells inhibits PAK-mediated c-Jun N-terminal kinase and nuclear factor κ B signaling pathways. Our results demonstrate that hPIP1 is a negative regulator of PAK and PAK signaling pathways.

Regulation of KiSS-1 Metastasis Suppressor Gene Expression in Breast Cancer Cells by Direct Interaction of Transcription Factors Activator Protein-2α and Specificity Protein-1

KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2α (AP-2α) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2α binding elements, AP-2α-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2α into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2α wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2α regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2α directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2α and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2α in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis.

GCIP/CCNDBP1, a helix–loop–helix protein, suppresses tumorigenesis

Deletions and/or loss of heterozygosity (LOH) on chromosome 15 (15q15 and 15q21) have been found in several human tumors, including carcinomas of the colorectum, breast, lung, prostate, and bladder, suggesting the presence of potential tumor suppressor gene(s) in this particular region of chromosome 15. GCIP also called CCNDBP1, DIP1, or HHM, localized at chromosome 15q15, is a recently identified helix–loop–helix leucine zipper (HLH‐ZIP) protein without a basic region like the Id family of proteins. In this study, we reported that the expression of GCIP was significantly downregulated in several different human tumors, including breast tumor, prostate tumor, and colon tumors. In human colon tumors, both mRNA and protein expression levels of GCIP were decreased significantly compared to the normal tissues. Treatment of colon cancer cells SW480 with sodium butyrate (NaB), which induces colon cancer cell differentiation, can induce the upregulation of GCIP expression, suggesting that the protein functions as a negative regulator in cell proliferation. Overexpression of GCIP in SW480 colon cancer cell line resulted in a significant inhibition on tumor cell colony formation, while silencing of GCIP expression by siRNA can promote cell colony formation. Furthermore, overexpression of GCIP inhibited the transcriptional activity of cyclin D1 promoter and the expression of cyclin D1 protein in the cell. Finally, we demonstrate that GCIP specifically interacts with one of the class III HDAC proteins, SirT6, which is important for maintaining genome stability. Together, our data suggest a possible function of GCIP in tumor suppression. J. Cell. Biochem. 100: 1376–1386, 2007.

GGAPs, a New Family of Bifunctional GTP-Binding and GTPase-Activating Proteins

ABSTRACT G proteins are molecular switches that control a wide variety of physiological functions, including neurotransmission, transcriptional activation, cell migration, cell growth. and proliferation. The ability of GTPases to participate in signaling events is determined by the ratio of GTP-bound to GDP-bound forms in the cell. All known GTPases exist in an inactive (GDP-bound) and an active (GTP-bound) conformation, which are catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), respectively. In this study, we identified and characterized a new family of bifunctional GTP-binding and GTPase-activating proteins, named GGAP. GGAPs contain an N-terminal Ras homology domain, called the G domain, followed by a pleckstrin homology (PH) domain, a C-terminal GAP domain, and a tandem ankyrin (ANK) repeat domain. Expression analysis indicates that this new family of proteins has distinct cell localization, tissue distribution, and even message sizes. GTPase assays demonstrate that GGAPs have high GTPase activity through direct intramolecular interaction of the N-terminal G domain and the C-terminal GAP domain. In the absence of the GAP domain, the N-terminal G domain has very low activity, suggesting a new model of GGAP protein regulation via intramolecular interaction like the multidomain protein kinases. Overexpression of GGAPs leads to changes in cell morphology and activation of gene transcription.

Breast Cancer Metastasis Suppressor-1 Differentially Modulates Growth Factor Signaling

That metastatic tumor cells grow in selective non-native environments suggests an ability to differentially respond to local microenvironments. BRMS1, like other metastasis suppressors, halts ectopic growth (metastasis) without blocking orthotopic tumor formation. BRMS1-expressing tumor cells reach secondary sites but do not colonize distant tissues, compelling the hypothesis that BRMS1 selectively restricts the ability of tumor cells to respond to exogenous regulators in different tissues. Here we report that BRMS1 expression in metastatic human breast cancer cells leads to a selective reduction in epidermal growth factor receptor expression and downstream (AKT) signaling. Signaling through another receptor tyrosine kinase, hepatocyte growth factor receptor (c-Met), remains unaltered despite reduced levels of the signaling intermediate phosphatidylinositol (4,5)-bisphosphate. Interestingly, reduced downstream calcium signaling is observed following treatment with platelet-derived growth factor, consistent with decreased phosphatidylinositol (4,5)-bisphosphate. However, platelet-derived growth factor receptor expression is unaltered. Thus, BRMS1 differentially attenuates cellular responses to mitogenic signals, not only dependent upon the specific signal received, but at varying steps within the same signaling cascade. Specific modulation of signaling responses received from the microenvironment may ultimately dictate which environments are permissive/restrictive for tumor cell growth and provide insights into the biology underlying metastasis.

Transcriptional regulation of KiSS-1 gene expression in metastatic melanoma by specificity protein-1 and its coactivator DRIP-130

Loss of the metastasis suppressor gene, KiSS-1 has been strongly correlated to the progression of metastases in numerous types of cancers. The mechanism through which KiSS-1 is lost during metastasis, however, is still not completely known. Previous studies have shown that genetic material on human chromosome 6q16.3–q23 is essential for KiSS-1 expression in normal tissues. Additionally, microcell-mediated transfer of this chromosome in cancerous tissue results in rescued expression of KiSS-1 and reduced metastatic phenotype. Here, we show that loss of Sp1-coactivator protein DRIP-130, which is encoded by human chromosome 6q16.3–q23, results in reduced KiSS-1 promoter activation in highly malignant melanoma cells. Co-expression of Sp1 and DRIP-130 not only rescues KiSS-1 expression, but also induces an inhibition of the invasive and migratory behavior in highly metastatic melanoma cells, similar to the overexpression of KiSS-1 metastasis suppressor gene in those cells. Furthermore, we demonstrate that KiSS-1 expression is regulated by Sp1 elements within the first 100-bp region of the KiSS-1 promoter and that targeted deletion of a single GC-rich region spanning −93 to −58 interrupts Sp1- and DRIP-130-modulated transcriptional control of KiSS-1 expression. Our results thus suggest that DRIP-130 is a key regulator in KiSS-1 transactivation in normal tissue, and that the loss of DRIP-130 expression, as a result of the gross loss of human chromosome 6q16.3–q23, provokes increased tumor metastasis.

Increased expression of prostate-specific G-protein-coupled receptor in human prostate intraepithelial neoplasia and prostate cancers

The G‐protein‐coupled receptors and signal transduction pathways represent important specific targets for a variety of human diseases, ranging from the control of blood pressure, allergic response, hormonal disorders and neurologic diseases to tumorigenesis. Most recently, we and others have identified a novel human prostate‐specific G‐protein coupled receptor (PSGR). To investigate the potential roles of PSGR in human normal prostate and prostate cancers, we examined the expression level of PSGR in 146 human prostate samples with real‐time quantitative reverse transcription‐PCR and in situ hybridization method. We significantly extended previous studies and demonstrated that PSGR is specifically expressed in human prostate tissues, not in any other normal and tumor samples tested. Compared to normal and benign prostatic hyperplasia tissues, the expression of PSGR increased significantly in human prostate intraepithelial neoplasia (PIN) and prostate tumors (approximately 10‐fold), especially in early prostate tumors, suggesting PSGR may play an important role in early prostate cancer development and progression. The sensitivity and specificity estimates for PSGR expression were calculated as the area under the receiver‐operating characteristics curve (0.902), indicating high‐level sensitivity and specificity for discriminating benign prostate tissues from malignant prostate tissues. The association of PSGR expression with clinical parameters (clinical stages, Gleason scores, recurrent status and metastasis) was also investigated in this study. Our data suggest that overexpression of PSGR in human PIN and prostate cancers have the potential for early prostate cancer detection and diagnosis.