Rajeev S Samant

Abstract: The microenvironment at the site of tumor metastasis plays a key role in determining the fate of the metastasizing tumor cells. This ultimately has a direct impact on the progression of cancer. Bone is the preferred site of metastasis of breast cancer. Painful, debilitating osteolytic lesions are formed as a result of crosstalk between breast cancer cells and cells in the bone, predominantly the osteoblasts and osteoclasts. In this paper, we have discussed the temporal and spatial role of hedgehog (Hh) signaling in influencing the fate of metastatic breast cancer cells in bone. By virtue of its secreted ligands, the Hh pathway is capable of homotypic and heterotypic signaling and consequently altering the microenvironment in the bone. We also have put into perspective the therapeutic implications of using Hh inhibitors to prevent and/or treat bone metastases of breast cancer.

Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) have been widely reported to display strong efficacy for cancer chemoprevention, although their mechanism of action is poorly understood. The most well-documented effects of NSAIDs include inhibition of tumor cell proliferation and induction of apoptosis, but their effect on tumor cell invasion has not been well studied. Here, we show that the NSAID, sulindac sulfide (SS) can potently inhibit the invasion of human MDA-MB-231 breast and HCT116 colon tumor cells in vitro at concentrations less than those required to inhibit tumor cell growth. To study the molecular basis for this activity, we investigated the involvement of microRNA (miRNA). A total of 132 miRNAs were found to be altered in response to SS treatment, including miR-10b, miR-17, miR-21 and miR-9, which have been previously implicated in tumor invasion and metastasis. We confirmed that these miRNA can stimulate tumor cell invasion and show that SS can attenuate their invasive effects by downregulating their expression. Employing luciferase and chromatin immunoprecipitation assays, NF-κB was found to bind the promoters of all four miRNAs to suppress their expression at the transcriptional level. We show that SS can inhibit the translocation of NF-κB to the nucleus by decreasing the phosphorylation of IKKβ and IκB. Analysis of the promoter sequences of the miRNAs suppressed by SS revealed that 81 of 115 sequences contained NF-κB-binding sites. These results show that SS can inhibit tumor cell invasion by suppressing NF-κB-mediated transcription of miRNAs.Oncogene advance online publication, 30 January 2012; doi:10.1038/onc.2011.655.

Abstract: Elevated levels of the oncoprotein, osteopontin (OPN), are associated with poor outcome of several types of cancers including melanoma. We have previously reported an important involvement of DNAJB6, a member of heat-shock protein 40 (HSP40) family, in negatively impacting tumor growth. The current study was prompted by our observations reported here which revealed a reciprocal relationship between DNAJB6 and OPN in melanoma specimens. The 'J domain' is the most conserved domain of HSP40 family of proteins. Hence, we assessed the functional role of the J domain in activities of DNAJB6. We report that the J domain of DNAJB6 is involved in mediating OPN suppression. Deletion of the J domain renders DNAJB6 incapable of impeding malignancy and suppressing OPN. Our mechanistic investigations reveal that DNAJB6 binds HSPA8 (heat-shock cognate protein, HSC70) and causes dephosphorylation of glycogen synthase kinase 3β (GSK3β) at Ser 9 by recruiting protein phosphatase, PP2A. This dephosphorylation activates GSK3β, leading to degradation of β-catenin and subsequent loss of TCF/LEF (T cell factor1/lymphoid enhancer factor1) activity. Deletion of the J domain abrogates assembly of this multiprotein complex and renders GSK3β inactive, thus, stabilizing β-catenin, a transcription co-activator for OPN expression. Our in-vitro and in-vivo functional analyses show that silencing OPN expression in the background of deletion of the J domain renders the resultant tumor cells less malignant despite the presence of stabilized β-catenin. Thus, we have uncovered a new mechanism for regulation of GSK3β activity leading to inhibition of Wnt/β-catenin signaling.Oncogene advance online publication, 23 January 2012; doi:10.1038/onc.2011.623.

Abstract: Dickkopf1 (DKK1), a secreted inhibitor of the Wnt/β-catenin pathway, is a negative regulator of bone formation. DKK1 acts as a switch that transitions prostate cancer bone metastases from osteolytic to osteoblastic and also is an active indicator of poor outcome for multiple myeloma. However, in other tumor types, DKK1 upregulation or overexpression suppresses tumor growth. Thus, the role of DKK1 in cancer appears to be diverse. This raises a question: Could the increased levels of DKK1 still be tumor protective when observed in high levels in the serum of patients? Here, we summarize the diverse, seemingly contradicting roles of DKK1 and attempt to explain the apparent dichotomy in its activity. We propose that DKK1 is a critical secreted factor that modulates microenvironment. Based on the location and components of the microenvironment DKK1 will support different outcomes.

Abstract: ABSTRACT: The metastasis suppressor, BRMS1, has been demonstrated to cause dramatic regression of metastatic lesions without blocking orthotopic tumor growth. The role of BRMS1 is well-documented for several non-melanoma malignancies such as breast cancer, ovarian cancer and non-small cell lung cancer. However, its role in melanoma is just beginning to be understood, with a recent article by Slipicevic et al. highlighting the levels of expression of BRMS1 in benign nevi, primary and metastatic melanoma samples. Their findings emphasize that the intracellular location of BRMS1 protein (cytoplasmic or nuclear), appears to have a significant impact upon the metastatic capacity of melanoma cells. Interestingly, this selective localization translates into a statistically significant decrease in the relapse-free period in melanoma patients, further associated with a thicker Breslow's depth of primary melanomas. However, and more importantly, this study begins to define a clearer role for BRMS1 in melanoma that is strictly dependent upon its cellular location, with nuclear expression associated with invasive and metastatic capacity and cytoplasmic expression resulting in repressive effects upon progression and metastasis. Please see related article: http://www.biomedcentral.com/1471-2407/12/73.

Abstract: The remodeling process in bone yields numerous cytokines and chemokines that mediate crosstalk between osteoblasts and osteoclasts and also serve to attract and support metastatic tumor cells. The metastatic tumor cells disturb the equilibrium in bone that manifests as skeletal complications. The Hedgehog (Hh) pathway plays an important role in skeletogenesis. We hypothesized that the Hh pathway mediates an interaction between tumor cells and osteoblasts and influences osteoblast differentiation in response to tumor cells. We have determined that breast tumor cells have an activated Hh pathway characterized by upregulation of the ligand, IHH and transcription factor GLI1. Breast cancer cells interact with osteoblasts and cause an enhanced differentiation of pre-osteoblasts to osteoblasts that express increased levels of the osteoclastogenesis factors, RANKL and PTHrP. There is sustained expression of osteoclast-promoting factors, RANKL and PTHrP, even after the osteoblast differentiation ceases and apoptosis sets in. Moreover, tumor cells that are deficient in Hh signaling are compromised in their ability to induce osteoblast differentiation and consequently are inefficient in causing osteolysis. The stimulation of osteoblast differentiation sets the stage for osteoclast differentiation and overall promotes osteolysis. Thus, in the process of developing newer therapeutic strategies against breast cancer metastasis to bone it would worthwhile to keep in mind the role of the Hh pathway in osteoblast differentiation in an otherwise predominant osteolytic phenomenon.

Abstract: DNAJB6 is a constitutively expressed member of the HSP40 family. It has been described as a negative regulator of breast tumor progression and a regulator of epithelial phenotype. Expression of DNAJB6 is reported to be compromised with tumor progression. However, factors responsible for its downregulation are still undefined. We used a knowledge-based screen for identifying miRNAs capable of targeting DNAJB6. In this work, we present our findings that hsa-miR-632 (miR-632) targets the coding region of DNAJB6. Invasive and metastatic breast cancer cells express high levels of miR-632 compared with mammary epithelial cells. Analysis of RNA from breast tumor specimens reveals inverse expression patterns of DNAJB6 transcript and miR-632. In response to exogenous miR-632 expression, DNAJB6 protein levels are downregulated and the resultant cell population shows significantly increased invasive ability. Silencing endogenous miR-632 abrogates invasive ability of breast cancer cells and promotes epithelial like characteristics noted by E-cadherin expression with concomitant decrease in mesenchymal markers such as Zeb2 and Slug. Thus, miR-632 is a potentially important epigenetic regulator of DNAJB6, which contributes to the downregulation of DNAJB6 and plays a supportive role in malignant progression.

Abstract: The expression of Nuclear Protein 1 (NUPR1) is associated with chemoresistance in multiple malignancies. We previously reported that NUPR1 functions as a transcriptional cofactor for the p300-p53 complex and transcriptionally regulates p21 expression. In the present study we investigated the activity of NUPR1 in p53-deficient, triple-negative, inflammatory SUM159 breast cancer cells. Our studies reveal that NUPR1 confers growth benefit and chemoresistance by causing Akt-mediated phosphorylation and subsequent cytoplasmic re-localization of p21 and activation of the anti-apoptotic Bcl-xL protein. Our findings elucidate a NUPR1-PI-3-K/Akt-phospho-p21 axis that functions in p53-negative, inflammatory breast cancer cells to enhance chemoresistance in breast cancer.

Abstract: HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntington's, Parkinson's diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S).

Abstract: DKK1 (dickkopf 1 homologue) is a secreted inhibitor of the Wnt signalling pathway and a critical modulator of tumour promotion and the tumour microenvironment. However, mechanisms regulating DKK1 expression are understudied. DNAJB6 {DnaJ [HSP40 (heat-shock protein 40 kDa)] homologue, subfamily B, member 6} is an HSP40 family member whose expression is compromised during progression of breast cancer and melanoma. Inhibition of the Wnt/β-catenin signalling pathway by up-regulation of DKK1 is one of the key mechanisms by which DNAJB6 suppresses tumour metastasis and EMT (epithelial-mesenchymal transition). Analysis of the DKK1 promoter to define the cis-site responsible for its up-regulation by DNAJB6 revealed the presence of two binding sites for a transcriptional repressor, MSX1 (muscle segment homeobox 1). Our investigations showed that MSX1 binds the DKK1 promoter and inhibits DKK1 transcription. Interestingly, silencing DNAJB6 resulted in up-regulation of MSX1 concomitant with increased stabilization of β-catenin. ChIP (chromatin immunoprecipitation) studies revealed that β-catenin binds the MSX1 promoter and stabilization of β-catenin elevates MSX1 transcription, indicating that β-catenin works as a transcription co-activator for MSX1. Functionally, exogenous expression of MSX1 in DNAJB6-expressing cells promotes the mesenchymal phenotype by suppression of DKK1. Thus we have identified a novel regulatory mechanism of DNAJB6-mediated DKK1 transcriptional up-regulation that can influence EMT. DKK1 is a feedback regulator of β-catenin levels and thus our studies also define an additional negative control of this β-catenin/DKK1 feedback loop by MSX1, which may potentially contribute to excessive stabilization of β-catenin.

Abstract:

Abstract: Bone integrity is maintained by a dynamic equilibrium between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. Osteolytic lesions are a painful consequence of metastasis of breast cancer cells to bone in an overwhelming majority of breast cancer patients. Factors secreted by breast cancer cells propel a cascade of events that trigger osteoclastogenesis and elevated bone resorption. In the present study, we show that the Hedgehog (Hh) ligands secreted by breast cancer cells promote osteoclast differentiation and potentiate the activity of mature osteoclasts. Paracrine Hh signaling induced by breast cancer cells mediates a detrimental chain of events by the up-regulation of osteopontin (OPN), which in turn enhances osteoclastic activity by up-regulating cathepsin K and MMP9. Hh signaling is essential for osteoclasts because blocking the Hh pathway using the pharmacological Hh inhibitor, cyclopamine, results in an overall decrease in osteoclastogenesis and resorptive activity. Our studies suggest that inhibiting Hh signaling interferes with the ability of pre-osteoclasts to respond to the stimulatory effects of the breast cancer cells, indicating that Hh signaling is vital to osteoclast activity.

Abstract: Since angiogenesis is critical for tumor growth and metastasis, anti-angiogenic treatment is a highly promising therapeutic approach. Thus, for over last couple of decades, there has been a robust activity aimed towards the discovery of angiogenesis inhibitors. More than forty anti-angiogenic drugs are being tested in clinical trials all over the world. This review discusses agents that have approved by the FDA and are currently in use for treating patients either as single-agents or in combination with other chemotherapeutic agents.

Abstract: In addition to its role in embryonic development, the Hedgehog pathway has been shown to be an active participant in cancer development, progression, and metastasis. Although this pathway is activated by autocrine signaling by Hedgehog ligands, it can also initiate paracrine signaling with cells in the microenvironment. This creates a network of Hedgehog signaling that determines the malignant behavior of the tumor cells. As a result of paracrine signal transmission, the effects of Hedgehog signaling most profoundly influence the stromal cells that constitute the tumor microenvironment. The stromal cells in turn produce factors that nurture the tumor. Thus, such a resonating cross-talk can amplify Hedgehog signaling, resulting in molecular chatter that overall promotes tumor progression. Inhibitors of Hedgehog signaling have been the subject of intense research. Several of these inhibitors are currently being evaluated in clinical trials. Here, we review the role of the Hedgehog pathway in the signature characteristics of cancer cells that determine tumor development, progression, and metastasis. This review condenses the latest findings on the signaling pathways that are activated and/or regulated by molecules generated from Hedgehog signaling in cancer and cites promising clinical interventions. Finally, we discuss future directions for identifying the appropriate patients for therapy, developing reliable markers of efficacy of treatment, and combating resistance to Hedgehog pathway inhibitors.

Abstract: The Hedgehog (Hh) pathway is well known for its involvement in angiogenesis and vasculogenesis during ontogeny. The ligand, Sonic Hh (SHH), has an important role in vascular formation during development. However, SHH expression is upregulated on tumor cells and can impact the tumor microenvironment. We have investigated the effects of autocrine as well as paracrine Hh signaling on tumor cells as well as on endothelial cells, respectively. Upon constitutive expression of SHH, breast cancer cells showed aggressive behavior and rapid xenograft growth characterized by highly angiogenic tumors that were spontaneously metastatic. In these cells, SHH caused activation of the Hh transcription factor, GLI1, leading to upregulated expression of the potent pro-angiogenic secreted molecule, CYR61 (cysteine-rich angiogenic inducer 61). Silencing of CYR61 from these SHH-expressing Hh activated cells blunted the malignant behavior of the tumor cells and resulted in reduced tumor vasculature and limited hematogenous metastases. Thus, CYR61 is a critical downstream contributor to the Hh influenced pro-angiogenic tumor microenvironment. We also observed concomitant upregulation of SHH and CYR61 transcripts in tumors from patients with advanced breast cancer, further ratifying the clinical relevance of our findings. In summary, we have defined a novel, VEGF-independent, clinically relevant, pro-angiogenic factor, CYR61, that is a transcriptional target of Hh-GLI signaling.Oncogene advance online publication, 7 November 2011; doi:10.1038/onc.2011.496.

Abstract: Unlike malignancies of the nervous system, there have been no mutations identified in Merlin in breast cancer. As such, the role of the tumor suppressor, Merlin, has not been investigated in breast cancer. We assessed Merlin expression in breast cancer tissues by immunohistochemistry and by real-time PCR. The expression of Merlin protein (assessed immunohistochemically) was significantly decreased in breast cancer tissues (although the transcript levels were comparable) simultaneous with increased expression of the tumor-promoting protein, osteopontin (OPN). We further demonstrate that the loss of Merlin in breast cancer is brought about, in part, due to OPN-initiated Akt-mediated phosphorylation of Merlin leading to its proteasomal degradation. Restoring expression of Merlin resulted in reduced malignant attributes of breast cancer, characterized by reduced invasion, migration, motility, and impeded tumor (xenograft) growth in immunocompromised mice. The possibility of developing a model using the relationship between OPN and Merlin was tested with a logistic regression model applied to immunohistochemistry data. This identified consistent loss of immunohistochemical expression of Merlin in breast tumor tissues. Thus, we demonstrate for the first time a role for Merlin in impeding breast malignancy, identify a novel mechanism for the loss of Merlin protein in breast cancer, and have developed a discriminatory model using Merlin and OPN expression in breast tumor tissues.

Abstract: Utilizing gene microarray profiling of melanoma samples, we have recently identified a novel gene overexpressed in both thick primary and metastatic melanomas. This gene, progestagen-associated endometrial protein (PAEP), has never before been implicated in the oncogenic processes of melanoma, with its true function in oncogenesis and tumour progression relatively unknown. Overexpression of the PAEP gene in freshly procured thick primary and metastatic melanoma samples (58%) and daughter cell lines (77%) is confirmed by quantitative RT-PCR, immunohistochemistry, Western blotting and mass spectrometric analysis. We suggest that PAEP gene overexpression is involved with melanoma tumour progression as well as an aggressive phenotype. Transfection of melanoma cells with PAEP small interfering RNA (siRNA) reveals a significant decrease in soft agar colony formation and a marked inhibition of both cell migration and cell invasion. Furthermore, we establish stable melanoma transfectants via PAEP lentiviral small hairpin RNA (shRNA), examine their growth characteristics in a murine xenograft model and reveal that tumour growth is significantly inhibited in two separate melanoma cell lines. Our data strongly implicate the PAEP gene as a tumour growth promoter with oncogenic properties and a potential therapeutic target for patients with advanced melanoma.

Abstract: Breast cancer metastasis suppressor 1 (BRMS1) has been shown to functionally reduce the metastatic potential of melanoma. We also previously reported that BRMS1 negatively regulates the expression of the oncoprotein osteopontin (OPN). This study was carried out to assess the clinical relevance of BRMS1 and OPN in melanoma.

Abstract: Osteopontin (OPN) is a matricellular protein that is produced by multiple tissues in our body and is most abundant in bone. It is also produced by cancer cells and plays a determinative role in the growth, progression and metastasis of cancer. Clinically, OPN has been reported to be upregulated in tumor cells per se; this is also reflected by increased levels of OPN in the circulation. Thus, increased OPN levels the plasma are an effect of tumor growth and progression. Functionally, high OPN levels are determinative of higher incidence of bone metastases in mouse models and are clinically correlated with metastatic bone disease and bone resorption in advanced breast cancer patients. Several research efforts have been made to therapeutically target and inhibit the activities of OPN. In this article we have reviewed OPN in its role as an effector of critical steps in tumor progression and metastasis, with a particular emphasis on its role in facilitating bone metastasis of breast cancer. We have also addressed the role of the host-derived OPN in influencing the malignant behavior of the tumor cells.

Abstract: We showed that expression of MRJ (DNAJB6) protein is lost in invasive ductal carcinoma, and restoration of MRJ(L) restricts malignant behavior of breast cancer and melanoma cells. However, the signaling pathways influenced by MRJ(L) are largely unknown. Our observations revealed that MRJ(L) expression causes changes in cell morphology concomitant with down-regulation of several mesenchymal markers, viz. vimentin, N-cadherin, Twist, and Slug, and up-regulation of epithelial marker keratin 18. Importantly, MRJ(L) expression led to reduced levels of beta-catenin, an epithelial mesenchymal transition marker, and a critical player in the Wnt pathway. We found that MRJ(L) up-regulates expression of DKK1, a well known Wnt/beta-catenin signaling inhibitor, that causes degradation of beta-catenin. Re-expression of DNAJB6 alters the Wnt/beta-catenin signaling in cancer cells, leading to partial reversal of the mesenchymal phenotype. Thus, MRJ(L) may play a role in maintaining an epithelial phenotype, and inhibition of the Wnt/beta-catenin pathway may be one of the potential mechanisms contributing to the restriction of malignant behavior by MRJ(L).

Abstract: The growth of cancer cells as multicellular spheroids has frequently been reported to mimic the in vivo tumour architecture and physiology and has been utilized to study antitumour drugs. In order to determine the distinctive characteristics of the spheroid-derived cells compared to the corresponding monolayer-derived cells, we enriched multicellular spheroid-forming subpopulations of cells from three human breast cancer cell lines (MCF7, MCF10AT and MCF10DCIS.com). These spheroid-derived cells were injected into female athymic nude mice to assess their tumorigenic potential and were profiled for their characteristic miRNA signature. We discovered that the spheroid-derived cells expressed increased levels of osteopontin (OPN), an oncogenic protein that has been clinically correlated with increased tumour burden and adverse prognosis in patients with breast cancer metastasis. Our studies further show that increased OPN levels are brought about in part, by decreased levels of hsa-mir-299-5p in the spheroid-forming population from all three cell lines. Moreover, the spheroid-forming cells can organize into vascular structures in response to nutritional limitation; these structures recapitulate a vascular phenotype by the expression of endothelial markers CD31, Angiopoeitin-1 and Endoglin. In this study, we have validated that hsa-mir-299-5p targets OPN; de novo expression of OPN in turn plays a critical role in enhancing proliferation, tumorigenicity and the ability to display vasculogenic mimicry of the spheroid-forming cells.

Abstract: The exact cellular and molecular mechanisms involved in melanoma tumorigenesis remain obscure. Previous gene expression profiling analyses performed upon NHEM and human melanoma samples identified WFDC1 as one of the most frequently down-regulated genes. Here we further showed that NHEM readily express WFDC1 but expression is reduced or completely lost in 80% of the patients-derived melanoma cell lines and tissue samples examined. Furthermore, we show that promoter hypermethylation accounts for the silencing of the WFDC1 gene in 20% of the melanoma cell lines examined. The over-expression of WFDC1 in two metastatic melanoma cell lines, A375 and LOX, resulted in a significant delay of tumor growth in a murine xenograft model, despite a non-significant difference in tumor cell growth in vitro. Gene expression microarray analysis and further expression validation suggests that the Dickkopf-1 (Dkk1) gene is up-regulated in WFDC1 over-expressing cell lines, suggesting that the tumor suppressive function of WFDC1 may be partially a result of up-regulated Dkk1 gene expression, which is known to be a potent inhibitor of the Wnt signaling pathway.

Abstract: HSP40 (DNAJ) is an understudied family of co-chaperones. The human genome codes for over 41 members of HSP40 family that reside at distinct intracellular locations. Despite their large numbers, little is known about their physiologic roles. Recent research has revealed involvement of some of the DNAJ family members in various types of cancers. In this article we summarize the information about the involvement of human DNAJ family members in various aspects of cancer biology. Furthermore we discuss the potential role of the J domain of DNAJ proteins in cancer biology.

Abstract: Osteopontin (OPN) is a matricellular protein that is produced by multiple tissues in our body and is most abundant in bone. It is also produced by cancer cells and plays a determinative role in the growth, progression and metastasis of cancer. Clinically, OPN has been reported to be upregulated in tumor cells per se; this is also reflected by increased levels of OPN in the circulation. Thus, increased OPN levels the plasma are an effect of tumor growth and progression. Functionally, high OPN levels are determinative of higher incidence of bone metastases in mouse models and are clinically correlated with metastatic bone disease and bone resorption in advanced breast cancer patients. Several research efforts have been made to therapeutically target and inhibit the activities of OPN. In this article we have reviewed OPN in its role as an effector of critical steps in tumor progression and metastasis, with a particular emphasis on its role in facilitating bone metastasis of breast cancer. We have also addressed the role of the host-derived OPN in influencing the malignant behavior of the tumor cells.

Abstract: We found that the expression levels of N-Myc interactor (Nmi) were low in aggressive breast cancer cell lines when compared with less aggressive cell lines. However, the lower levels in the aggressive lines were inducible by interferon-gamma (IFN-gamma). Because Nmi has been reported to be a transcription cofactor that augments IFN-gamma induced transcription activity, we decided to test whether Nmi regulates expression of Dkk1, which is also inducible by IFN-gamma. We established stable clones constitutively expressing Nmi in MDA-MB-231 (breast) and MDA-MB-435 (melanoma) cell lines. Dkk1 was significantly up-regulated in the Nmi expressing clones concurrent with reduced levels of the critical transcription cofactor of Wnt pathway, beta-catenin. Treatment of the Nmi expressors with blocking antibody to Dkk1 restored beta-catenin protein levels. c-Myc is a known downstream target of activated beta-catenin signaling. Treatment of Nmi expressors with the proteosome inhibitor MG132, resulted in elevated beta-catenin levels with concomitant elevation of c-Myc levels. Our functional studies showed that constitutive expression of Nmi reduced the ability of tumor cells for the invasion, anchorage independent growth and tumor growth in vivo. Collectively, the data suggest that overexpression of Nmi inhibits the Wnt/beta-catenin signaling via up-regulation of Dkk1 and retards tumor growth.

Abstract: The role of Hedgehog (Hh) signaling as a developmental pathway is well established. Several recent studies have implicated a role for this pathway in multiple cancers. In this study we report that expression of GLI1 and osteopontin (OPN) increase progressively with the progression of melanoma from primary cutaneous cancer to metastatic melanoma in clinically derived specimens. We have further determined that OPN is a direct transcriptional target of GLI1. We have observed that OPN expression is stimulated in the presence of Hh ligands and inhibited in the presence of the Smoothened (SMO) inhibitor, cyclopamine. Transcriptional silencing of GLI1 negatively impacts OPN expression and compromises the ability of cancer cells to proliferate, migrate, and invade in vitro and interferes with their ability to grow as xenografts and spontaneously metastasize in nude mice. These altered attributes could be rescued by re-expressing OPN in the GLI1-silenced cells, suggesting that OPN is a critical downstream effector of active GLI1 signaling. Our observations lead us to conclude that the GLI1-mediated up-regulation of OPN promotes malignant behavior of cancer cells.

Abstract: NUPR1, or p8 or com1, was first identified from rat pancreas during acute pancreatitis and later as a gene whose expression was upregulated in metastatic breast cancer cells. NUPR1 is a molecule whose expression is upregulated in response to stress and is hence influenced by the host microenvironment. While NUPR1 has been implicated in several diseases, there is no singular biochemical pathway that can be attributed to its role in cancer. NUPR1 has been found to aid the establishment of metastasis and to play a key role in the progression of several malignancies including those of breast, thyroid, brain and pancreas. NUPR1 has been implicated in inducing chemoresistance in pancreatic and breast cancer cells, protecting them from apoptosis and making tumor cells genetically unstable. In prostate cancer, however, NUPR1 appears to have tumor suppressive activity. Understanding the mechanism of action of the multifaceted functions of NUPR1 may open up new dimensions towards creating novel therapies against cancer as well as other pathologies. This review draws on several published studies on NUPR1, mainly in cancer biology, and assesses NUPR1 from the perspective of its functional role in making cancer cells resistant to the action of conventional chemotherapeutic drugs.

Abstract: Ser/Thr protein phosphatase 5 (PP5) regulates several signaling-cascades that suppress growth and/or facilitate apoptosis in response to genomic stress. The expression of PP5 is responsive to hypoxia inducible factor-1 (HIF-1) and estrogen, which have both been linked to the progression of human breast cancer. Still, it is not clear if PP5 plays a role in the development of human cancer. Here, immunostaining of breast cancer tissue-microarrays (TMAs) revealed a positive correlation between PP5 over-expression and ductal carcinoma in situ (DCIS; P value 0.0028), invasive ductal carcinoma (IDC; P value 0.012) and IDC with metastases at the time of diagnosis (P value 0.0001). In a mouse xenograft model, the constitutive over-expression of PP5 was associated with an increase in the rate of tumor growth. In a MCF-7 cell culture model over-expression correlated with both an increase in the rate of proliferation and protection from cell death induced by oxidative stress, UVC-irradiation, adriamycin, and vinblastine. PP5 over-expression had no apparent effect on the sensitivity of MCF-7 cells to taxol or rapamycin. Western analysis of extracts from cells over-expressing PP5 revealed a decrease in the phosphorylation of known substrates for PP5. Together, these studies indicate that elevated levels of PP5 protein occur in human breast cancer and suggest that PP5 over-expression may aid tumor progression.

Abstract: The BRMS1 metastasis suppressor interacts with the protein AT-rich interactive domain 4A (ARID4A, RBBP1) as part of SIN3.histone deacetylase chromatin remodeling complexes. These transcriptional co-repressors regulate diverse cell phenotypes depending upon complex composition. To define BRMS1 complexes and their roles in metastasis suppression, we generated BRMS1 mutants (BRMS1(mut)) and mapped ARID4A interactions. BRMS1(L174D) disrupted direct interaction with ARID4A in yeast two-hybrid genetic screens but retained an indirect association with ARID4A in MDA-MB-231 and -435 human breast cancer cell lines by co-immunoprecipitation. Deletion of the first coiled-coil domain (BRMS1(DeltaCC1)) did not disrupt direct interaction in yeast two-hybrid screens but did prevent association by co-immunoprecipitation. These results suggest altered complex composition with BRMS1(mut). Although basal transcription repression was impaired and the pro-metastatic protein osteopontin was differentially down-regulated by BRMS1(L174D) and BRMS1(DeltaCC1), both down-regulated the epidermal growth factor receptor and suppressed metastasis in MDA-MB-231 and -435 breast cancer xenograft models. We conclude that BRMS1(mut), which modifies the composition of a SIN3.histone deacetylase chromatin remodeling complex, leads to altered gene expression profiles. Because metastasis requires the coordinate expression of multiple genes, down-regulation of at least one important gene, such as the epidermal growth factor receptor, had the ability to suppress metastasis. Understanding which interactions are necessary for particular biochemical/cellular functions may prove important for future strategies targeting metastasis.

Abstract: INTRODUCTION: Mammalian relative of DnaJ (MRJ [DNAJB6]), a novel member of the human DnaJ family, has two isoforms. The smaller isoform, MRJ(S), is studied mainly for its possible role in Huntington's disease. There are no reports of any biologic activity of the longer isoform, MRJ(L). We investigated whether this molecule plays any role in breast cancer. Our studies were prompted by interesting observations we made regarding the expression of MRJ in breast cancer cell lines and breast cancer tissue microarrays, as described below. METHODS: Expression of MRJ(L) from several breast cancer cell lines was evaluated using real-time PCR. Relative levels of the small and large isoforms in breast cancer cell lines were studied using Western blot analysis. A breast cancer progression tissue microarray was probed using anti-MRJ antibody. MRJ(L) was ectopically expressed in two breast cancer cell lines. These cell lines were evaluated for their in vitro correlates of tumor aggressiveness, such as invasion, migration, and anchorage independence. The cell lines were also evaluated for in vivo tumor growth and metastasis. The secreted proteome of the MRJ(L) expressors was analyzed to elucidate the biochemical changes brought about by re-expression of MRJ(L). RESULTS: We found that MRJ(L) is expressed at a significantly lower level in aggressive breast cancer cell lines compared with normal breast. Furthermore, in clinical cases of breast cancer expression of MRJ is lost as the grade of infiltrating ductal carcinoma advances. Importantly, MRJ staining is lost in those cases that also had lymph node metastasis. We report that MRJ(L) is a protein with a functional nuclear localization sequence. Expression of MRJ(L) via an exogenous promoter in breast cancer cell line MDA-MB-231 and in MDA-MB-435 (a cell line that metastasizes from the mammary fat pad) decreases their migration and invasion, reduces their motility, and significantly reduces orthotopic tumor growth in nude mice. Moreover, the secreted proteome of the MRJ(L)-expressing cells exhibited reduced levels of tumor progression and metastasis promoting secreted proteins, such as SPP1 (osteopontin), AZGP1 (zinc binding alpha2-glycoprotein 1), SPARC (osteonectin), NPM1 (nucleophosmin) and VGF (VGF nerve growth factor inducible). On the other hand, levels of the secreted metastasis-suppressor KiSS1 (melanoma metastasis suppressor) were increased in the secreted proteome of the MRJ(L)-expressing cells. We confirmed by quantitative RT-PCR analysis that the secreted profile reflected altered transcription of the respective genes. CONCLUSION: Collectively, our data indicate an important role for a totally uncharacterized isoform of DNAJB6 in breast cancer. We show that MRJ(L) is a nuclear protein that is lost in breast cancer, that regulates several key players in tumor formation and metastasis, and that is functionally able to retard tumor growth.

Abstract: Various in vitro and in vivo experimental models have been used for the discovery of genes and pathways involved in melanoma and other types of cancer. However, in many cases, the results from various tumor models failed to be validated successfully in clinical studies. Limited information is available on how closely these models reflect the in vivo physiological conditions. In this study, a comprehensive genomics approach was used to systematically compare the expression patterns of snap frozen samples obtained from patients with primary melanoma, lymph node metastasis, and distant metastases, and compare these patterns to those of their corresponding cell lines and tumor xenografts in nude mice. The GE Healthcare 20k human genome array was used and the expression data was normalized and analyzed using GeneSpring 7.2 software. Based on the expression analysis, the correlation rate between the snap frozen primary patient samples vs. derived cell lines was 66%, with 1687 differentially expressed genes. The correlation rate between the snap frozen primary patient samples and the tumor xenografts was 75%, with 1,374 differentially expressed genes, and the correlation rate comparing tumor xenografts to derived cell lines ranged between 58% and 84%. These results demonstrated significant gene expression differences between tumor materials with different in vitro and in vivo growth microenvironments. Such studies can help us to distinguish between genes up- or down-regulated as a result of the microenvironment and those stably expressed independently of the tumor milieu. With the extensive use of cell lines and xenografts in cancer research, the information obtained using our approach may help to better interpret results generated from different tumor models by understanding common differences, as well as similarities at the gene expression level, information that may have important practical and biological implications.

Abstract: Nuclear protein 1 (NUPR1/com1/p8) has been shown to interact with transcriptional regulators such as p300, PTIP, estrogen receptor-beta, and SMAD. NUPR1 also has been implicated in the regulation of cell cycle and apoptosis. An increase in NUPR1 expression has been seen with serum starvation and in response to compounds such as cycloheximide, ceramide, and staurosporine. There are several overtly conflicting reports about the exact role of NUPR1 in tumor biology. This work investigates the nature of the relationship between NUPR1 and the cdk-inhibitor p21 (Waf1/Cip1) expression. We show that the expression of resident and doxorubicin-induced p21 paralleled that of endogenous NUPR1 levels. NUPR1 formed a complex with p53 and p300 and bound the p21 promoter and transcriptionally upregulated p21 expression. Moreover, NUPR1 allowed cells to progress through cell cycle in presence of doxorubicin. Since NUPR1 upregulated p21, concomitant with phosphorylation of Rb and upregulation of the anti-apoptotic protein, Bcl-x(L) we propose that NUPR1 expression imparts a cell growth and survival advantage. Importantly, we also report that NUPR1 conferred resistance to two chemotherapeutic drugs, Taxol and doxorubicin.

Abstract: Breast Cancer Metastasis Suppressor 1 (BRMS1) suppresses metastasis of human breast cancer, ovarian cancer and melanoma in athymic mice. Studies have also shown that BRMS1 is significantly downregulated in some breast tumors, especially in metastatic disease. However, the mechanisms which regulate BRMS1 expression are currently unknown. Upon examination of the BRMS1 promoter region by methylation specific PCR (MSP) analysis, we discovered a CpG island (-3477 to -2214), which was found to be hypermethylated across breast cancer cell lines. A panel of 20 patient samples analyzed showed that 45% of the primary tumors and 60% of the matched lymph node metastases, displayed hypermethylation of BRMS1 promoter. Furthermore, we found a direct correlation between the methylation status of the BRMS1 promoter in the DNA isolated from tissues, with the loss of BRMS1 expression assessed by immunohistochemistry. There are several studies investigating the mechanism by which BRMS1 suppresses metastasis; however thus far there is no study that reports the cause(s) of loss of BRMS1 expression in aggressive breast cancer. Here we report for the first time that BRMS1 is a novel target of epigenetic silencing; and aberrant methylation in the BRMS1 promoter may serve as a cause of loss of its expression.

Abstract: BACKGROUND: The process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples. METHODS: We have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes. RESULTS: We find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including SPRR1A/B, KRT16/17, CD24, LOR, GATA3, MUC15, and TMPRSS4, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as MAGE, GPR19, BCL2A1, MMP14, SOX5, BUB1, RGS20, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (SPP-1, MITF, CITED-1, GDF-15, c-Met, HOX loci) and suppressor genes (PITX-1, CST-6, PDGFRL, DSC-3, POU2F3, CLCA2, ST7L), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype. CONCLUSION: The gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches.

Abstract: Molecular mechanisms underlying the different capacity of two in vivo selected human melanoma cell variants to form experimental metastases were studied. The doubling times of the FEMX-I and FEMX-V cell sublines in vitro were 15 and 25 h, respectively. The invasive capacity of FEMX-I cells was 8-fold higher than FEMX-V cells, and the time to form approximately 10 mm s.c. tumors in nude mice was 21 versus 35 days. FEMX-I displayed a spindle-like formation in vitro, whereas FEMX-V cells had a rounded shape. Hence, we examined known determinants of cell shape and proliferation, the small GTPases. The four studied showed equal expression in both cell types, but Rac1 activity was significantly decreased in FEMX-V cells. Rac1 stimulates NFkappaB, and we found that endogenous NFkappaB activity of FEMX-V cells was 2% of that of FEMX-I cells. Inhibition of Rac1 resulted in blocked NFkappaB activity. Specific inhibition of either Rac1 or NFkappaB significantly reduced proliferation and invasion of FEMX-I cells, the more pronounced effects observed with Rac1 inhibition. These data indicate that Rac1 activity in FEMX cells regulates cell proliferation and invasion, in part via its effect on NFkappaB, signifying Rac1 as a key molecule in melanoma progression and metastasis.

Abstract: BACKGROUND: Osteopontin (OPN), a secreted phosphoglycoprotein, has been strongly associated with tumor progression and aggressive cancers. MDA-MB-435 cells secrete very high levels of OPN. However metastasis-suppressed MDA-MB-435 cells, which were transfected with breast cancer metastasis suppressor 1 (BRMS1), expressed significantly less OPN. BRMS1 is a member of mSin3-HDAC transcription co-repressor complex and has been shown to suppress the metastasis of breast cancer and melanoma cells in animal models. Hence we hypothesized that BRMS1 regulates OPN expression. RESULTS: The search for a BRMS1-regulated site on the OPN promoter, using luciferase reporter assays of the promoter deletions, identified a novel NF-kappaB site (OPN/NF-kappaB). Electrophoretic mobility shift assays and chromatin immunoprecipitations (ChIP) confirmed this site to be an NF-kappaB-binding site. We also show a role of HDAC3 in suppression of OPN via OPN/NF-kappaB. CONCLUSION: Our results show that BRMS1 regulates OPN transcription by abrogating NF-kappaB activation. Thus, we identify OPN, a tumor-metastasis activator, as a crucial downstream target of BRMS1. Suppression of OPN may be one of the possible underlying mechanisms of BRMS1-dependent suppression of tumor metastasis.

Abstract: The murine ortholog (Brms1) of human breast cancer metastasis suppressor 1 shares 95% identity to the human metastasis suppressor, BRMS1, in amino acid structure. We tested Brms1 for suppression of metastasis of mouse mammary carcinoma cell line 4T1 in syngenic BALB/c mice, using orthotopic (mammary fat pad) injection as well as intravenous injection. As observed for BRMS1, transfection with Brms1 did not inhibit 4T1 primary tumor formation, but significantly suppressed lung colonization. We also show that Brms1 protein interacts with histone deacetylases, indicating involvement of Brms1 in murine Sin3-HDAC complex, like its human counterpart. Thus, because of similarities with its human ortholog, the results suggest that Brms1 will be useful as a model for studying mechanism of action of BRMS1.

Abstract: Breast cancer metastasis suppressor 1 (BRMS1) is a member of the mSin3-HDAC transcription co-repressor complex. However, the proteins associated with BRMS1 have not been fully identified. Yeast two-hybrid screen, immuno-affinity chromatography, and co-immunoprecipitation experiments were performed to identify BRMS1 interacting proteins (BIPs). In addition to known core mSin3 transcriptional complex components RBBP1 and mSDS3, BRMS1 interacted with other proteins including three chaperones: DNAJB6 (MRJ), Hsp90, and Hsp70. Hsp90 is a known target of HDAC6 and reversible acetylation is one of the mechanisms that is implicated in regulation of Hsp90 chaperone complex activity. BRMS1 interacted with class II HDACs, HDAC 4, 5, and 6. We further found that BRMS1 is stabilized by Hsp90, and its turnover is proteasome dependent. The stability of BRMS1 protein may be important in maintaining the functional role of BRMS1 in metastasis suppression.

Abstract: Elevated expression of osteopontin (OPN), a secreted phosphoglycoprotein, is frequently associated with many transformed cell lines. Various studies suggest that OPN may contribute to tumor progression as well as metastasis in multiple tumor types. High levels of OPN have been reported in patients with metastatic cancers, including breast. We found that the expression of OPN corroborates with the aggressive phenotype of the breast cancer cells i.e. the expression of OPN is acquired as the breast cancer cells become more aggressive. To assess the role(s) of OPN in breast carcinoma, expression of endogenous OPN was knocked down in metastatic MDA-MB-435 human breast carcinoma cells using RNA interference. We targeted multiple regions of the OPN transcript for RNA interference, along with 'scrambled' and 'non-targeting siRNA pool' controls to distinguish between target-specific and potential off-target effects including interferon-response gene (PeIF2-alpha) induction. The OPN knockdown by shRNA suppressed tumor take in immunocompromised mice. The 'silenced' cells also showed significantly lower invasion and migration in modified Boyden chamber assays and reduced ability to grow in soft agar. Thus, in addition to the widely reported roles of OPN in late stages of tumor progression, these results provide functional evidence that OPN contributes to breast tumor growth as well.

Abstract: Several molecules that suppress metastasis without suppressing tumorigenicity have been identified, but their mechanisms of action have not yet been determined. Many block growth at the secondary site, suggesting involvement in how cells respond to signals from the extracellular milieu. Breast cancer metastasis suppressor 1 (BRMS1)-transfected MDA-MB-435 cells were examined for modifications of phosphoinositide signaling as a potential mechanism for metastasis suppression. 435/BRMS1 cells expressed <10% of phosphatidylinositol-4, 5-bisphosphate compared with parental cells, whereas levels of the PtdIns(4)P and phosphatidylinositol-3-phosphate were unchanged. Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] were decreased in 435/BRMS1 cells by approximately 50%. Phosphatidylinositol-3,4,5-trisphosphate levels were undetectable in 435/BRMS1 cells, even when stimulated by exogenous insulin or platelet-derived growth factor. Immunofluorescence microscopy to examine cellular distribution confirmed that phosphatidylinositol-4,5-bisphosphate distribution with cells was unchanged but was uniformly decreased throughout the cell. Although the gross morphology of 435/BRMS1 cells is similar to the parent, filamentous actin was more readily apparent in 435/BRMS1. Intracellular calcium, measured using Fluo-3 and Fura-2 fluorescent calcium indicator dyes, was somewhat lower, but not statistically different in 435/BRMS1 compared with parental cell. However, when stimulated with platelet-derived growth factor, MDA-MB-435 cells, but not 435/BRMS1 cells mobilized intracellular calcium. Taken together, these results implicate signaling through phosphoinositides in the regulation of breast cancer metastasis, specifically metastasis that can be suppressed by BRMS1.

Abstract: Our previous studies demonstrate that introduction of a approximately 70 cM region (now estimated at 63.75 Mb by the Human Genome Project) of human chromosome 12 into the highly metastatic Dunning rat prostate cancer cell line AT6.1 results in >30-fold (>/=90%) reduction in the number of overt metastases in spontaneous metastasis assays. We report the further localization and biological characterization of the metastasis-suppressor activity encoded by a reduced region of chromosome 12. To localize this metastasis-suppressor activity, a panel of AT6.1 microcell hybrids that retain varying portions of human chromosome 12 was constructed and subjected to sequence-tagged site (STS)-based PCR analysis and assessment of in vivo metastatic ability. Data from these complementary approaches localized the metastasis-suppressor activity to a approximately 4.2 Mb portion of human chromosome 12q24.3 comprised of 3 separate regions. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting were used for differential expression analyses to identify which characterized genes, predicted gene sequences and expressed sequence tags (ESTs) within this region could be responsible for the observed metastasis suppression. Comprehensive in vivo studies showed that suppressed AT6.1-12 hybrids that retain the metastasis-suppressor region on 12q24.3 are capable of arriving at the secondary site, but are not able to persist there. Thus, unlike other metastasis-suppressor genes characterized to date, the metastasis-suppressor gene encoded by this region appears to utilize a different biologic mechanism to suppress the growth of overt metastases at the secondary site.

Abstract: Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple human and murine cancer cells without inhibiting tumorigenicity. By yeast two-hybrid and co-immunoprecipitation, BRMS1 interacts with retinoblastoma binding protein 1 and at least seven members of the mSin3 histone deacetylase (HDAC) complex in human breast and melanoma cell lines. BRMS1 co-immunoprecipitates enzymatically active HDAC proteins and represses transcription when recruited to a Gal4 promoter in vivo. BRMS1 exists in large mSin3 complex(es) of approximately 1.4-1.9 MDa, but also forms smaller complexes with HDAC1. Deletion analyses show that the carboxyl-terminal 42 amino acids of BRMS1 are not critical for interaction with much of the mSin3 complex and that BRMS1 appears to have more than one binding point to the complex. These results further show that BRMS1 may participate in transcriptional regulation via interaction with the mSin3.HDAC complex and suggest a novel mechanism by which BRMS1 might suppress cancer metastasis.

Abstract: The breast cancer metastasis-suppressor gene BRMS1 is downregulated in metastatic breast cancer cells. Previous reports have shown restoration of gap junctional intercellular communication (GJIC) in the metastatic human breast carcinoma cell line MDA-MB-435 (435) transfected with BRMS1 cDNA. Metastasis, to a large extent in most breast cancers, occurs to bone. However, the reason for this preferential metastasis is not known. We explored cell-to-cell communication between 435 carcinoma cells and a human osteoblastic cell line, hFOB1.19, to determine whether carcinoma cells can form gap junctions with bone cells and to explore the role of these heterotypic gap junctions and the BRMS1 gene in breast cancer metastasis to bone. 435 cells displayed greater cell-to-cell communication with hFOB 1.19 cells than with themselves. Transfection of BRMS1 into 435 cells increased homotypic gap junctional communication but did not significantly affect heterotypic communication with hFOBs. However, heterotypic communication of BRMS1 transfectants with hFOB cells was reduced relative to homotypic communication. In contrast, parental 435 cells displayed greater heterotypic communication with hFOBs relative to homotypic communication. Our results suggest that there are differences in the relative homotypic and heterotypic GJIC of metastasis-capable and -suppressed cell lines.

Abstract: INTRODUCTION: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The alpha(v)beta3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the alpha(v)beta3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. MATERIALS AND METHODS: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded. RESULTS AND CONCLUSIONS: IC50 for S247 adhesion to alpha(v)beta3 or alpha(IIB)beta3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 microM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25-50% of vehicle-only-treated mice; P = 0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the alpha(v)beta3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that alpha(v)beta3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.

Abstract: BRMS1 (breast cancer metastasis suppressor 1) was recently identified as a novel breast cancer metastasis suppressor gene. To further characterize BRMS1-mediated metastasis suppression, we applied two-dimensional proteomic and mass spectrometry (LC-tandem MS and MALDI-TOF) analysis to identify proteins differentially expressed between highly metastatic MDA-MB-435 cells and metastasis-suppressed BRMS1-transfected MDA-MB-435 cells. Quadruplicate independent 2D gels were run and analyzed under identical conditions. Following in-gel trypsin digestion of seven differentially expressed proteins, amino acid sequence and mass profiles of the peptides were generated. Proteins were identified from the NCBI non-redundant database using the search program TurboSequest. Differential expression was confirmed for five proteins, including annexin I and alpha B-crystallin, by Northern blot analysis and immunostaining. Furthermore, we showed that both proteins were expressed in vivo in lungs containing metastasized MDA-MB-435 cells but not expressed in normal lung tissue of athymic mice. Our results suggest that annexin I and alpha B-crystallin are important cellular proteins that are down regulated through BRMS1 mediated metastasis suppression.

Abstract: We recently identified a novel metastasis suppressor gene, BRMS1, in breast cancer. Since the BRMS1 gene maps to chromosome 11q13.1-q13.2 and since chromosome 11q defects have been described in various stages of human melanoma progression, we hypothesized that BRMS1 may function as a tumor or metastasis suppressor in melanomas as well. Quantitative real-time RT-PCR revealed that BRMS1 mRNA expression was high in melanocytes, considerably reduced in early melanoma-derived cell lines, and barely detectable in advanced/metastatic cell lines. Stable transfectants of BRMS1 in the human melanoma cell lines MelJuSo and C8161.9 did not alter the tumorigenicity of either cell line, but significantly suppressed metastasis compared to vector-only transfectants. Orthotopic tumors continued to express BRMS1, but expression was lost in lung metastases. In vitro morphology, growth rate, and histology of BRMS1 transfectants were similar to controls. BRMS1 transfectants were less invasive in a collagen sandwich assay and had restored homotypic gap junctional intercellular communication (GJIC). Thus, BRMS1 functions as a metastasis suppressor in more than one tumor type (i.e., breast carcinoma and cutaneous melanoma) by modifying several metastasis-associated phenotypes.

Abstract: We have cloned a novel metastasis-suppressor gene (BRMS1) by differential display, comparing metastatic human breast carcinoma cell line MDA-MB-435 to its metastasis-suppressed human chromosome 11 microcell hybrid. Screening of a murine cDNA library led to the identification of a 1.4 kb cDNA with a sequence revealing 85% homology to human BRMS1 within the open reading frame. The predicted protein sequence for the murine ortholog is 95% identical, suggesting that it is strongly conserved across these 2 species. The cloned cDNA was used to screen a murine strain SV129 BAC library to obtain brms1 genomic DNA. Three BAC clones [226(I4), 226(H4) and 239(N7)] were confirmed to encode the entire brms1 gene. Detailed analysis of BAC clone 226(I4) shows that the gene spans 8.5 kb and, like the human gene, is organized into 10 exons and 9 introns. While the exons share a high degree of homology, there are greater differences when comparing intron structures between the human and murine genes. The 5' upstream region shares about 64% homology with its human counterpart, retaining several of the many putative regulatory elements. Like the human genomic BRMS1, the murine ortholog of the iGnT gene is found upstream of brms1 and the murine ortholog of the RIN1 gene is found downstream of brms1. brms1 was then tested for suppression of metastasis of mouse mammary carcinoma cell line 66cl4 in syngeneic BALB/c mice. Transfection with brms1 did not inhibit 66cl4 primary tumor formation but significantly suppressed its metastatic capability. This suggests that the murine ortholog functions similarly to BRMS1.

Abstract: Increased cellular activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) synthesis, is an independent adverse prognostic factor for overall survival in human breast cancer, thus suggesting an important role for PA in tumor progression. The experiments presented here were designed to investigate the role of PA in invasion and metastasis, using the highly aggressive MDA-MB-435 and MDA-MB-231 human breast cancer cell lines. Administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, significantly reduced, in a dose-dependent manner, the invasiveness in matrigel of both MDA-MB-435 and MDA-MB-231 cells by approximately 70%. DFMO treatment also inhibited (P < 0.0001) 'stellate' colony formation (an indicator of aggressive phenotype) by MDA-MB-435 cells plated in the matrigel outgrowth assay. Administration of DFMO (2% in drinking water) reduced the growth rate of both cell lines implanted orthotopically in nude mice. To evaluate metastasis while minimizing effects on proliferation, DFMO-treated mice were sacrificed later to allow their tumors to reach the same size of the tumors in the control mice. The most striking finding was that DFMO, while ineffective in reducing local invasion, nearly totally abolished (P = 0.0152) pulmonary metastasis in mice bearing MDA-MB-435 xenografts. These results support a role of PA in promoting breast cancer aggressiveness, particularly with regard to the development of distant metastasis. Furthermore, the data suggest that PA involvement is distal to local invasion in the metastatic cascade.

Abstract:

Abstract: Due to a lack of effective treatments, the development of metastases remains the most lethal aspect of prostate cancer. In order to help overcome this problem there has been an ongoing effort to develop strategies for early intervention. This includes the development of strategies that allow histologic lesions and disseminated cells that are highly likely to cause metastatic disease to be distinguished from those that are not, as well as therapeutic approaches to specifically target bone metastases. Such approaches will be expedited by the identification of genes and signaling cascades that regulate metastatic growth. Genes that specifically suppress metastasis are strong candidates for these studies. This review will focus on metastasis-suppressor genes that have been identified functionally, particularly those found to play a role in prostate cancer, and discuss how the identification and study of these genes has influenced our overall understanding of the metastatic process.

Abstract: Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.

Abstract: We previously showed that introduction of a normal, neomycin-tagged human chromosome 11 reduces the metastatic capacity of MDA-MB-435 (435) human breast carcinoma cells by 70-90% without affecting tumorigenicity, suggesting the presence of one or more metastasis suppressor genes encoded on human chromosome 11. To identify the gene(s) responsible, differential display comparing chromosome 11-containing (neo11/ 435) and parental, metastatic cells was done. We describe the isolation and functional characterization of a full-length cDNA for one of the novel genes, designated breast-cancer metastasis suppressor 1 (BRMS1), which maps to human chromosome 11q13.1-q13.2. Stably transfected MDA-MB-435 and MDA-MB-231 breast carcinoma cells still form progressively growing, locally invasive tumors when injected into mammary fat pads but are significantly less metastatic to lungs and regional lymph nodes. These data provide compelling functional evidence that breast-cancer metastasis suppressor 1 is a novel mediator of metastasis suppression in human breast carcinoma.