Transforming growth factor ␤ type II receptor (T␤RII) is a tumor suppressor gene that can be transcriptionally silenced by histone deacetylases (HDACs) in cancer cells. In this report, we demonstrated the mechanism by which trichostatin A (TSA), an inhibitor of HDAC, induces the expression of T␤RII in human pancreatic cancer cell lines by modulating the transcriptional components that bind a specific DNA region of the T␤RII promoter. This region of the T␤RII promoter possesses Sp1 and NF-Y binding sites in close proximity (located at ؊102 and ؊83, respectively). Treatment of cells with TSA activates the T␤RII promoter in a time-dependent manner through the recruitment of p300 and PCAF into a Sp1⅐NF-Y⅐HDAC complex that binds this DNA element. The recruitment of p300 and PCAF into the complex is associated with a concomitant acetylation of Sp1 and an overall decrease in the amount of HDAC associated with the complex. Transient overexpression of p300 or PCAF potentiated TSA-induced T␤RII promoter activity. The effect of PCAF was dependent on its histone acetyltransferase activity, whereas that of p300 was independent. Stable transfection of PCAF caused an increase in T␤RII mRNA expression, the association of PCAF with T␤RII promoter, and the acetylation of Sp1. Taken together, these results showed that TSA treatment of pancreatic cancer cells leads to transcriptional activation of the T␤RII promoter through modulation of the components of a Sp1⅐NF-Y⅐p300⅐PCAF⅐HDAC-1 multiprotein complex. Moreover, the interaction of NF-Y with the Sp1-associated complex may further explain why this specific Sp1 site mediates transcriptional responsiveness to TSA. TGF-␤1 plays a significant role in the growth inhibition of most normal epithelial and some cancer cells (1). TGF-␤ mediates its biological effects through cell surface receptors known as TGF-␤ type I receptor (T␤RI) and TGF-␤ type II receptor (T␤RII). Its intracellular signaling is initiated upon the selective binding of the active cytokine to the T␤RII homodimer. T␤RII is a ubiquitously expressed and constitutively active serine/threonine kinase. Ligand binding to T␤RII induces the assembly of a heterotetrameric complex consisting of T␤RI and T␤RII. Once the receptor complex is formed, T␤RII phosphorylates and thereby activates the T␤RI serine/threonine kinase. Activation of T␤RI propagates downstream signaling via Smad family proteins. T␤RI directly interacts with and phosphorylates Smad2 and Smad3. These Smads bind Smad4 and then result in the translocation of this complex to the nucleus and modulate TGF-␤-responsive gene expression (2-4).The TGF-␤ signaling pathway is inactivated in many tumors. Loss of negative growth regulation by TGF-␤ affords cells a selective growth advantage associated with decreased dependence of exogenous growth factor and increased tumorigenicity. Frequently, inhibition of TGF-␤ signaling occurs by either abolition of the function of a common mediator, Smad4, or interference with T␤RII function (5, 6). Smad4 and T␤RII are tumor suppressor gene...

Sp3 transcription factor can either activate or repress target gene expression. However, the molecular event that controls this dual function is unclear. We previously reported (Ammanamanchi, S., and Brattain, M. G.

. Northern analysis indicated that 5-aza-2-dC treatment did not affect the Sp1 transcript levels. Western blot analysis revealed an increase of Sp1 protein in the 5-aza-2-dCtreated MCF-7L cells, but there was no change in the c-Jun levels. Studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5-aza-2-dCtreated MCF-7L extracts compared with untreated control extracts. These results indicate that the transcriptional repression of RII in the ER ؉ breast cancer cells is caused by suboptimal activity of Sp1, whereas treatment with 5-aza-2-dC stabilizes the protein thus increasing steady-state Sp1 levels and thereby leads to enhanced RII transcription and subsequent restoration of TGF-␤ sensitivity.

. This report demonstrates that inappropriate overexpression of Sp3 is a mechanism that contributes to repression of TGF-␤ receptors. TGF-␤1 plays a vital role in the regulation of cell proliferation, differentiation, and extracellular matrix re-modeling in various cell types (1, 2). TGF-␤ carries out its biological effects through three cell surface receptors, which are referred to as type I (RI), type II (RII), and type III (RIII). RI and RII are serine/threonine kinases, and an active receptor complex consists of two molecules each of RI and RII, which are essential for TGF-␤ signal transduction and inhibition of cell growth (3-6).One of the crucial roles of TGF-␤ is the growth inhibition of normal epithelial cells as well as some cancer cells. Because RI and RII are necessary for TGF-␤-mediated growth arrest, mutational inactivation of either receptor has been reported to generate TGF-␤ resistance and hence the loss of the tumor suppressive function of TGF-␤ in cancer cells (7-9). DNA methylation of the RI promoter has been reported in a subset of gastric cancer cells (10). Another mechanism for loss of TGF-␤-mediated tumor suppression is transcriptional repression of RII due to decreased binding of stimulatory nuclear proteins to the RII promoter in keratinocytes and breast cancer cells (11,12). RI and RII replacement in cells that lack, or show reduced levels of, TGF-␤ receptors led to restoration of TGF-␤ response and subsequent reversal of malignancy, as seen in breast and colon cancer cells (13,14).The promoters for RI and RII have been characterized (15, 16). Both RI and RII promoters lack distinct TATA boxes and are highly GC-rich and depend on Sp1 transcription factor for the initiation of transcription. The RI promoter contains four consensus and several putative Sp1 sites, whereas the RII promoter contains two Sp1 sites. The Sp gene family of transcription factors consists of four members, which are referred to as Sp1-Sp4. Whereas Sp1, Sp2, and Sp4 are known to be activators of gene transcription, Sp3 is generally considered to be a repressor (17). Sp1 and Sp3 transcription factors recognize the same DNA element and have similar binding affinities. Sp3 has been shown to repress Sp1-mediated trans-activation of several genes (18 -20).MCF-7 early passage (MCF-7E) breast cancer cells express RI and RII and are responsive to the growth inhibitory effects of TGF-␤. However, MCF-7 late passage cells (MCF-7L) lack RII, show reduced levels of RI, and are TGF-␤-resistant. Loss or reduced expression of TGF-␤ receptors was due to low Sp1 protein levels in MCF-7L cells in comparison to 22). Sp1 deficiency was reversed by 5-aza-2Ј-deoxycytidine treatment of these cells, leading to the restoration of TGF-␤ receptor expression and signal transduction (12). We now show that in addition to Sp1 deficiency, MCF-7L breast cancer cells express higher levels of Sp3 than do MCF-7E cells, which express adequate amounts of both receptors and are consequently sensitive to TGF-␤. Sp3 acts as a transcriptional repressor of TG...

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The loss of transforming growth factor-␤ (TGF-␤) response due to the dysregulation of TGF-␤ receptors type I (RI) and type II (RII) is well known for its contribution to oncogenesis. Estrogen receptor-expressing breast cancer cells are refractory to TGF-␤-mediated growth control because of the reduced expression of TGF-␤ receptors. Although RII is required for the binding of TGF-␤ to RI, RI is responsible for directly transducing TGF-␤ signals through the Smad protein family. Treatment of estrogen receptor-expressing MCF-7L and ZR75 breast cancer cells with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) led to a dramatic induction of RI. Accumulation of acetylated histones H3 and H4 was observed in the SAHAtreated cells. Chromatin immunoprecipitation analysis followed by PCR with RI promoter-specific primers indicated an accumulation of acetylated histones in chromatin associated with the RI gene, suggesting that histone deacetylation was involved in the transcriptional inactivation of RI. SAHA treatment stimulated RI promoter activity through the inhibition of Sp1/Sp3-associated HDAC activity. Histone acetyltransferase p300 stimulated RI promoter activity, thus further confirming the involvement of HDAC activity in the transcriptional repression of RI. Significantly, SAHA-mediated RI regeneration restored the TGF-␤ response in breast cancer cells.Transforming growth factor-␤ (TGF-␤), 1 a 25-kDa homodimeric polypeptide, plays an important role in the growth inhibition of most normal epithelial and some cancer cells (1). TGF-␤ functions through cell surface receptors referred to as type I (RI) and type II (RII). RI requires RII for the binding of TGF-␤. However, RI is a direct player in the TGF-␤ signaling pathway as it conveys signals from TGF-␤ through the activation of Smad protein family members (2). The loss of TGF-␤ response is well known for its contribution to oncogenesis and tumor progression. Direct involvement of RI in TGF-␤ signal transduction would suggest that loss or reduced expression of RI could contribute to TGF-␤ resistance resulting in a growth advantage that contributes to tumor progression.TGF-␤ resistance due to methylation of the RI promoter or RI promoter repression by Sp1 deficiency was reported to be a cause of TGF-␤ resistance in gastric and colon carcinomas (3,4). The RI gene is frequently mutated in ovarian carcinomas (5). Decreased expression of RI is associated with poor prognosis in bladder transitional cell carcinomas (6). Reduced RI expression is also associated with unfavorable prognosis in esophageal squamous cell carcinoma (7). RI*6A, a polymorphic allele of RI, is emerging as a high frequency, low penetrance tumor susceptibility allele that predisposes to the development of breast, ovarian, and colorectal cancer, as well as hematologic malignancies (8). Polymorphisms in the microsatellite region of the RI gene were reported in head and neck cancers as well as non-small cell lung cancer (9, 10). Mutations in the kinase domain of the RI gene we...

Hypoxia-inducible factor-1␣ (HIF-1␣The hypoxic response is mainly regulated by the hypoxiainducible factor-1 (HIF-1), 2 a basic helix-loop-helix transcription factor composed of two subunits HIF-1␣ and HIF-1␤ (1). HIF-1␣ forms heterodimers with HIF-1␤, and this complex binds to hypoxia-responsive element (HRE: 5Ј-RCGTG-3Ј) within the promoter regions of target genes. Multiple studies of HIF-1␣ and breast cancer have shown a significant association between HIF-1␣ overexpression and poor prognosis coupled to increased patient mortality (2-6). The levels of HIF-1␣ in human primary breast tumors increased with the progression of the pathologic stage (7). In a large retrospective study of 745 patients with high levels of HIF-1␣ at diagnosis, early relapse and metastatic disease were predicted (5). HIF-1␣ expression is closely linked to an aggressive phenotype in breast cancer, and HIF-1␣ expression enhanced osteolytic bone metastasis of breast cancer (8, 9). After prolonged treatment hormone-sensitive breast tumors frequently become resistant to hormonal therapy, and it was hypothesized that hypoxia may promote estrogen-independent growth. Deletion of HIF-1␣ in the mammary epithelium resulted in delayed tumor onset and retarded tumor growth as well as decreased pulmonary metastasis (10). These results suggest that HIF-1␣ is a negative prognostic factor in breast cancer progression. The HIF-1␤ subunit is constitutively expressed, whereas expression of HIF-1␣ is regulated by oxygen tension. HIF-1␣ protein is not detected in cells under normoxic conditions (20 -22% O 2 ) and is rapidly induced by hypoxic conditions (1-2% O 2 ). However, in the invasive carcinoma cells, including breast, steady-state HIF-1␣ expression can be detected even under normoxia. The synthesis of HIF-1␣ protein has been shown to be regulated in an O 2 -independent fashion, for example, through activation of the receptor tyrosine kinase pathways (11,12). The molecular targets of HIF-1␣ that contribute to breast tumorigenesis are under active investigation.Macrophage-stimulating protein (MSP) is the only known ligand for recepteur d'origine nantais (RON), a tyrosine kinase receptor. MSP is an 80-kDa heterodimer consisting of a 53-kDa ␣-chain and a 30-kDa ␤-chain linked by a disulfide bond. The ␤-chain of MSP binds to RON (13). RON is initially synthesized as a single chain precursor, 170-kDa pro-RON, which is subsequently cleaved into 40-kDa alpha chain and 150-kDa beta chain. The alpha chain is completely extracellular, whereas the beta chain traverses the cell membrane and contains the intracellular tyrosine kinase (13). The RON receptor also participates in cross-talk with other receptor tyrosine kinases such as MET and epidermal growth factor receptor. Several human tumor tissues show increased RON expression, including tumors of the breast, colon, lung, liver, kidney, ovary, stomach, pancreas, bladder, and prostate (14). Gene expression analyses indicated increase in RON expression is associated with metastatic disease. Transgenic mice that ov...

Macrophage-stimulating protein (MSP)2 is the only known ligand for RON (recepteur d'origine nantais). MSP is an 80 kDa heterodimer consisting of a 53 kDa ␣ chain and a 30-kDa ␤ chain linked by a disulfide bond. The ␤ chain of MSP binds to RON. MSP belongs to the plasminogen-prothrombin gene family (1, 2). MSP gene knock-out in mice is not lethal, indicating that MSP is not required for embryonic development and growth (3). Besides macrophages, MSP is expressed in a variety of epithelial cells. RON is initially synthesized as a single chain precursor, 170-kDa pro-RON, which is subsequently cleaved into 40-kDa ␣ chain and 150-kDa ␤ chain. The ␣ chain is completely extracellular, whereas the ␤ chain traverses the cell membrane and contains the intracellular tyrosine kinase (1). The C-terminal of RON regulates its kinase activity (4). RON forms either homodimers or heterodimers with other receptors such as c-Met and epidermal growth factor receptor (5-7). In addition to macrophages, RON is also expressed in multiple epithelial cells both malignant and nonmalignant. Homozygous deletion of RON was embryonically lethal. However, RON heterozygous mice mature normally except for an inappropriate inflammatory response (8, 9). The RON protein is regulated through c-Cbl ubiquitin ligase binding to phosphorylated RON leading to endocytosis and the subsequent degradation of RON (10).Abnormal expression of RON was reported in various cancers of epithelial origin. However, fibroblasts do not express RON. RON is moderately expressed in normal colorectal mucosa but significantly elevated in a majority of primary human colorectal adenocarcinoma samples (11). RON protein accumulation was reported to induce autophosphorylation of RON tyrosine kinase receptor and transduces signals that regulate tumorigenic activities of colon cancer cells (12). In nonsmall cell lung cancer cell lines, RON overexpression was reported in a majority of the cell lines examined. In addition, these cell lines expressed high levels of MSP ligand (13). The combination of RON overexpression and activation by MSP leads to increased invasion and resistance to apoptosis. These tumors supported by either autocrine or paracrine effects may acquire a survival advantage because of increased activation of the RON receptor by the local secretion of MSP.Altered RON expression was noticed in bladder and ovarian cancers. RON expression was positively associated with tumor size, stage, and grade in bladder carcinomas (14). The majority of ovarian carcinoma samples showed up-regulation in RON expression with a mix of cytoplasmic and membrane staining (5). Co-expression of MSP with RON was observed in ovarian carcinomas, providing a selective growth advantage and subsequent tumor progression. RON overexpression and not mutations is associated with head and neck squamous cell carcinomas (15). Normal breast cells and benign lesions (adenomas and papillomas) express relatively low levels of RON. However, RON is highly expressed in tumor specimens (1). Further, increased RON e...