The Role of Sp1 in the Differential Expression of Transforming Growth Factor-β Receptor Type II in Human Breast Adenocarcinoma MCF-7 Cells

Abstract

Progression of MCF-7 cells from early passage (MCF-7E, <200 passage) to late passage (MCF-7L, >500 passage) correlates with a loss of sensitivity to exogenous TGF␤1. We have previously shown that loss of TGF␤ sensitivity is due to decreased expression of the transforming growth factor receptor type II (T␤RII) and is associated with increased tumorigenicity in nude mice. Reduced T␤RII expression in MCF-7L cells is caused by decreased T␤RII promoter activity in this cell line. Our previous studies using 5 deletion constructs of this promoter revealed that MCF-7L cells were unable to support transcription of the minimal promoter (؊47 to ؉2) to the same levels as the MCF-7E cells. This region of the promoter contains an Sp1 element at position ؊25 from the major transcription start site. In this study, we in- T␤RIII is thought to play a role in presenting the ligand to the other receptors (6). Receptor type I (T␤RI), a 50 -60-kDa glycoprotein, and receptor type II (T␤RII), a 75-85-kDa glycoprotein, belong to a superfamily of receptor serine/threonine kinases. The presence of both T␤RI and T␤RII is necessary to effect a TGF␤ response, and both kinase activities must be functional for proper signal transduction (7-10). Therefore, cells defective in one or the other receptor are refractory to the effects of TGF␤ and escape its autocrine-negative effects on growth.The loss of TGF␤ sensitivity has been correlated to tumor progression. This loss has been most often associated with a loss of T␤RII expression (11). Replication error-positive colorectal cancer cells show a mutation in T␤RII coding region, which results in message instability and loss of protein (12)(13)(14). Deletion of the T␤RII gene, loss of T␤RII message, and expression of a truncated T␤RII message have also been observed in other cancer cell lines (15)(16)(17)(18)(19)(20).The human breast cancer MCF-7 cells have no detectable cell surface T␤RII and are refractory to the effects of TGF␤ (18,19). The re-expression of T␤RII in these cells restores their sensitivity to the growth inhibitory effects of TGF␤ (19). In addition, the transfectants showed decreased anchorage-independent growth, and a decreased tumorigenicity in nude mice compared with vector-transfected cells. These results suggest that T␤RII behaves as a tumor suppressor in the MCF-7 cells.Due to the significant role of T␤RII in determining tumorigenicity, it is likely that defects of T␤RII expression can lead to malignant progression. MCF-7 cells represent a well established model system for studying the biology of breast cancer cells. Our earlier work using MCF-7 early passage (MCF-7E) and MCF-7 late passage (MCF-7L) cells demonstrated that MCF-7L cells' resistance to exogenous TGF␤ correlated with the low, or undetectable level of T␤RII in this cell line (18).

Reduced expression of transforming growth factor‐β receptors is an unfavorable prognostic factor in human esophageal squamous cell carcinoma

“…34 The presence of both TGF-␤R-I and TGF-␤R-II is necessary to effect a TGF-␤ response, and both kinase activities must be functional for proper signal transduction. 14 We demonstrated immunohistochemically that reduced expression of TGF-␤R-I and TGF-␤R-II was correlated with depth of invasion, lymph node metastasis and pathologic stage and that overexpression of TGF-␤1 was correlated with tumor invasion and closely correlated with reduced expression of both receptors. In addition, reduced expression of TGF-␤R-I and TGF-␤R-II was correlated with a decreased probability of cancer-specific survival.…”

“…DISCUSSION TGF-␤ is a prototypical member of a superfamily of multifunctional cytokines that plays an important role in the inhibition of epithelial cell proliferation. TGF-␤1 elicits its effects by binding to specific cell-surface type II receptors, 14 which are constitutively active transmembrane serine/threonine kinases that recruit TGF-␤R-I and phosphorylate 1 or more substrates to initiate a signal cascade such as that of Smad proteins. 34 The presence of both TGF-␤R-I and TGF-␤R-II is necessary to effect a TGF-␤ response, and both kinase activities must be functional for proper signal transduction.…”

“…Replication error-positive colorectal cancer cells show a mutation in the TGF-␤R-II coding region, which results in message instability and loss of protein. 14,16,17 Deletion of the TGF-␤R-II gene, loss of the TGF-␤R-II message and expression of a truncated TGF-␤R-II message have also been observed in other cancer cell lines. 18 -23 Structural mutations of TGF-␤R-I have been observed less frequently compared to TGF-␤R-II mutations.…”

“…8 Many carcinoma cells, such as colon carcinoma, 9 breast carcinoma, 10 pancreas carcinoma, 11 prostate carcinoma, 12 gastric carcinoma 13 and bladder carcinoma, 3 show resistance to growth inhibition by TGF-␤1. This loss of response to the inhibitory effect of TGF-␤1 has been associated with functional inactivation of either TGF-␤ receptor expression 14 or signal transducers of the Smad family 15 as well as with cancer progression. Replication error-positive colorectal cancer cells show a mutation in the TGF-␤R-II coding region, which results in message instability and loss of protein.…”

Identification of a novel SP3 binding site in the promoter of human IGFBP4 gene: role of SP3 and AP-1 in regulating promoter activity in CaCo2 cells

“…The novel Sp3 binding sequence of 5 0 -TGCTGCA-3 0 , located at À861/À855 in the IGFBP4 promoter has, to the best of our knowledge, not been reported. More than 30 binding sequences have been identified for the Sp family of proteins; all of them are GC-rich sequences that range from 6-13 bp (Boisclair et al, 1993;Kao et al, 1997;Suske, 1999;Liu et al, 2000;Maor et al, 2000;Zhu et al, 2000). In a recent report, a 13 bp sequence on the Nkx2.1 promoter region was identified to be transactivated by Sp1 and Sp3 factors ; the sequence 5 0 -GCCTCCGGGAGGC-3 0 was specifically bound by Sp1 and Sp3, and an H441 cell nuclear factor.…”