Overexpression of Transforming Growth Factor ( TGF ) 31 Type II Receptor Restores TGF-1 Sensitivity and Signaling in Human Prostate Cancer Cells ’

TGF-fil is a potent negative regulator of cell growth that transduces signals through interaction with type I and type II receptors that form a heteroduplex. Abnormal expression and mutational alterations of these receptors have recently been shown in several human malignancies. In previous studies, we have demonstrated reduced expression of both types of transforming growth factor (TGF) i1 receptors in human prostate tumors. In this study, using the human prostate cancer cell line, LNCaP, which is refractory to TGF-fil and lacks type II receptor (R-ll), we investigated whether overexpression of the R-Il receptor can restore sensitivity to the negative growth effects of TGF31. LNCaP cells were transfected with plasmid containing the full length of human TGFR-Il receptor cDNA sequence. Stable transfectant clones were selected for R-Il mRNA and protein expression by Northern and Western analyses, respectively. The effect of TGF-I3 on LNCaP R-II overexpressing clones was examined on the basis ot (a) growth inhibition (cell number); (b) DNA synthesis using the H]thymidlne incorporation assay; (c) induction of cyclin-dependent-kinase inhibitors, p21 WAF 1/CIPI, p27 1, and p15; and (d) colony-forming ability in soft agar. Both the cell number and the rate of DNA synthesis of R-ll-overexpressing clones were significantly suppressed by exogenous TGF1 in a dose-dependent manner, compared with control cell lines. Treatment of R-ll cloned transfectants with TGFflu induced a G1 arrest, which was accompanied by a transient increase in p21WAFi/CIPI and p27’ expression at the mRNA and protein level. Furthermore, the LNCaP R-Il transfectants analyzed exhibited a markedly reduced colony-forming ability. Our results indicate that overexpression of TGF-fil R-Il receptor in LNCaP prostate cancer cells caused tumor growth inhibition by restoring the TGF3 signaling mechanism and TGF-fil sensitivity. Introduction Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer mortality in American males (1). Prostate cancer vanes in its clinical aggressiveness. Histologically detected localized prostatic cancers are heterogeneous, with only a small subset having undergone all of the malignant changes required to produce clinically aggressive tumors (2). Therefore, because not all histological prostate cancers are universally destined to produce clinical disease, there is an immediate need to develop new diagnostic methods for the identification of prostatic tumors that will progress to a stage that requires immediate surgicaVtherapeutic intervention. Development of such methods requires the characterization of the molecular and cellular changes associated with prostatic tumorigenesis and progression to metastatic disease. In the normal prostate, androgens exert their growthregulatory mechanisms via growth factor signaling pathways, escape from which would ultimately lead to tumongenic growth (3). TGF3-f3 is a negative growth regulator that has been postulated to play a significant role in the control of normal prostate growth via its ability to inhibit cell proliferation and induce apoptosis (4, 5). TGF3l has multifunctional properties in regulating cell growth, differentiation and apoptosis, control of extracellular matrix formation, and immunosuppression (6) in different cell types. TGF-f31 generates these biological responses by interacting with two major cell surface receptors, type I (R-I) and type II (A-Il), which are serine/threonine kinases (7, 8). The signal transduction cascade mediated by TGF-(31 is initiated by the binding of receptor R-II to the ligand, TGF-pl (8). Once bound to TGF31 receptor R-II, TGF-(3l can recruit receptor R-I to form a ternary complex (9). The direct involvement of both TGF3 receptors in the TGF-j31 signal transduction pathway suggests that the loss of functional TGF-f3 receptors may contribute to the loss of growth-inhibitory activity of TGF-f3. An expanding body of evidence indicates that a dysfunctional TGF-f3 signaling mechanism, due to loss of TGF-j3 receptors, leads to the development and progression of 5everal human malignancies, including breast cancer (1 0), T-celI