Autocrine human growth hormone enhancement of human mammary carcinoma cell spreading is Jak2 dependent.

We investigated the role of autocrine production of human (h) GH in the attachment and spreading of mammary carcinoma cells in vitro. We used a previously described model system for the study of the autocrine/paracrine role of GH in which the hGH gene (MCF-hGH) or a translation-deficient hGH gene (MCF-MUT) was stably transfected into MCF-7 cells. No differences in attachment to a collagen matrix between MCF-hGH and MCF-MUT cells were observed in either serum-free medium (SFM) or medium containing exogenous hGH, 5% serum, or 10% serum. In contrast, MCF-hGH cells spread more rapidly on a collagen matrix than did MCF-MUT cells. Exogenous hGH and 10% serum interacted with autocrine production of hGH in an additive manner to increase cell spreading. MCF-hGH cells formed filipodia and stress fibers earlier than MCF-MUT cells during the process of cell spreading and possessed marked differences in morphology after spreading. MCF-MUT cells displayed uniform and symmetrical formation of stress fibers, whereas MCF-hGH cells displayed irregular and elongated stress fiber formation. The level of cytoplasmic phosphotyrosine was increased in MCF-hGH compared with MCF-MUT cells during spreading and displayed colocalization with Janus kinase 2 (JAK2). Basal JAK2 tyrosine phosphorylation was increased, and it increased further on spreading in MCF-hGH cells compared with MCF-MUT cells. Transient transfection of JAK2 complementary DNA resulted in interaction with autocrine hGH to increase the rate of cell spreading in MCF-hGH cells compared with MCF-MUT cells. Treatment with a selective JAK2 tyrosine kinase inhibitor (AG 490) reduced the rate of MCF-hGH cell spreading to the rate of MCF-MUT cell spreading. Thus, we conclude that autocrine production of hGH enhances the rate of mammary carcinoma cell spreading in a JAK2-dependent manner.

The effects of autocrine human growth hormone (hGH) on human mammary carcinoma cell behavior are mediated via the hGH receptor.

The human GH (hGH) antagonist B2036 combines a single amino acid substitution impairing receptor binding site 2 (G120K) with eight additional amino acid substitutions that improve binding site 1 affinity. B2036 does not bind, activate, or antagonize the human PRL receptor and therefore is suitable to determine cellular effects mediated specifically through the hGH receptor. We have used this hGH receptor specific antagonist in MCF-7 cells stably transfected with either the hGH gene (MCF-hGH) or a translation deficient hGH gene (MCF-MUT) to determine whether the effects of autocrine hGH on mammary carcinoma cell behavior are mediated via the hGH receptor. Enhanced JAK2 tyrosine phosphorylation observed in MCF-hGH cells compared with MCF-MUT cells is abrogated by B2036 as is the autocrine hGH stimulated increase in total cell number and DNA synthesis. Interestingly, autocrine hGH functions as a potent inhibitor of apoptosis induced by serum withdrawal compared with exogenously added hGH, and the protection against apoptosis afforded by autocrine hGH is abrogated by B2036. B2036 also inhibited autocrine hGH stimulated transcriptional activation mediated by either STAT5, CHOP (p38 MAP kinase specific) or Elk-1 (p44/42 MAP kinase specific). Finally, B2036 inhibited the autocrine hGH-dependent enhancement of the rate of mammary carcinoma cell spreading on a collagen matrix. Thus, the effects of autocrine hGH on human mammary carcinoma cell behavior are mediated via the hGH receptor.

Serum IGF-binding protein-6 and prostate specific antigen in breast cancer.

OBJECTIVE: Recent studies have demonstrated the presence of the IGF-binding proteins (IGFBPs) and prostate specific antigen (PSA), an IGFBP protease. in human breast tissue. We sought to investigate the differences in serum IGFs, IGFBP-1, -3 and -6, and PSA between patients with surgically proven breast cancer and patients with benign breast disease. DESIGN AND METHODS: Concentrations of IGFs, IGFBP-1, -3 and -6, and PSA were determined in the sera from 57 patients with breast cancer (CA), and 46 women with benign breast disease (BBD) using immunoassays for IGFs and IGFBPs and an ultrasensitive ELISA for PSA. RESULTS: The mean (+/- S.E.M.) serum IGFBP-6 level in the CA group, 127 (16) ng/ml, was statistically significantly lower than in the BBD group, 157 (10) ng/ml (P = 0.016). Patients with CA had an elevated geometric mean serum PSA level of 0.018 (range: 0.0015-0.107) ng/ml, compared with 0.007 (range: 0.0015-0.019) ng/ml in women with BBD (P = 0.025). Mean serum IGFBP-1 concentrations were significantly lower in the CA group, 16 (2) ng/ml, versus 37 (4) ng/ml in the BBD group (P = 0.001). Mean serum IGFBP-3 concentrations were also lower in the CA group versus the BBD group, at 1981 (65) ng/ml, versus 2603 (140) ng/ml (P = 0.002) respectively. In the CA group, statistically significant correlations between PSA and IGFBP-6 (r = 0.413; P = 0.001), and between PSA and IGFBP-1 (r = -0.329; P = 0.021) were seen. Differences in IGF-I and -II between the two groups were not statistically significant. CONCLUSION: Lower serum concentrations of IGFBP-6, -3 and -1, but higher PSA concentrations were seen in the breast cancer group, and collectively these would suggest that there is an increase in bioavailable IGF-I in breast cancer.

Autocrine stimulation of human mammary carcinoma cell proliferation by human growth hormone.

Here we have investigated the role of autocrine production of human growth hormone (hGH) in the proliferation of mammary carcinoma cells (MCF-7) in vitro. MCF-7 cells were stably transfected with an expression plasmid encoding the hGH gene, and these cells (designated MCF-hGH) synthesized hGH in the cell and secreted hGH to the medium. For control purposes, a MCF cell line was generated (MCF-MUT) in which the start codon of the hGH gene was disabled, and these cells transcribed the hGH gene without translation to hGH protein. The MCF-hGH cell number increased at a rate significantly greater than that of MCF-MUT under serum-free conditions. Autocrine hGH also synergized with 10% serum and insulin-like growth factor-1 but not 17-beta-estradiol to increase cell number. The increased proliferation of MCF-hGH cells in both serum-free and serum-containing media could be completely abrogated by the use of the nonreceptor dimerizing hGH antagonist, hGH-G120R. Increased mitogenesis as a consequence of autocrine production of hGH was prevented by inhibition of either the p38 MAPK or p42/44 MAPK pathways. MCF-hGH cells also possessed a higher level of STAT5 (but not STATs 1 and 3) mediated transcriptional activation in both serum-free and serum-containing conditions than MCF-MUT cells. Thus we conclude that hGH can act in an autocrine/paracrine manner in human mammary carcinoma cells to promote cell proliferation and transcriptional activation.