Involvement of Shc in the signaling response of human prostate tumor cell lines to epidermal growth factor.

Autocrine growth factors for the epidermal growth factor receptor (EGFR) have been identified in prostate tumors, implicating a role for EGFR in the progression of prostate cancer. To investigate early signaling mechanisms used by the EGFR in prostate tumor cells, we have characterized the involvement of the Shc (src homology 2/x-collagen related) adapter protein in EGFR signaling in several human prostate tumor cell lines. In androgen-responsive lymph node-prostate cancer (LNCaP) cells and androgen-insensitive PC3, DU145 and PPC-I cells, Shc was identified as one of the most prominent phosphotyrosine proteins to be elevated in response to EGF. Equivalent levels of the 46- and 52-kDa Shc isoforms were detected in all of the tumor cell lines tested. However, levels of the 66-kDa isoform were variable among the cell lines. In all of the tumor cell lines, EGF caused an association between Shc and Grb2, another adapter protein linked to cellular ras activation. Additionally, several phosphotyrosine proteins, including a 115-120-kDa protein in EGF-treated LNCaP cells, co-associated with Shc. The profile of these Shc-associating proteins, however, differed among the tumor cell lines. Our results indicate that Shc is a common downstream element of EGFR signaling in prostate tumor cells and suggest multiple functions for Shc in prostate tumorigenesis.

Overexpression of EMS1/cortactin in NIH3T3 fibroblasts causes increased cell motility and invasion in vitro.

Cortactin, a p80/85 protein first identified as a src kinase substrate, is thought to be involved in the signaling pathway of mitogenic receptors and adhesion molecules mediating cytoskeletal reorganization. The cortactin gene, EMS1, maps to chromosome 11q13, a region amplified in head and neck squamous cell carcinomas (HNSCC) and breast cancer, which display lymph node metastasis and an unfavorable clinical outcome. To further address the role of cortactin in the malignant phenotype of cells, we stably overexpressed cortactin in NIH3T3 fibroblasts and evaluated the effects of elevated cortactin on cellular proliferation, motility and invasiveness. Cortactin overexpressing cells did not display any striking morphological changes, nor any significant differences in cell proliferation or saturation density as compared to control NIH3T3 cells. Furthermore, the cortactin overexpressing cells were anchorage dependent for growth. Interestingly, cortactin overexpressing cells were more motile and invasive in modified Boyden chamber assays. These results suggest that overexpression of cortactin may play a role in tumor progression by influencing tumor cell migration and invasion.

Attenuation of LPA-mediated calcium signaling and inositol polyphosphate production in rat-1 fibroblasts transformed by the v-src oncogene.

Alterations in cellular signaling underlie the transforming actions of many oncogenes. The vsrc oncogene tyrosine kinase, pp60vsrc, is known to alter multiple signal transduction pathways, including those involving phosphatidylinositol (PI) metabolism. In this study, we investigated the effects of vsrc-transformation on lysophosphatidic acid (LPA) receptor coupling to intracellular free calcium [Ca2+]i and PI turnover in rat-1 fibroblasts. In normal rat-1 cells, LPA rapidly elevated [Ca2+]i (EC50 = 10nM). In contrast, the ability of LPA to mobilize calcium was markedly attenuated in rat-1-vsrc cells. Further study revealed that the LPA-mediated generation of inositol (1,4,5)P3 and other inositol polyphosphates was also markedly attenuated in the vsrc-transformed cells. Although LPA caused a transient reduction in the level of PI(4,5)P2 in normal rat-1 cells, the agonist elevated the level of PI(4,5)P2 in the vsrc-transformed cells. These findings demonstrate that vsrc-transformation alters the coupling of LPA receptors to PI turnover and calcium signaling in rat-1 cells, and point to G protein-coupled receptor systems as targets for modulation by the vsrc kinase.

Calcium signaling in prostate cancer cells: evidence for multiple receptors and enhanced sensitivity to bombesin/GRP.

BACKGROUND: Cellular calcium is an important second messenger for growth regulation. We sought to identify potentially important receptors on prostate tumor cells by screening over 20 agonists for their ability to increase intracellular free calcium ([Ca2+]i) in several human prostate tumor cell lines. METHODS: Intracellular calcium mobilization was detected using fura-2. RESULTS: We found bombesin, GRP, ATP/UTP, lysophosphatidic acid, thrombin, endothelin, histamine, and bradykinin increased [Ca2+]i in the advanced tumor cell lines DU-145, PC3, and PPC-1. Bombesin failed to elevate [Ca2+]i in an immortalized human prostate cell line. Rank-order of potency studies suggested the presence of P2U nucleotide receptors for ATP/UTP on prostate epithelial cells. Potency studies also revealed GRP > > bombesin > > neuromedin B at elevating [Ca2+]i in responding tumor cells. CONCLUSIONS: These findings indicate that androgen independent prostate tumor cell lines express multiple receptors capable of elevating intracellular calcium, and suggest that GRP receptors may be selectively expressed and/or coupled to calcium signaling during prostate tumor progression. Calcium sensitive cellular events may therefore contribute to the progression of prostate cancer.

Inhibition of head and neck squamous cell carcinoma growth and invasion by the calcium influx inhibitor carboxyamido-triazole.

Local invasion and lymph node metastasis are correlated with a decreased overall survival in head and neck cancer patients and warrant new strategies to intervene in the metastatic cascade. One approach is to focus on the intracellular signaling pathways underlying the metastatic process. A common regulatory point in several signal transduction pathways is intracellular calcium homeostasis. We assessed the effect of a novel calcium influx inhibitor, carboxyamido-triazole (CAI), on the growth and invasive phenotype of cell lines derived from head and neck squamous cell carcinoma (HNSCC). CAI inhibited the growth of FaDu and EVSCC17M cells in a dose-dependent (IC50, 13-15 microM) and reversible manner. CAI also caused a generalized attenuation of receptor-mediated calcium elevation to several calcium mobilization agonists, including epidermal growth factor and bradykinin. The effects of CAI on the invasive phenotype of HNSCC cell lines were assessed by a chemo-invasion assay. HNSCC cell lines exhibited a range of invasive potential as measured by the capacity of tumor cells to penetrate a reconstituted basement membrane of Matrigel. HNSCCs were classified as highly invasive (EVSCC14M and EVSCC17M) or weakly invasive (EVSCC18, EVSCC19M, UMSCC10A, and FaDu). Treatment of HNSCC cell lines with 10 microM CAI for 24 h reduced invasion 2-14-fold in a dose-dependent manner. HNSCCs also exhibited different motilities as measured by a chemotaxis assay. EVSCC14M and EVSCC17M were highly motile, whereas EVSCC18, EVSCC19M, UMSCC10A, and FaDu were less motile. CAI reduced the migration of all cell lines. Conditioned medium from HNSCC cell lines was analyzed by zymography for production of Mr 72,000 type IV collagenase [matrix metalloproteinase (MMP)-2)] and Mr 92,000 type IV collagenase (MMP-9). All HNSCC cell lines secreted MMP-2 and/or MMP-9 into conditioned medium. Treatment of cells with 10 microM CAI for 24 h resulted in a reduction of both MMP-2 and MMP-9 production. The results demonstrate that CAI blocks cellular proliferation, migration, chemoinvasion, and MMP production by HNSCC in vitro and identify calcium-dependent signaling as a new target for inhibition of the malignant phenotype of HNSCC.

Effects of the calcium influx inhibitor carboxyamido-triazole on the proliferation and invasiveness of human prostate tumor cell lines.

Aberrant cellular signaling is a central feature of malignant cells and a potential target for anti-cancer therapy. Carboxyamido-triazole (CAI) is a calcium influx inhibitor that alters calcium-sensitive signal transduction pathways and suppresses the proliferative and metastatic potential of malignant cells. We have examined the effects of CAI on several tumor-associated parameters in human prostate cancer cell lines to evaluate the potential of CAI as a signal-transduction therapy agent for advanced-stage prostate cancer. Measuring anchorage-dependent cell growth, continuous application of CAI inhibited the growth of DU-145, PPC-1, PC3 and LNCaP tumor cells with 50% inhibitory concentrations ranging 10-30 microM. Direct cell enumeration assays revealed that the growth-suppressing activity of CAI toward DU-145 cells was reversible, indicating a cytostatic effect of the drug on tumor cells. The drug also inhibited the proliferation of several immortalized human prostatic epithelial cell lines. The proliferation of HaCaT- and RHEK-1-immortalized keratinocyte cell lines was relatively insensitive to CAI. Additionally, invasion by DU-145, PC3 and PPC-1 cells through Matrigel in vitro was reduced approximately 60-70% by 10 microM CAI. Other cellular effects of CAI included an attenuation of the elevation of intracellular free calcium in response to bombesin and carbachol in PC3 cells and a marked dose-dependent inhibition of prostate-specific antigen secretion in LNCaP cell cultures.

Amplification and expression of EMS-1 (cortactin) in head and neck squamous cell carcinoma cell lines.

Amplification of chromosome 11q13 DNA sequences is detected in approximately 30% of primary head and neck squamous cell carcinomas (HNSCC). The amplified region includes genes for cyclin D1, hst-1, int-2, and more recently, ems-1. Ems-1 encodes an 80/85kd cytoskeletal associated protein termed cortactin, which has been shown to bind F-actin and is a pp60src substrate. We investigated 16 HNSCC cell lines for ems-1 DNA amplification and gene expression by western blotting and immunofluorescence using mAb 4F11. Amplification of ems-1 DNA was detected in 8/16 (50%) cell lines and was related directly to over-expression of cortactin by western blotting and immunofluorescence. Western blotting detected both forms of cortactin, p80 and p85, at equal intensity. Immunofluorescent staining revealed low levels of cortactin localized to the cytoplasm and surface membrane in normal bronchial epithelial cells and tumor cell cultures with single copy ems-1 DNA. In contrast, tumor cell cultures with ems-1 DNA amplification demonstrated intense, homogeneous cortactin cytoplasmic staining. These results suggest that overexpression of p80/85 may be a useful marker to identify 11q13 amplification, a molecular alteration correlated with the presence of lymph node metastasis in head and neck cancer.

Selective amplification of endothelin-stimulated inositol 1,4,5-trisphosphate and calcium signaling by v-src transformation of rat-1 fibroblasts.

The effects of the expression of the protein tyrosine kinase pp60v-src on endothelin- and thrombin-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production and calcium responses were investigated in Rat-1 fibroblasts. The ability of endothelin-1 to induce the accumulation of these second messengers was dramatically amplified by v-src transformation, with 6- and 3-fold enhancements of the peak Ins(1,4,5)P3 and peak calcium responses, respectively. In contrast, thrombin-dependent responses were slightly reduced following v-src transformation, demonstrating that the augmentation of endothelin-stimulated signal transduction is a selective effect. The magnitude of the stimulated accumulation of Ins(1,4,5)P3 presumably depends upon both the functional activation of phospholipase C to produce Ins(1,4,5)P3, and the activity of the enzymes that metabolize Ins(1,4,5)P3. Although the metabolism of Ins(1,4,5)P3 was strikingly altered by expression of pp60v-src, with a bias towards the production of higher inositol polyphosphates that is consistent with an activated Ins(1,4,5)P3 3-kinase, this change could not account for the marked increase in endothelin-stimulated signaling induced by v-src transformation. This suggests that an effect of pp60v-src is expressed at the level of the plasma membrane, through an interaction with one or more components in the receptor/guanine nucleotide binding protein (G protein)/phospholipase C system that transduces the endothelin signal into Ins(1,4,5)P3 production. Preparation of membranes from normal and v-src-transformed cells showed that, while there was no change in the number of high-affinity endothelin binding sites, the release of Ins(1,4,5)P3 in response to guanine nucleotides and endothelin-1 was significantly increased following v-src transformation. In contrast, the Ins(1,4,5)P3 responses to thrombin and high Ca2+ concentrations were unaffected by transformation. Thus the selective interactions within the G protein system that couples the endothelin receptor to phospholipase C are potential sites at which the v-src transformation process may act to amplify endothelin-dependent Ins(1,4,5)P3 production.

Phosphoinositides and cell growth.

The findings described above illustrate how the src kinase can influence several new pathways of inositol phosphate metabolism, both at the membrane level with the production of novel D-3 phosphoinositides and the activation of PI-3 kinase, and at the cytosolic level by altering the expression of certain inositol polyphosphates, in particular Ins(1,4,5,6)P4. At present, it is difficult to speculate on the role these phenomena play in cellular transformation by src, since the functions of D-3 phosphoinositides and most inositol polyphosphates are unclear. There is evidence, however, that these new pathways of phosphoinositide metabolism occur in response to other types of cellular stimulations besides src transformation. Novel D-3 phosphoinositides are expressed in a variety of nonneoplastic cells, including human platelets treated with thrombin, smooth muscle cells and stimulated neutrophils. In addition, unusual InsP4 isomers such as D/L-Ins(1,4,5,6)P4 are found in chicken erythrocytes, murine macrophages, AR4-2J rat pancreatoma cells and adrenal glomerulosa cells, to name only a few. Recently, associations have been reported between PI-3 kinases and cytoskeletal elements in thrombin- stimulated platelets, and between activated ras proteins in rat liver epithelial cells. The latter discovery is particularly intriguing since GTP-binding proteins such as ras are known to influence cell shape and serve as downstream effector proteins in the signal transduction pathways of numerous growth factor receptors. Thus, one function of novel phosphoinositides and their metabolites may lie at the level of cytoskeletal and cell shape regulation. Clearly, additional roles for phosphoinositides exist in cells besides their traditional use as precursors for the generation of Ins(1,4,5)P3 and diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene.

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.

Inhibition of epidermal growth factor receptor biosynthesis caused by the src oncogene product, pp60v-src.

We have previously shown that an intracellular mechanism down regulates epidermal growth factor (EGF) receptor levels in rodent fibroblasts transformed by the src oncogene (W. J. Wasilenko, L. K. Shawver, and M. J. Weber, J. Cell. Physiol. 131:450-457, 1987). We now report that this down regulation is due to an inhibition of EGF receptor biosynthesis. With Rat-1 (R1) cells infected with a temperature-sensitive src mutant, we found that 125I-labeled EGF binding to cells began to decrease soon after the activation of pp60v-src by shift down to the permissive temperature for transformation. This effect of src on EGF receptors was reversible. Pulse-chase studies with [35S]methionine-labeled cells revealed that the tyrosine protein kinase activity of pp60v-src had little if any effect on EGF receptor degradation rate. By contrast, the expression of pp60v-src caused a large reduction in the apparent rate of EGF receptor biosynthesis. Northern (RNA) blot analysis demonstrated that pp60v-src also caused marked reductions in the steady-state level of EGF receptor mRNA. These data indicate that one way the expression of the src oncogene can affect the machinery of growth control is by affecting the expression of specific genes for growth factor receptors.

Fibroblasts transformed with v-src show enhanced formation of an inositol tetrakisphosphate.

The tyrosine kinase pp60v-src, encoded by the v-src oncogene, seems to regulate phosphatidylinositol metabolism. The effect of pp60v-src on control points in inositol phosphate production was examined by measuring the amounts of inositol polyphosphates in Rat-1 cells expressing wild-type or mutant forms of the protein. Expression of v-src-resulted in a five- to sevenfold elevation in the steady-state amount of an isomer of inositol tetrakisphosphate, whereas the concentrations of inositol trisphosphates or other inositol tetrakisphosphates were not affected. The activity of a key enzyme in the formation of inositol tetrakisphosphates, inositol (1,4,5)-trisphosphate 3-kinase, was increased six- to eightfold in cytosolic extracts prepared from the v-src-transformed cells, suggesting that this enzyme may be one target for the pp60v-src kinase and that it may participate in the synthesis of novel, higher order inositol phosphates.

Down-modulation of EGF receptors in cells transformed by the src oncogene.

The effects of src oncogene expression on epidermal growth factor (EGF) receptors have been investigated in mouse 3T3 and rat-1 fibroblasts. Transformation of both cell types with src resulted in marked reductions in cellular EGF receptor levels, as assayed by either 125I-EGF binding or immunoprecipitation of receptor protein from radiolabeled cell lysates. In contrast to cells transformed by other types of retroviral oncogenes, the loss of EGF receptors in the src-transformed cells did not appear to be due to secreted transforming growth factor-alpha (TGF-alpha), since such factors were undetectable in culture fluids from the src-transformed cells. By several criteria of transformation, an EGF-receptorless cell line infected with src was shown to be transformed, suggesting that EGF receptors themselves are not obligatory to the src transformation process. We suggest that pp60src down-modulates EGF receptors by an intracellular mechanism and that the loss of the receptors is symptomatic of more general effects of pp60src on the machinery of growth regulation.

Utilization of pyruvate and pyruvate precursors by normal and carcinogen-altered rat tracheal epithelial cells in culture.

The metabolism of [14C]pyruvate, [14C]glucose, [14C]glutamine and [14C]alanine was compared between normal rat tracheal epithelial cells and carcinogen-altered cells derived from dimethylbenz(a)anthracene-exposed tracheal implants. Normal primary cultures (NPC) of tracheal cells are distinguished by their need for pyruvate-supplemented medium for growth and survival. The altered cells were selected out by their survival in the unsupplemented medium. Compared to the selected primary cultures (SPC), the NPC showed a three- to four-fold higher incorporation of radioactivity from [2-14C]pyruvate in all the macromolecular fractions, as well as in all the metabolites isolated from the acid soluble fraction and from lactic acid isolated from the medium. [U-14C]glucose was also incorporated at higher levels into lactic acid isolated from the acid soluble fraction and the medium of NPC. These data indicate a higher rate of glycolysis in the normal tracheal cells. This was supported by the findings of a two-fold greater glucose consumption and two-fold higher production of lactic acid isolated from the NPC medium. Lactate dehydrogenase activity was also two-fold higher in NPC. Thus, despite the apparently higher level of pyruvate production in the NPC, exogenous pyruvate is necessary to satisfy the metabolic needs of NPC. The utilization of [U-14C]glutamine or [U-14C]alanine was not markedly different between NPC and SPC. Furthermore, radioactivity from both of the amino acids was recovered in lactic acid in the medium, indicating that both cell types can derive pyruvic acid from either glutamine or alanine. SPC apparently do not use these routes to supply higher levels of pyruvic acid for survival in culture. The oxidation of none of the radioactive metabolites into CO2 was distinctly different between NPC and SPC except for the 1.7-fold higher utilization of [1-14C]glucose along the oxidative arm of the pentose cycle in the normal cells.

Malic enzyme and malate dehydrogenase activities in rat tracheal epithelial cells during the progression of neoplasia.

Malic enzyme and malate dehydrogenase (MDH) activities were radiometrically assayed in digitonin fractionated normal primary cultures (NPC), preneoplastic selected primary cultures (SPC) and tumor-derived primary cultures (TPC) of rat tracheal epithelial cells. Carcinogen-altered SPC and TPC selectively grow in the absence of pyruvate, which is required by NPC for survival. Mitochondrial-containing particulate fractions from TPC and especially SPC had markedly higher levels of NADP+-dependent malic enzyme than NPC in the presence or absence of pyruvate. This suggests that induction of mitochondrial malic enzyme activity occurs early in the progression of neoplasia. Malic enzyme activities in the soluble fractions from the various populations were not distinctly different. In contrast, particulate-bound MDH activity was higher in NPC and SPC than TPC in most cases, indicating a decrease in this enzyme late in tumorigenesis.

Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells.

These studies examined the effect of exogenous pyruvate on the growth and differentiation of primary cell cultures of rat tracheal epithelial cells. The cell cultures were derived from outgrowths of tracheal explants, and require pyruvate for survival and growth in the presence of 10% FBS. In pyruvate-supplemented (2 mM) medium, the number of cells attached to the dish increased rapidly, while exfoliation of cells into the medium as well as formation of cornified envelopes were relatively low. The growth response to pyruvate was concentration-dependent in these cell cultures. In the absence of pyruvate, the extent of terminal differentiation to keratinization gradually increased. This was characterized by a cessation of growth after one week, and an increase in exfoliation until all cells had sloughed from the dish. Accompanying these changes was a marked increase in the formation of cornified envelopes. Cells undergoing DNA synthesis were present throughout 2 weeks of culture in pyruvate-deprived medium, even as the total number of cells was diminishing. Several compounds, including other 2-oxocarboxylic acids, were ineffective growth substitutes for pyruvate. These results indicate that the requirement for pyruvate is quite stringent in these cultures and that one way pyruvate promotes the growth of tracheal epithelial cells is by inhibiting terminal differentiation.

Hexose uptake in 7,12-dimethylbenz(a)anthracene-preexposed rat tracheal epithelial cells during the progression of neoplasia.

Hexose uptake during the progression of neoplasia in rat tracheal epithelial cells was studied by measuring the uptake of 2-deoxy[3H]glucose (2-dGlc) in nontumorigenic (C-18) and tumorigenic (T-8, 1000-WT) rat tracheal epithelial cell lines with varying degrees of cell association as well as in: normal primary cell cultures (NPC) derived from explants of nonexposed tracheas; selected primary cell cultures (SPC) generated from explants of 7,12-dimethylbenz(a)anthracene-treated tracheal implants; and primary tumor cell cultures (TPC) derived from explants of 7,12-dimethylbenz(a)anthracene-induced tracheal carcinomas. The latter two groups represented cells from earlier and late stages in the progression of neoplasia, respectively, and each displayed an in vitro growth advantage that allowed for their survival and growth in medium devoid of supplements of pyruvate and insulin. This property was used in this study to select the carcinogen-altered cells from neighboring normal cells. Uptake of 2-dGlc per microgram of DNA was similar in subconfluent cultures of all cell lines. At confluency, uptake per microgram of DNA was reduced markedly (greater than 3-fold) in C-18 cells but it was reduced only 1.3-fold in T-8 cells and 1.6-fold in 1000-WT cells. Hexose uptake was further reduced in T-8 and 1000-WT cell cultures generated as outgrowths from explants of denuded tracheas bearing a reestablished epithelium from each cell line. Under these conditions, T-8 cells retained higher 2-dGlc uptake than did C-18, but uptake by 1000-WT was lower, indicating that tissue-like cell associations have a profound effect on hexose uptake in these epithelial cells. Results were generally similar when uptake was expressed per mg of protein although, in several instances, the interpretation of uptake data was affected by differences in the protein content between cultures (assessed by comparing protein:DNA ratios). Compared to NPC, hexose uptake was lower in SPC and one group of TPC. A second group of TPC, characterized by loose cell associations and much cell overlapping, had distinctly higher 2-dGlc uptake than did controls. Comparable results in these primary cultures were also observed when the number of cells per culture was used as a reference for 2-dGlc uptake. Under conditions of glucose deprivation, hexose uptake was increased in NPC and SPC. The production of lactic acid in each type of culture was dependent on the level of glucose in the medium, and this was nearly 2-fold greater in NPC than in SPC.(ABSTRACT TRUNCATED AT 400 WORDS)