Increased protein kinase C (PKC) activity in malignant breast tissue and positive correlations between PKC activity and expression of a more aggressive phenotype in breast cancer cell lines suggest a role for this signal transduction pathway in the pathogenesis and/or progression of breast cancer. To examine the role of PKC in the progression of breast cancer, human MCF-7 breast cancer cells were transfected with PKC-a, and a group of heterogenous cells stably overexpressing PKC-a were isolated (MCF-7-PKCa). MCF-7-PKC-a cells expressed fivefold higher levels of PKC-a as compared to parental or vector-transfected MCF-7 cells. MCF-7-PKC-a cells also displayed a substantial increase in endogenous expression of PKC-8 and decreases in expression of the novel 6-and q-PKC isoforms.MCF-7-PKC-a cells displayed an enhanced proliferative rate, anchorage-independent growth, dramatic morphologic alterations including loss of an epithelioid appearance, and increased tumorigenicity in nude mice. MCF-7-PKCa cells exhibited a significant reduction in estrogen receptor expression and decreases in estrogen-dependent gene expression. These findings suggest that the PKC pathway may modulate progression of breast cancer to a more aggressive neoplastic process. (J. Clin. Invest. 1995. 95:1906-1915

Protein kinase C (PKC) modulates growth, differentiation, and apoptosis in a cell-specific fashion. Overexpression of PKC-a in MCF-7 breast cancer cells (MCF-7-PKC-a cell) leads to expression of a more transformed phenotype. The response of MCF-7 and MCF-7-PKC-c cells to phorbol esters (TPA) was examined. TPA-treated MCF-7 cells demonstrated a modest cytostatic response associated with a G, arrest that was accompanied by Cipl expression and retinoblastoma hypophosphorylation. While p53 was detected in MCF-7 cells, evidence for TPA-induced stimulation of p53 transcriptional activity was not evident. In contrast, TPA treatment induced death of MCF-7-PKC-a cells. Bryostatin 1, another PKC activator, exerted modest cytostatic effects on MCF-7 cells while producing a cytotoxic response at low doses in MCF-7-PKC-a cells that waned at higher concentrations. TPA-treated MCF-7-PKC-a cells accumulated in G2/M, did not express p53, displayed decreased Cipl expression, and demonstrated a reduction in retinoblastoma hypophosphorylation. TPA-treated MCF-7-PKC-a cells expressed gadd45 which occurred before the onset of apoptosis. Thus, alterations in the PKC pathway can modulate the decision of a breast cancer cell to undergo death or differentiation. In addition, these data show that PKC activation can induce expression ofgadd45 in a p53-independent fashion. (J.

Insulin receptor substrate-1 (IRS-1) is involved in insulin signal transduction distal to receptor occupation. Targeted disruption of IRS-1 leads to insulin resistance and hyperglycemia in mice, which suggests that altered IRS-1 expression could contribute to the insulin resistance seen in non-insulin-dependent diabetes mellitus. In vitro studies using phorbol esters have implicated the protein kinase C (PKC) pathway as being involved in the pathogenesis of insulin resistance. Using the MCF-7 breast cancer cell, a role for PKC in regulating IRS-1 expression was examined. In an MCF-7 cell line (MCF-7-PKC-␣) that exhibits multiple alterations in PKC isoform expression, IRS-1 content was reduced to negligible levels relative to parental MCF- Insulin receptor substrate-1 (IRS-1) 1 is a cytoplasmic protein phosphorylated on tyrosine residues by ligand-induced activation of the insulin receptor (1). Insulin receptor-induced tyrosine phosphorylation of IRS-1 stimulates its binding to the 85-kDa subunit of phosphatidylinositol-3-kinase and other Src homology 2 domain-containing proteins (2-4). These phosphorylation-dependent interactions involving the IRS-1 molecule are essential in mediating signal transduction distal to occupation of the insulin receptor. Evidence of the importance of IRS-1 in insulin signal transduction is the insulin resistance, hyperinsulinemia, and mild hyperglycemia seen in mice harboring a targeted disruption of the IRS-1 gene (5, 6).

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