Understanding the role of keratins 8 and 18 in neoplastic potential of breast cancer derived cell lines.

BACKGROUND: Breast cancer is a complex disease which cannot be defined merely by clinical parameters like lymph node involvement and histological grade, or by routinely used biomarkers like estrogen receptor (ER), progesterone receptor (PGR) and epidermal growth factor receptor 2 (HER2) in diagnosis and prognosis. Breast cancer originates from the epithelial cells. Keratins (K) are cytoplasmic intermediate filament proteins of epithelial cells and changes in the expression pattern of keratins have been seen during malignant transformation in the breast. Expression of the K8/18 pair is seen in the luminal cells of the breast epithelium, and its role in prognostication of breast cancer is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have modulated K8 expression to understand the role of the K8/18 pair in three different breast epithelium derived cell lines: non-transformed MCF10A, transformed but poorly invasive MDA MB 468 and highly invasive MDA MB 435. The up-regulation of K8 in the invasive MDA MB 435 cell line resulted in a significant decrease in proliferation, motility, in-vitro invasion, tumor volume and lung metastasis. The down-regulation of K8 in MDA MB 468 resulted in a significant increase in transformation potential, motility and invasion in-vitro, while MCF10A did not show any changes in cell transformation assays. CONCLUSIONS/SIGNIFICANCE: These results indicate the role of K8/18 in modulating invasion in breast cancer -its presence correlating with less invasive phenotype and absence correlating with highly invasive, dedifferentiated phenotype. These data may have important implications for prognostication of breast cancer.

Staurosporine-induced growth inhibition of glioma cells is accompanied by altered expression of cyclins, CDKs and CDK inhibitors.

Staurosporine was found to bring about complete growth inhibition of human glioma cell lines. U87 MG cells were arrested in S phase while U373 MG cells in G2/M phase on staurosporine treatment. Consistent with this observation, no change in G1 phase regulators viz., Cyclin D1, D3 and CDK4 was seen on staurosporine treatment. The levels of CDK2, CDC2, Cyclin A and Cyclin B proteins decreased, while the levels of CDK inhibitors viz., p21 and p27 were found to increase on staurosporine treatment. The mRNA levels of CDK2 and CDC2 genes were also found to decrease on staurosporine treatment. Thus apart from staurosporine's known direct inhibitory effect on CDK2 and CDC2 activities, staurosporine was found to down-regulate activities of these two kinases by modulating the expression of the kinases themselves as well that of their activating partners (Cyclins) and their inhibitors.

Induction of BAALC and down regulation of RAMP3 in astrocytes treated with differentiation inducers.

Genes controlling proliferation and differentiation of astrocytes are likely to play an important role in the pathogenesis of astrocytic gliomas and could serve as therapeutic targets. mRNA differential display analysis identified two genes, viz. BAALC and RAMP3, whose expression is altered in primary astrocytes treated with differentiation inducers. BAALC, which has been reported to be expressed in neural progenitor cells, was found to be up regulated in both normal and malignant astrocytes on inhibition of proliferation. RAMP3 functions as a modulator of the receptor for adrenomedullin (AM). AM has been suggested to act as autocrine/paracrine growth factor for gliomas. Our studies show RAMP3 down regulation on staurosporine treatment of astrocytes, suggesting protein kinase C inhibition as a possible strategy for inhibiting AM activity and thereby growth of glioma cells.

Activated JNK brings about accelerated apoptosis of Bcl-2-overexpressing C6 glioma cells on treatment with tamoxifen.

Tamoxifen causes apoptosis of malignant glial cells at a concentration that does not kill normal astrocytes. C6 glioma cells were stably transfected with a vector expressing Bcl-2 under the control of metallothionin promoter. Low leaky Bcl-2 expression offered complete protection against tamoxifen-induced apoptosis. High Bcl-2 levels, on the other hand, accelerated the apoptosis, with Bcl-2-overexpressing clones dying within 48 h of tamoxifen treatment as compared to 6 days for parental C6 cells. Overexpressed Bcl-2 is localized primarily in mitochondria and to a much lower extent in endoplasmic reticulum (ER). Only a minor fraction of the overexpressed Bcl-2 gets phosphorylated in tamoxifen-treated cells and the phosphorylation does not affect its binding to Bax. Tamoxifen treatment of Bcl-2-overexpressing clones was found to result in activation of c-Jun N-terminal kinase (JNK) and p38 kinase. Inhibition of JNK but not p38 kinase completely abrogated the accelerated apoptosis. Constitutively expressed endogenous c-Jun was found to be phosphorylated, resulting in increased activator protein 1 (AP-1) DNA-binding activity. Expression of Fas ligand (FasL), an AP-1 transcriptional target, increased during accelerated cell death. This presumably brought about activation of caspase 8, as inhibition of caspase 8 blocked the apoptosis. The JNK/c-Jun/AP-1/FasL pathway could be considered as a potential target for the therapy of gliomas.

Overexpression of the immediate early gene fra-1 inhibits proliferation, induces apoptosis, and reduces tumourigenicity of c6 glioma cells.

Hexamethylene bisacetamide (HMBA)-induced growth inhibition and differentiation of the rat C6 glioma cell line were found to be accompanied by down-regulation of the constitutively expressed fra-1 gene. In order to check if the fra-1 gene down-regulation was essential for HMBA's growth inhibitory effect, C6 cells were stably transfected with vector expressing fra-1 cDNA under CMV promoter in either sense or antisense orientation. Contrary to the expectations, fra-1 overexpression was found to inhibit proliferation and induce morphological differentiation of C6 cells. Furthermore, all three differentiation inducers studied viz. dibutyryl cyclic AMP (dbcAMP), staurosporine, and HMBA have greater growth inhibitory effect on fra-1 overexpressing clones as compared to the parental C6 cells. dbcAMP and staurosporine not only inhibit proliferation but bring about complete apoptosis of fra-1 overexpressing clones. Spontaneous apoptosis is seen in fra-1 overexpressing clones especially in confluent cultures. fra-1 overexpression also results in substantial reduction in anchorage-independent growth and tumourigenicity of C6 cells. Overexpression of fra-1 leading to proliferation inhibition of C6 glioma cells is consistent with the concept that fra-1 functions as a negative regulator of AP-1 activity.