Kaladhar B. Reddy

Role of MAP kinase in tumor progression and invasion.

Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is a frequent event in tumorigenesis. MAPKs have been implicated in cell migration, proteinase-induction, regulation of apoptosis, and angiogenesis, events that are essential for successful completion of metastasis. In this review, we discuss the potential role that MAPKs play in metastasis by regulating cell migration, proteinase-induction and apoptosis.

Epidermal growth factor and amphiregulin up‐regulate matrix metalloproteinase‐9 (MMP‐9) in human breast cancer cells

The EGF family of proteins encompasses several polypeptides such as epidermal growth factor (EGF), transforming growth factor alpha (TGFα), amphiregulin (AR) and heregulin (HRG‐β1). These polypeptides regulate proliferation in breast cancer cells through interaction with membrane receptors. It has been previously shown that high EGF receptor number correlates with aggressive behavior and increased metastasis in human breast cancer. In the present study, we investigated the association between EGF and EGF‐like ligand‐induced DNA synthesis and secretion of MMP‐9 and MMP‐2 in metastatic SKBR‐3 cells and non‐metastatic MCF‐7 breast cancer cells. Exposure of SKBR‐3 cells to EGF or Ar induces expression of MMP‐9 but has no effect on MMP‐2 secretion. In contrast to EGF and AR, HRG had no effect on gelatinase induction. None of the EGF polypeptides had any effect on gelatinase induction in MCF‐7 non‐metastatic breast cancer cells. While a relatively specific inhibitor of EGF receptor tyrosine kinase, PD 153035, inhibited EGF‐, AR‐ and HRG‐induced cell proliferation, it had no effect on MMP‐9 induced by EGF and AR. Experimental evidence suggest that signalling mechanisms for cell proliferation and MMP‐9 induction are mediated by different pathways down‐stream of EGF receptor autophosphorylation or that low levels of EGF‐ induced signal that escape inhibition are sufficient to induce MMP‐9 but unable to support cell proliferation. In addition, our results suggest that EGF and AR may modulate invasion of metastatic breast cancer cells by increasing the expression of MMPs. Int. J. Cancer 70:722–726, 1997.

Temporal and quantitative regulation of mitogen-activated protein kinase (MAPK) modulates cell motility and invasion

We have shown that ER-negative and invasive human breast cancer cell lines MDA-MB-468 and MDA-MB-231 have constitutively higher mitogen activated protein kinase (ERK1&2/MAPK) when compared to the ER-positive and non-invasive MCF-7 human breast cancer cells. In MCF-7 cells, TGFα stimulation induced only transient MAPK activation, leading to a transient increase in cell migration. However, MDA 231 and MDA 468 cells, TGFα stimulation induced sustained MAPK activation, which correlated with enhanced cell motility and in vitro invasion. Serum stimulation activates ERK/MAPK activity persistently in both ER-positive and ER-negative breast cancer cells, leading to enhanced and sustained cell migration. Inhibition of MAPK activation by anti-sense MEK expression in MDA-MB-468 cells significantly inhibits cell migration and in vitro invasion. In contrast, MCF-7 cells expressing constitutively activated MEK show a significant increase in MAPK activity and cell migration, but this failed to enhance in vitro invasion. The kinetic profiles of MAPK activation and inhibition show a relationship between the duration and magnitude of MAPK activation and cell migration in both ER-positive and ER-negative human breast cancer cells. These studies show that cell motility is modulated by the magnitude and the duration of MAPK activation; but increased activation of MAPK may not be sufficient to allow in vitro invasion in non-invasive MCF-7 breast cancer cells.

Mitogen-activated protein kinase (MAPK) regulates the expression of progelatinase B (MMP-9) in breast epithelial cells

Mitogen‐activated protein kinases (MAPKs) play a major role in the mitogenic signal transduction pathway and are essential components of both growth and differentiation. Constitutive activation of the MAPK cascade is associated with the carcinogenesis and metastasis of human breast and renal cell carcinomas. The gelatinases B (MMP‐9) and A (MMP‐2) are 2 members of the matrix metalloproteinase (MMPs) family which are expressed in human cancers and thought to play a critical role in tumor cell invasion and metastasis. In a previous study, we have shown that EGF and amphiregulin upregulate MMP‐9 in metastatic SKBR‐3 cells but have no effect on MMP‐2 secretion. We now investigated specific step(s) in EGF‐induced signalling associated with regulation of cell proliferation and MMP‐9 induction. EGF‐induced signalling in SKBR‐3 cells was blocked by relatively specific inhibitors either on ras (FPT inhibitor‐I) or PI3 kinase (Wortmannin) or by reduction in EGF‐induced tyrosine kinase activity (RG 13022). Blocking these signalling pathways significantly inhibited of EGF‐induced cell proliferation but only partially reduced in EGF‐induced MMP‐9 secretion. In contrast, when SKBR‐3 cells were exposed to MEK inhibitor (PD 98059) or MAPK inhibitors (Apigenin or MAPK antisense phosphorothioate oligodeoxynucleotides), EGF‐induced cell proliferation, MMP‐9 induction and invasion through reconstituted basement membrane were significantly reduced. Our results suggest that interfering with MAPK activity may provide a novel means of controlling growth and invasiveness of tumors in which the signalling cascade is activated. Int. J. Cancer 82:268–273, 1999.

MAP kinase/estrogen receptor cross-talk enhances estrogen-mediated signaling and tumor growth but does not confer tamoxifen resistance.

The estrogen receptor alpha (ERα) signaling plays an essential role in breast cancer progression and endocrine therapy. Mitogen-activated protein kinase (MAPK/Erk1/2) has been implicated in ligand-independent activation of ER, resulting in the cross-talk between growth factor and ER mediated signaling. In this study, we examined the effect of the cross-talk on estradiol (E2)-mediated signaling, tumor growth and its effect on anti-estrogen therapy. Our findings demonstrate that expression of constitutively activated mitogen activated kinase kinase (MEK1), an immediate upstream activator of MAPK in estrogen receptor positive MCF-7 breast cancer cells (MEK/MCF-7), showed an increase in ERα-driven transcriptional activation. In MEK/MCF-7 cells maximal transactivation levels were achieved in response to treatment with much lower E2 concentrations (10−10 M E2) when compared to MCF-7 control cells (10−8 M E2). Furthermore, we have seen an increased association between ERα and its nuclear coactivators AIB1 or TIF-2, in MEK/MCF-7 cells relative to those seen in MCF-7 control cells. In addition, in vivo studies show that MEK/MCF-7 cell tumors are ∼threefold larger than those of MCF-7 cell, in the presence of E2. Immunohistochemical staining demonstrates that progesterone receptor (PR) and pS2, two E2-regulated gene products, are significantly increased in MEK/MCF-7 cell tumors compared to those of MCF-7 control tumors, suggesting that activation of ERα by MAPK enhances the expression of E2-regulated genes and accelerates tumor growth. Remarkably, the antiestrogens tamoxifen and ICI 182,780, were shown both in vitro and in vivo studies to efficiently antagonize the stimulatory effects of E2 on ER regulated transactivation and tumor growth in MEK/MCF-7 as well as MCF-7 cell lines. Taken together, these data suggest that MAPK/ER cross-talk enhances ERα-mediated signaling and accelerates E2-dependent tumor growth without diminishing sensitivity to the inhibitory effects of anti-estrogens.

MicroRNA (miRNA) in cancer

In recent years, there has been a tremendous and growing interest among researchers to investigate the role of mircoRNA (miRNA) in normal cellular as well as in disease processes. miRNAs are a family of small non-coding RNAs which were reported to regulate the expression of various oncogenes or tumor suppressor genes. The expression profiling of miRNAs has already entered into cancer clinics as diagnostic and prognostic biomarkers to assess tumor initiation, progression and response to treatment in cancer patients. This review summarizes: (i) the current understanding of interactions between miRNAs and their target genes, (ii) recent advances in the regulatory mechanisms that control the expression of genes related to carcinogenesis, and (iii) the role of miRNAs in cancer diagnosis and therapy.

Upregulation of PKC-δ contributes to antiestrogen resistance in mammary tumor cells

Acquired resistance to tamoxifen (Tam) in breast cancer patients is a serious therapeutic problem. We have previously reported that protein kinase C-delta (PKC-δ) plays a major role in estrogen (E2)-mediated cell proliferation. To determine if PKC-δ is one of the major alternate signaling pathways that supports cell growth in the presence of Tam, we determined the levels of PKC isoforms in four different models of antiestrogen-resistant cells. Three out of four antiestrogen resistance cell lines (Tam/MCF-7, ICI/MCF-7 and HER-2/MCF-7) expressed significantly high levels of both total and activated PKC-δ levels compared to sensitive cells. Estrogen receptor (ER) alpha content and function are maintained in all the antiestrogen-resistant cell lines. Overexpressing active PKC-δ in Tam-sensitive MCF-7 cells (PKC-δ/MCF-7) led to Tam resistance both in vitro and in vivo. Inhibition of PKC-δ by rottlerin (a relatively specific inhibitor of PKC-δ) or siRNA significantly inhibited estrogen- and Tam-induced growth in antiestrogen-resistant cells. PKC-δ levels are significantly higher in Tam-resistant tumors compared to Tam-sensitive tumors in xenograft model (P<0.05). Taken together, these data suggest that PKC-δ plays a major role in antiestrogen resistance in breast tumor cells and thus provides a new target for treatment.

Clinicopathologic analysis of amphiregulin and heregulin immunostaining in breast neoplasia

We observed no association between neoplastic epithelial immunostainingfor either amphiregulin (AR) or heregulin (HRG) and presence of ER,EGFR/ERBB-2 overexpression, nodal status, or disease recurrence in 34 breastcarcinomas. However, stromal cell staining for both correlated with outcome;29% of stromal cell AR⊖ cases recurred vs. 85% forAR⊕ cases (p=0.001), and 41% of stromal cell HRG⊖cases recurred vs. 82% of HRG⊕ cases (p=0.01). Weconclude that both HRG and AR have significant biologic roles in breastcarcinoma growth or progression via mediation of host-tumor interactionswhich favor aggressive tumor behavior.

Tyrosine kinase inhibitor as a novel signal transduction and antiproliferative agent: prostate cancer

In prostate cancer cells, the binding of peptide growth factors to specific receptors increases tyrosine kinases (TK) activity to regulate cell proliferation, cell differentiation, and signaling processes. To determine whether inhibition of receptor TK activity inhibits tumor growth, we studied the effects of a tyrosine kinase inhibitor, RG-13022 (tyrphostin), on cultured human prostate cancer cells. RG-13022 significantly inhibited TGF alpha-induced phosphorylation of EGF receptor (EGFR). This compound inhibited TGF alpha-stimulated [3H]thymidine incorporation in a dose-dependent manner with IC50 being 30 microM. Clonogenicity in soft agar was reduced in the presence of RG-13022. Inhibitory effects were also observed in androgen-positive LNCaP cells and androgen-negative PC3 cells. RG-13022 not only inhibited TGF alpha-induced growth but also growth stimulated by epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF) and serum. In addition, RG-13022 also blocked androgen-stimulated cell proliferation, suggesting that functioning TK pathways are required for androgen-induced growth. This novel synthetic inhibitor may be useful in providing a new strategy for future therapeutic intervention for prostate cancer.

Enhanced Anticancer Effect of the Combination of Cisplatin and TRAIL in Triple-Negative Breast Tumor Cells

Women with triple-negative breast cancer (TNBC) have a worse prognosis compared with other breast cancer subtypes. Hormonal or Herceptin-based therapies were found to be ineffective because of the loss of target receptors, such as ER, PR, and HER-2 amplification. Conventional chemo- and/ or radiation therapy also seems to have limited efficacy in TNBC patients. We studied the effects of cisplatin plus TRAIL on 1 normal and 2 TNBC cells in vitro. The in vitro studies indicate that cisplatin plus TRAIL significantly enhanced cell death in TNBC cell lines CRL2335 and MDA-MB-468 by approximately 60%–70% compared with approximately 10%–15% in CRL8799 normal breast cell line. Treatment with cisplatin/TRAIL also inhibited the expression of EGFR, p63, survivin, Bcl-2, and Bcl-xL in TNBC cells. Specific inhibition of EGFR and/or p63 protein in TNBC cells by small interfering RNA (siRNA) does not increase TRAIL-induced apoptosis. However, inhibition of survivin by siRNA enhances TRAIL-induced apoptosis. These observations suggested the possibility that survivin played an important role in cisplatin plus TRAIL-induced apoptosis in TNBC cells. In vivo experiments, treatment of mice with cisplatin plus TRAIL resulted in a significant inhibition of CRL2335 xenograft tumors compared with untreated control tumors. Taken together the data suggest that cisplatin plus TRAIL treatment have the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients. Mol Cancer Ther; 10(3); 550–7. ©2011 AACR.