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.

Activation of mitogen-activated protein kinase (Erk/ MAPK) is a critical signal transduction event for estrogen (E 2 )-mediated cell proliferation. Recent studies from our group and others have shown that persistent activation of Erk plays a major role in cell migration and tumor progression. The signaling mechanism(s) responsible for persistent Erk activation are not fully characterized, however. In this study, we have shown that E 2 induces a slow but persistent activation of Erk in MCF-7 breast carcinoma cells. The E 2 -induced Erk activation is dependent on new protein synthesis, suggesting that E 2 -induced growth factors play a major role in Erk activation. When MCF-7 cells were treated with E 2 in the presence of an anti-HER-2 monoclonal antibody (herceptin), 60 -70% of E 2 -induced Erk activation is blocked. In addition, when untreated MCF-7 cells were exposed to conditioned medium from E 2 -treated cells, Erk activity was significantly enhanced. Furthermore Erk activity was blocked by an antibody against HER-2 or by heregulin (HRG) depletion from the conditioned medium through immunoprecipitation. In contrast, epidermal growth factor receptor (Ab528) antibody only blocked 10 -20% of E 2 -induced Erk activation, suggesting that E 2 -induced Erk activation is predominantly mediated through the secretion of HRG and activation of HER-2 by an autoctine/paracrine mechanism. Inhibition of PKC-␦-mediated signaling by a dominant negative mutant or the relatively specific PKC-␦ inhibitor rottlerin blocked most of the E 2 -induced Erk activation but had no effect on TGF␣-induced Erk activation. By contrast inhibition of Ras, by inhibition of farnesyl transferase (Ftase-1) or dominant negative (N17)-Ras, significantly inhibited both E 2 -and TGF␣-induced Erk activation. This evaluation of downstream signaling revealed that E 2 -induced Erk activation is mediated by a HRG/HER-2/PKC-␦/Ras pathway that could be crucial for E 2 -dependent growth-promoting effects in early stages of tumor progression.

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.

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 ERpositive and non-invasive MCF-7 human breast cancer cells. In MCF-7 cells, TGFa stimulation induced only transient MAPK activation, leading to a transient increase in cell migration. However, MDA 231 and MDA 468 cells, TGFa 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 signi®cantly inhibits cell migration and in vitro invasion. In contrast, MCF-7 cells expressing constitutively activated MEK show a signi®cant increase in MAPK activity and cell migration, but this failed to enhance in vitro invasion. The kinetic pro®les 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 su cient to allow in vitro invasion in noninvasive MCF-7 breast cancer cells. Oncogene (2001) 20, 4209 ± 4218.

The estrogen receptor alpha (ERa) 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 e ect of the cross-talk on estradiol (E 2 )-mediated signaling, tumor growth and its e ect on anti-estrogen therapy. Our ®ndings 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 ERadriven transcriptional activation. In MEK/MCF-7 cells maximal transactivation levels were achieved in response to treatment with much lower E 2 concentrations (10 710 M E 2 ) when compared to MCF-7 control cells (10 78 M E 2 ). Furthermore, we have seen an increased association between ERa 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 E 2 . Immunohistochemical staining demonstrates that progesterone receptor (PR) and pS2, two E 2 -regulated gene products, are signi®-cantly increased in MEK/MCF-7 cell tumors compared to those of MCF-7 control tumors, suggesting that activation of ERa by MAPK enhances the expression of E 2 -regulated genes and accelerates tumor growth. Remarkably, the antiestrogens tamoxifen and ICI 182,780, were shown both in vitro and in vivo studies to e ciently antagonize the stimulatory e ects of E 2 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 ERa-mediated signaling and accelerates E 2 -dependent tumor growth without diminishing sensitivity to the inhibitory e ects of anti-estrogens.

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. © 1999 Wiley‐Liss, Inc.

The EGF family of proteins encompasses several polypeptides such as epidermal growth factor (EGF), transforming growth factor alpha (TGFa), amphiregulin (AR) and heregulin (HRG-b1). 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 ligandinduced DNA synthesis and secretion of MMP-9 and MMP-2 in metastatic SKBR-3 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 HRGinduced cell proliferation, it had no effect on MMP-9 induced by EGF and AR. Experimental evidence suggests that signaling mechanisms for cell proliferation and MMP-9 induction are mediated by different pathways down-stream of EGF receptor autophosphorylation or that low levels of EGFinduced 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.

The triple negative breast cancer (TNBCs) and non-small cell lung cancers (NSCLCs) often acquire mutations that contribute to failure of drugs in clinic and poor prognosis, thus presenting an urgent need to develop new and improved therapeutic modalities. Here we report that CARP-1 functional mimetic (CFMs) compounds 4 and 5, and 4.6, a structurally related analog of CFM-4, are potent inhibitors of TNBC and NSCLC cells in vitro. Cell growth suppression by CFM-4 and -4.6 involved interaction and elevated expression of CARP-1/CCAR1 and Death Effector Domain (DED) containing DNA binding (DEDD)2 proteins. Apoptosis by these compounds also involved activation of pro-apoptotic stress-activated kinases p38 and JNK1/2, cleavage of PARP and loss of mitotic cyclin B1. Both the CFMs inhibited abilities of NSCLC and TNBC cells to migrate, invade, and form colonies in suspension, while disrupting tubule formation by the human umbilical vein endothelial cells (HUVECs). Nano-lipid formulation of CFM-4 (CFM-4 NLF) enhanced its serum bioavailability when compared with the free CFM-4. Oral administration of CFM-4 NLF reduced weights and volume of the xenografted tumors derived from A549 NSCLC and MDA-MB-231 TNBC cells. Although no gross tissue or histological toxicities were noticed, the immuno-histochemical analysis revealed increased CARP-1 and DNA fragmentation in tumors of the CFM-4 NLF-treated animals. In conclusion, while stimulation of pro-apoptotic CARP-1 and DEDD2 expression and their binding underscore a novel mechanism of apoptosis transduction by CFM compounds, our proof-of-concept xenograft studies demonstrate therapeutic potential of CFM-4 for TNBC and NSCLC.