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, TGFalpha stimulation induced only transient MAPK activation, leading to a transient increase in cell migration. However, MDA 231 and MDA 468 cells, TGFalpha 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.     

CSF-1 activates MAPK-dependent and p53-independent pathways to induce growth arrest of hormone-dependent human breast cancer cells

The CSF-1 receptor (CSF-1R) is expressed in >50% of human breast cancers. To investigate the consequence of CSF-1R expression, hormone-dependent human breast cancer cell lines, MCF-7 and T-47D, were transfected with CSF-1R. Unexpectedly, CSF-1 substantially inhibited estradiol (E2) and insulin-dependent proliferation of MCF-7 transfectants (MCF-7fms) and prevented cyclin E/cdk2 and cyclin A/cdk2 activation, consistent with a G1 arrest. In contrast, CSF-1 increased DNA synthesis in T-47D transfectants (T-47Dfms) alone and with E2 or insulin. In response to CSF-1, there was a marked and sustained upregulation of the cyclin-dependent kinase inhibitor, p21Waf1/Cip1, in MCF-7fms but not T-47Dfms. CSF-1 also markedly upregulated cyclin D1 in MCF-7fms. The coordinate increase in cyclin D1 and p21 had the effect of decreasing the specific but not absolute activity of cyclin D1/cdk4. p53 was not involved since CSF-1 induction of p21 was unaffected by dominant-negative p53 expression. ERK activation by CSF-1 was robust and sustained in MCF-7fms and to a much lesser extent in T-47Dfms. Using pharmacological and transient transfection approaches, we showed that ERK activation was necessary and sufficient for p21 induction in MCF-7fms. Moreover, activated MEK inhibited E2-stimulated cdk2 activity. Our findings indicate that the consequence of CSF-1R-mediated signals in human breast cancer cells is dependent on the genetic background of the particular tumor.     

TGFBR1*6A enhances the migration and invasion of MCF-7 breast cancer cells through RhoA activation

TGFBR1*6A is a common hypomorphic variant of the type 1 transforming growth factor beta receptor (TGFBR1), which has been associated with increased cancer risk in some studies. Although TGFBR1*6A is capable of switching TGF-beta growth-inhibitory signals into growth-stimulatory signals when stably transfected into MCF-7 breast cancer cells, the biological effects of TGFBR1*6A are largely unknown. To broadly explore the potential oncogenic properties of TGFBR1*6A, we assessed its effects on NIH-3T3 cells as well as its effect on the migration and invasion of MCF-7 cells. We found that TGFBR1*6A has decreased oncogenic properties compared with TGFBR1. However, TGFBR1*6A significantly enhances MCF-7 cell migration and invasion in a TGF-beta signaling-independent manner. Gene expression profiling studies identified two down-regulated genes involved in cell migration and invasion: ARHGAP5, encoding ARHGAP5, and FN1, encoding fibronectin-1 (FN1). ARHGAP5 and FN1 expression was similarly down-regulated in MCF-7 cells stably transfected with a kinase-inactivated TGFBR1*6A construct. Functional assays show that TGFBR1*6A-mediated decreased ARHGAP5 expression is associated with higher RhoA activation, a crucial mediator of cell migration. Extracellular signal-regulated kinase (ERK) activation is also higher in cells that harbor the TGFBR1*6A allele. We conclude that TGFBR1*6A is not an oncogene but enhances MCF-7 cell migration and invasion through RhoA and ERK pathway activation and down-regulates two crucial mediators of this phenotype. These results provide the first evidence that TGFBR1*6A may contribute to cancer progression in a TGF-beta signaling-independent manner.     

Kruppel-like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion

Kruppel-like factor 4 (KLF4) is highly expressed in more than 70% of breast cancers and functions as an oncogene. However, an exact mechanism by which KLF4 enhances tumorigenesis of breast cancer remains unknown. In this study, we show that KLF4 was highly expressed in cancer stem cell (CSC)-enriched populations in mouse primary mammary tumor and breast cancer cell lines. Knockdown of KLF4 in breast cancer cells (MCF-7 and MDA-MB-231) decreased the proportion of stem/progenitor cells as demonstrated by expression of stem cell surface markers such as aldehyde dehydrogenase 1, side population and by in vitro mammosphere assay. Consistently KLF4 overexpression led to an increase of the cancer stem cell population. KLF4 knockdown also suppressed cell migration and invasion in MCF-7 and MDA-MB-231 cells. Furthermore, knockdown of KLF4 reduced colony formation in vitro and inhibited tumorigenesis in immunocompromised non-obese diabetic/severe combined immunodeficiency mice, supporting an oncogenic role for KLF4 in breast cancer development. Further mechanistic studies revealed that the Notch signaling pathway was required for KLF4-mediated cell migration and invasion, but not for CSC maintenance. Taken together, our study provides evidence that KLF4 has a potent oncogenic role in mammary tumorigenesis likely by maintaining stem cell-like features and by promoting cell migration and invasion. Thus, targeting KLF4 may provide an effective therapeutic approach to suppress tumorigenicity in breast cancer.     

Loss of Nrdp1 enhances ErbB2/ErbB3-dependent breast tumor cell growth

Dysregulation of ErbB receptor tyrosine kinases is thought to promote mammary tumor progression by stimulating tumor cell growth and invasion. Overexpression and aberrant activation of ErbB2/HER2 confer aggressive and malignant characteristics to breast cancer cells, and patients displaying ErbB2-amplified breast cancer face a worsened prognosis. Recent studies have established that ErbB2 and ErbB3 are commonly co-overexpressed in breast tumor cell lines and in patient samples. ErbB2 heterodimerizes with and activates the ErbB3 receptor, and the two receptors synergize in promoting growth factor-induced cell proliferation, transformation, and invasiveness. Our previous studies have shown that the neuregulin receptor degradation protein-1 (Nrdp1) E3 ubiquitin ligase specifically suppresses cellular ErbB3 levels by marking the receptor for proteolytic degradation. Here, we show that overexpression of Nrdp1 in human breast cancer cells results in the suppression of ErbB3 levels, accompanied by the inhibition of cell growth and motility and the attenuation of signal transduction pathways. In contrast, either Nrdp1 knockdown or the overexpression of a dominant-negative form enhances ErbB3 levels and cellular proliferation. Additionally, Nrdp1 expression levels inversely correlate with ErbB3 levels in primary human breast cancer tissue and in a mouse model of ErbB2 mammary tumorigenesis. Our observations suggest that Nrdp1-mediated ErbB3 degradation suppresses cellular growth and motility, and that Nrdp1 loss in breast tumors may promote tumor progression by augmenting ErbB2/ErbB3 signaling.     

Human chorionic gonadotropin β induces cell motility via ERK1/2 and MMP-2 activation in human glioblastoma U87MG cells

Human chorionic gonadotropin β (hCGβ) promotes tumorigenesis in a variety of tumors including glioblastoma, breast and prostate cancer cells, etc. However, the involved mechanisms remain elusive. Distinct from the other tumors, glioblastoma is a highly invasive brain tumor; invasion causes high recurrence and mortality. Characterization of hCGβ signaling is to determine therapeutic targets to inhibit invasion and lower recurrence. Through both a stable cell line over-expressing hCGβ and hCGβ standards, we tested hCGβ signaling, migration and invasion in human glioblastoma U87MG cells. ELISA showed that hCGβ secreted into culture medium at an amount of 237.8 ± 7.8 ng/10⁷ cells in hCGβ transfected stable cells after the cells were grown for 24 h. Through Western blot and Gelatin zymography, we found that hCGβ standards phosphorylated ERK1/2 and upregulated MMP-2 expression in dose- and time-dependent manners. Meanwhile, overexpressed hCGβ phosphorylated ERK1/2, and upregulated MMP-2 expression and activity, whereas ERK1/2 blocker PD98059 (25 μM) significantly decreased both ERK1/2 and MMP-2 expression and activity. In addition, in the same conditions as the signaling test, hCGβ promoted cell migration and invasion, whereas the PD98059 diminished these effects. These findings demonstrated that hCGβ phosphorylated ERK1/2 upregulating MMP-2 expression and activity leading to cell migration and invasion, suggesting that hCGβ, ERK1/2 and MMP-2 are the potential targets to inhibit glioblastoma invasion.     

Persistent transactivation of EGFR and ErbB2/HER2 by protease-activated receptor-1 promotes breast carcinoma cell invasion

Hyperactivation of ErbB signaling is implicated in metastatic breast cancer. However, the mechanisms that cause dysregulated ErbB signaling and promote breast carcinoma cell invasion remain poorly understood. One pathway leading to ErbB activation that remains unexplored in breast carcinoma cell invasion involves transactivation by G-protein-coupled receptors (GPCRs). Protease-activated receptor-1 (PAR1), a GPCR activated by extracellular proteases, is overexpressed in invasive breast cancer. PAR1 is also proposed to function in breast cancer invasion and metastasis, but how PAR1 contributes to these processes is not known. In this study, we report that proteolytic activation of PAR1 by thrombin induces persistent transactivation of EGFR and ErbB2/HER2 in invasive breast carcinoma, but not in normal mammary epithelial cells. PAR1-stimulated EGFR and ErbB2 transactivation leads to prolonged extracellular signal-regulated kinase-1 and -2 signaling and promotes breast carcinoma cell invasion. We also show that PAR1 signaling through Galpha(i/o) and metalloprotease activity is critical for ErbB transactivation and cellular invasion. Finally, we demonstrate that PAR1 expression in invasive breast carcinoma is essential for tumor growth in vivo assessed by mammary fat pad xenografts. These studies reveal a critical role for PAR1, a receptor activated by tumor-generated proteases, in hyperactivation of ErbB signaling that promotes breast carcinoma cell invasion.     

c-Src modulates ErbB2 and ErbB3 heterocomplex formation and function

Overexpression and/or gene amplification of c-Src and members of the epidermal growth factor receptor (EGFR/ErbB) family have been implicated in the pathogenesis of breast cancer. Although members of the EGFR family are known to form heterocomplexes with one another, c-Src has also been shown to physically interact with members of this family in breast cancer cell lines and tumors. This paper investigates the role of c-Src in modulating the physical and functional interaction between ErbB2 and ErbB3, two family members that preferentially associate with one another and together exhibit high oncogenic potential. We show that overexpressed wild-type c-Src enhances heterocomplex formation of ErbB2 and ErbB3 that results in increased basal and/or heregulin-induced activation of receptors, and their downstream intracellular effectors. Expression of a kinase-inactive form of c-Src (K(-) c-Src) or pharmacological inhibition of c-Src by PP2 negatively affects these events. Furthermore, cellular motility and anchorage-independent growth promoted by the ErbB2/ErbB3 heterocomplex are dependent upon c-Src, as demonstrated by the effects of K(-) c-Src overexpression or treatment with PP2. In contrast to previous studies that defined a role for c-Src downstream of ErbB2/ErbB3, the current work suggests an upstream mechanism, whereby c-Src enhances ErbB2/ErbB3 signaling and biological functions by positively modulating the association between ErbB2 and ErbB3.     

Prostate-derived ETS factor is a mediator of metastatic potential through the inhibition of migration and invasion in breast cancer

Cell migration and invasion are critical events during the progression to metastasis. Without motile function, cancer cells are unable to leave the primary tumor site, invade through the basement membrane, and form secondary tumors. Expression of the epithelial-specific ETS factor prostate-derived ETS factor (PDEF) is reduced in human invasive breast tissue and lost in invasive breast cancer cell lines. Gain-of-function studies that examine different aspects of cell migration show that constitutive or inducible PDEF reexpression inhibits migration and invasion in multiple breast cancer cell lines, and loss-of-function studies show a stimulation of migration in noninvasive breast cancer cells. Furthermore, the introduction of PDEF into invasive breast cancer cells led to a remodeling of the actin cytoskeleton and altered focal adhesion localization and adherence levels. Cells expressing PDEF no longer form the defined morphologic polarity required for efficient, directional migration. Collectively, these data indicate that PDEF down-regulation in invasive breast cancer may promote actin-mediated cell migration through the extracellular matrix.