Darcie D. Seachrist

HER2/ErbB2-induced Breast Cancer Cell Migration and Invasion Require p120 Catenin Activation of Rac1 and Cdc42

Breast cancers that overexpress the receptor tyrosine kinase ErbB2/HER2/Neu result in poor patient outcome because of extensive metastatic progression. Herein, we delineate a molecular mechanism that may govern this malignant phenotype. ErbB2 induction of migration requires activation of the small GTPases Rac1 and Cdc42. The ability of ErbB2 to activate these small GTPases necessitated expression of p120 catenin, which is itself up-regulated by signaling through ErbB2 and the tyrosine kinase Src. Silencing p120 in ErbB2-dependent breast cancer cell lines dramatically inhibited migration and invasion as well as activation of Rac1 and Cdc42. In contrast, overexpression of constitutively active mutants of these GTPases reversed the effects of p120 silencing. Lastly, ectopic expression of p120 promoted migration and invasion and potentiated metastatic progression of a weakly metastatic, ErbB2-dependent breast cancer cell line. These results suggest that p120 acts as an obligate intermediate between ErbB2 and Rac1/Cdc42 to modulate the metastatic potential of breast cancer cells.

Gene expression profiling of cancer progression reveals intrinsic regulation of transforming growth factor-β signaling in ErbB2/Neu-induced tumors from transgenic mice

Upregulation of HER2/ErbB2/Neu occurs in 15–30% of human breast cancers and correlates with poor prognosis. Identification of ErbB2/Neu transcriptional targets should facilitate development of novel therapeutic approaches. Development of breast cancer is a multistep process; thus, to identify the transcriptomes associated with different stages of progression of tumorigenesis, we compared expression profiles of mammary tumors and preneoplastic mammary tissue from MMTV-Neu transgenic mice to expression profiles of wild-type mammary glands using Affymetrix microarrays. We identified 324 candidate genes that were unique to ErbB2/Neu-induced tumors relative to normal mammary gland tissue from wild-type controls. Expression of a subset of these genes (82) was also changed in the preneoplastic mammary glands compared to wild-type controls, indicating that they may play a pivotal role during early events of ErbB2/Neu-initiated mammary tumorigenesis. Further analysis of the microarray data revealed that expression of several known transforming growth factor (TGF)-β target genes was altered, suggesting that the TGF-β signaling cascade is downregulated in ErbB2/Neu-induced tumors. Western blot analysis for TGF-β-Receptor-I/ALK5 and immunohistochemistry for TGF-β-Receptor-I/ALK5 and phosphorylated/activated Smad2 confirmed that the Smad-dependent TGF-β signaling cascade was inactive in these tumors. Although absent in most of the tumor, phosphorylated Smad2 was present in the periphery of tumors. Interestingly, presence of phosphorylated/activated Smad2 correlated with expression of Activin-Receptor-IB/ALK4, suggesting that although Smad-dependent TGF-β signaling is absent in ErbB2/Neu-induced tumors, Activin signaling may be active at the leading edge of these tumors. Cumulatively, these data indicate that the TGF-β pathway is intrinsically suppressed in ErbB2/Neu tumors via a mechanism involving loss of TGF-β-Receptor-I/ALK5.

Fatty Acid-binding Protein 5 and PPARβ/δ Are Critical Mediators of Epidermal Growth Factor Receptor-induced Carcinoma Cell Growth

Epidermal growth factors and their receptors (EGFRs) promote breast cancer cell proliferation and can drive tumorigenesis. However, the molecular mechanisms that mediate these effects are incompletely understood. We previously showed that mammary tumor development in the mouse model of breast cancer MMTV-neu, a model characterized by amplification of the EGFR ErbB2 in mammary tissue, correlates with a marked up-regulation of fatty acid-binding protein 5 (FABP5). FABP5 functions to deliver ligands to and enhance the transcriptional activity of the nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a receptor whose target genes include genes involved in cell growth and survival. We show here that in MCF-7 mammary carcinoma cells, EGFR signaling directly up-regulates the expression of FABP5. The data demonstrate that treatment of these cells with the EGFR ligand heregulin-β1 signals through the ERK and the phophatidylinositol-3-kinase cascades, resulting in activation of the transcription factor NF-κB. In turn, NF-κB induces the expression of FABP5 through two cognate response elements in the promoter of this gene. The observations further demonstrate that FABP5 and PPARβ/δ are critical mediators of the ability of EGFR to enhance cell proliferation, indicating that this transcriptional pathway plays a key role in EGFR-induced tumorigenesis. Additional observations indicate that the expression of FABP5 is down-regulated by the Krüppel-like factor KLF2, suggesting a tumor suppressor activity for this factor.

Genetic ablation of the fatty acid binding protein FABP5 suppresses HER2-induced mammary tumorigenesis

The fatty acid-binding protein FABP5 shuttles ligands from the cytosol to the nuclear receptor PPARβ/δ (encoded for by Pparδ), thereby enhancing the transcriptional activity of the receptor. This FABP5/PPARδ pathway is critical for induction of proliferation of breast carcinoma cells by activated epidermal growth factor receptor (EGFR). In this study, we show that FABP5 is highly upregulated in human breast cancers and we provide genetic evidence of the pathophysiologic significance of FABP5 in mammary tumorigenesis. Ectopic expression of FABP5 was found to be oncogenic in 3T3 fibroblasts where it augmented the ability of PPARδ to enhance cell proliferation, migration, and invasion. To determine whether FABP5 is essential for EGFR-induced mammary tumor growth, we interbred FABP5-null mice with MMTV-ErbB2/HER2 oncomice, which spontaneously develop mammary tumors. FABP5 ablation relieved activation of EGFR downstream effector signals, decreased expression of PPARδ target genes that drive cell proliferation, and suppressed mammary tumor development. Our findings establish that FABP5 is critical for mammary tumor development, rationalizing the development of FABP5 inhibitors as novel anticarcinogenic drugs.

Rapamycin inhibits multiple stages of c-Neu/ErbB2-induced tumor progression in a transgenic mouse model of HER2-positive breast cancer

Amplification of the HER2 (ErbB2, c-Neu) proto-oncogene in breast cancer is associated with poor prognosis and high relapse rates. HER2/ErbB2, in conjunction with ErbB3, signals through the Akt/phosphatidylinositol 3-kinase pathway and leads to the activation of mammalian target of rapamycin (mTOR), a critical mRNA translation regulator that controls cell growth. Gene expression analysis of mammary tumors collected from mouse mammary tumor virus-c-Neu transgenic mice revealed that mRNA levels of several mTOR pathway members were either up-regulated (p85/phosphatidylinositol 3-kinase and p70S6 kinase) or down-regulated (eIF-4E-BP1) in a manner expected to enhance signaling through this pathway. Treatment of these mice with the mTOR inhibitor rapamycin caused growth arrest and regression of primary tumors with no evidence of weight loss or generalized toxicity. The treatment effects were due to decreased proliferation, associated with reduced cyclin D1 expression, and increased cell death in primary tumors. Whereas many of the dead epithelial cells had the histopathologic characteristics of ischemic necrosis, rapamycin treatment was not associated with changes in microvascular density or apoptosis. Rapamycin also inhibited cellular proliferation in lung metastases. In summary, data from this preclinical model of ErbB2/Neu-induced breast cancer show that inhibition of the mTOR pathway with rapamycin blocks multiple stages of ErbB2/Neu-induced tumorigenic progression. [Mol Cancer Ther 2007;6(8):2188–97]

LMO4 is an essential mediator of ErbB2/HER2/Neu-induced breast cancer cell cycle progression.

ErbB2/HER2/Neu-overexpressing breast cancers are characterized by poor survival due to high proliferation and metastasis rates and identifying downstream targets of ErbB2 should facilitate developing novel therapies for this disease. Gene expression profiling revealed the transcriptional regulator LIM-only protein 4 (LMO4) is upregulated during ErbB2-induced mouse mammary gland tumorigenesis. Although LMO4 is frequently overexpressed in breast cancer and LMO4-overexpressing mice develop mammary epithelial tumors, the mechanisms involved are unknown. In this study, we report that LMO4 is a downstream target of ErbB2 and PI3K in ErbB2-dependent breast cancer cells. Furthermore, LMO4 silencing reduces proliferation of these cells, inducing a G2/M arrest that was associated with decreased cullin-3, an E3-ubiquitin ligase component important for mitosis. Loss of LMO4 subsequently results in reduced Cyclin D1 and Cyclin E. Further supporting a role for LMO4 in modulating proliferation by regulating cullin-3 expression, we found that LMO4 expression oscillates throughout the cell cycle with maximum expression occurring during G2/M and these changes precede oscillations in cullin-3 levels. LMO4 levels are also highest in high-grade/less differentiated breast cancers, which are characteristically highly proliferative. We conclude that LMO4 is a novel cell cycle regulator with a key role in mediating ErbB2-induced proliferation, a hallmark of ErbB2-positive disease.

Embryonic Expression of the Luteinizing Hormone β Gene Appears to Be Coupled to the Transient Appearance of p8, a High Mobility Group-related Transcription Factor

A comparison between two pituitary-derived cell lines (αT3-1 and LβT2) that represent gonadotropes at early and late stages of development, respectively, was performed to further elucidate the genomic repertoire required for gonadotrope specification and luteinizing hormone β (LHβ) gene expression. One isolated clone that displayed higher expression levels in LβT2 cells encodes p8, a high mobility group-like protein with mitogenic potential that is up-regulated in response to proapoptotic stimuli and in some developing tissues. To test the functional significance of this factor in developing gonadotropes, a knockdown of p8 in LβT2 cells was generated. The loss of p8 mRNA correlated with loss of endogenous LHβ mRNA and the loss of activity of a transfected LHβ promoter-driven reporter, even upon treatment with gonadotropin-releasing hormone. In addition, expression of p8 mRNA in developing mouse pituitary glands mirrored its expression in the gonadotrope-derived cell lines and coincided with the first detectable appearance of LHβ mRNA. In contrast, p8 mRNA was undetectable in the pituitary glands of normal adults. Taken together, our data indicate that p8 is a stage-specific component of the gonadotrope transcriptome that may play a functional role in the initiation of LHβ gene expression during embryonic cellular differentiation.

The double-stranded RNA-binding protein, PACT, is required for postnatal anterior pituitary proliferation

PACT is a double-stranded RNA-binding protein that also binds and activates the latent protein kinase, PKR, which plays a major role in cellular antiviral defense in mammals. For evaluating PACTs contribution to the innate immune system, Pact−/− mice have been generated; these mice exhibit notable developmental abnormalities including microtia, with craniofacial, ear, and hearing defects. Here we report that, in addition, Pact−/− mice had smaller body size and fertility defects, both of which were caused by defective pituitary functions. Pact−/− mice exhibited anterior pituitary lobe (AL) hypoplasia, which developed postnatally, when the second phase of pituitary expansion occurs. Among the 5 cell types in AL, the numbers of corticotrophs, gonadotrophs, and somatotrophs were equally decreased in Pact−/− mice with a greater impact on lactotrophs and a lesser impact on thyrotrophs. PACT mRNA and protein were highly expressed in the pituitary of wild-type (Wt) mice during the postnatal wave of AL proliferation, the same period in which the hypoplasia developed in Pact−/− mice. During this time, the pituitaries of Pact−/− mice did not exhibit significantly increased apoptosis compared with Wt mice but showed a decrease in cell proliferation. The inhibition of cell proliferation observed in vivo could be recapitulated in vitro in GH3 somato/lactotroph and LβT2 gonadotroph cell lines; knockdown of PACT expression with siRNA diminished the rate of proliferation of these cells. Our study revealed a physiologically significant role for PACT in cell proliferation and an essential role of a dsRNA-binding protein in mammalian pituitary expansion.

Sustained trophism of the mammary gland is sufficient to accelerate and synchronize development of ErbB2/Neu-induced tumors

Epidemiological studies indicate that parity enhances HER2/ErbB2/Neu-induced breast tumorigenesis. Furthermore, recent studies using multiparous, ErbB2/Neu-overexpressing mouse mammary tumor virus (MMTV-Neu) mice have shown that parity induces a population of cells that are targeted for ErbB2/Neu-induced transformation. Although parity accelerates mammary tumorigenesis, the pattern of tumor development in multiparous MMTV-Neu mice remains stochastic, suggesting that additional events are required for ErbB2/Neu to cause mammary tumors. Whether such events are genetic in nature or reflective of the dynamic hormonal control of the gland that occurs with pregnancy remains unclear. We postulated that young age at pregnancy initiation or chronic trophic maintenance of mammary epithelial cells might provide a cellular environment that significantly increases susceptibility to ErbB2/Neu-induced tumorigenesis. MMTV-Neu mice that were maintained pregnant or lactating beginning at 3 weeks of age demonstrated accelerated tumorigenesis, but this process was still stochastic, indicating that early pregnancy does not provide the requisite events of tumorigenesis. However, bitransgenic mice that were generated by breeding MMTV-Neu mice with a luteinizing hormone-overexpressing mouse model of ovarian hyperstimulation developed multifocal mammary tumors in an accelerated, synchronous manner compared to virgin MMTV-Neu animals. This synchrony of tumor development in the bitransgenic mice suggests that trophic maintenance of the mammary gland provides the additional events required for tumor formation and maintains the population of cells that are targeted by ErbB2/Neu for transformation. Both the synchrony of tumor appearance and the ability to characterize a window of commitment by ovariectomy/palpation studies permitted microarray analysis to evaluate changes in gene expression over a defined timeline that spans the progression from normal to preneoplastic mammary tissue. These approaches led to identification of several candidate genes whose expression changes in the mammary gland with commitment to ErbB2/Neu-induced tumorigenesis, suggesting that they may either be regulated by ErbB2/Neu and/or contribute to tumor formation.

Aberrant expression of LMO4 induces centrosome amplification and mitotic spindle abnormalities in breast cancer cells

The LIM‐only protein, LMO4, is a transcriptional modulator overexpressed in breast cancer. It is oncogenic in murine mammary epithelium and is required for G2/M progression of ErbB2‐dependent cells as well as growth and invasion of other breast cancer cell types. However, the mechanisms underlying the oncogenic activity of LMO4 remain unclear. Herein, we show that LMO4 is expressed in all breast cancer subtypes examined and its expression level correlates with the degree of proliferation of such tumours. In addition, we have determined that LMO4 silencing induces G2/M arrest in cells from various breast cancer subtypes, suggesting that LMO4 action in the cell cycle is not restricted to a single breast cancer subtype. This arrest was accompanied by increased cell death, amplification of centrosomes, and formation of abnormal mitotic spindles. Consistent with its ability to positively and negatively regulate the formation of active transcription complexes, overexpression of LMO4 also resulted in an increase in centrosome number. Centrosome amplification has been shown to prolong the G2/M phase of the cell cycle and induce apoptosis; thus, we conclude that supernumerary centrosomes mediate the G2/M arrest and cell death in LMO4‐deficient cells. Furthermore, the correlation of centrosome amplification with genomic instability suggests that the impact of dysregulated LMO4 on the centrosome cycle may promote LMO4‐induced tumour formation. Copyright