PELP1: a key mediator of oestrogen signalling and actions in the brain.

Proline-, glutamic acid- and leucine-rich protein 1 (PELP1) is an oestrogen receptor (ER) coregulator protein identified by our collaborative group. Work from our laboratory and others has shown that PELP1 is a scaffold protein that interacts with ERs and kinase signalling factors, as well as proteins involved in chromatin remodelling and DNA repair. Its role in mediating 17ß-oestradiol (E2 ) signalling and actions has been studied in detail in cancer cells, although only recently has attention turned to its role in the brain. In this review, we discuss the tissue, cellular and subcellular localisation of PELP1 in the brain. We also discuss recent evidence from PELP1 forebrain-specific knockout mice demonstrating a critical role of PELP1 in mediating both extranuclear and nuclear ER signalling in the brain, as well as E2 -induced neuroprotection, anti-inflammatory effects and regulation of cognitive function. Finally, the PELP1 interactome and unique gene network regulated by PELP1 in the brain is discussed, especially because it provides new insights into PELP1 biology, protein interactions and mechanisms of action in the brain. As a whole, the findings discussed in the present review indicate that PELP1 functions as a critical ER coregulator in the brain to mediate E2 signalling and actions.

Novel KDM1A inhibitors induce differentiation and apoptosis of glioma stem cells via unfolded protein response pathway.

Glioma stem cells (GSCs) have a central role in glioblastoma (GBM) development and chemo/radiation resistance, and their elimination is critical for the development of efficient therapeutic strategies. Recently, we showed that lysine demethylase KDM1A is overexpressed in GBM. In the present study, we determined whether KDM1A modulates GSCs stemness and differentiation and tested the utility of two novel KDM1A-specific inhibitors (NCL-1 and NCD-38) to promote differentiation and apoptosis of GSCs. The efficacy of KDM1A targeting drugs was tested on purified GSCs isolated from established and patient-derived GBMs using both in vitro assays and in vivo orthotopic preclinical models. Our results suggested that KDM1A is highly expressed in GSCs and knockdown of KDM1A using shRNA-reduced GSCs stemness and induced the differentiation. Pharmacological inhibition of KDM1A using NCL-1 and NCD-38 significantly reduced the cell viability, neurosphere formation and induced apoptosis of GSCs with little effect on differentiated cells. In preclinical studies using orthotopic models, NCL-1 and NCD-38 significantly reduced GSCs-driven tumor progression and improved mice survival. RNA-sequencing analysis showed that KDM1A inhibitors modulate several pathways related to stemness, differentiation and apoptosis. Mechanistic studies showed that KDM1A inhibitors induce activation of the unfolded protein response (UPR) pathway. These results strongly suggest that selective targeting of KDM1A using NCL-1 and NCD-38 is a promising therapeutic strategy for elimination of GSCs.

Proline, glutamic acid and leucine-rich protein-1 is essential for optimal p53-mediated DNA damage response.

Proline-, glutamic acid- and leucine-rich protein-1 (PELP1) is a scaffolding oncogenic protein that functions as a coregulator for a number of nuclear receptors. p53 is an important transcription factor and tumor suppressor that has a critical role in DNA damage response (DDR) including cell cycle arrest, repair or apoptosis. In this study, we found an unexpected role for PELP1 in modulating p53-mediated DDR. PELP1 is phosphorylated at Serine1033 by various DDR kinases like ataxia-telangiectasia mutated, ataxia telangiectasia and Rad3-related or DNAPKc and this phosphorylation of PELP1 is important for p53 coactivation functions. PELP1-depleted p53 (wild-type) breast cancer cells were less sensitive to various genotoxic agents including etoposide, camptothecin or γ-radiation. PELP1 interacts with p53, functions as p53-coactivator and is required for optimal activation of p53 target genes under genomic stress. Overall, these studies established a new role of PELP1 in DDRs and these findings will have future implications in our understanding of PELP1's role in cancer progression.

Significance of PELP1/HDAC2/miR-200 regulatory network in EMT and metastasis of breast cancer.

Tumor metastasis is the leading cause of death among breast cancer patients. PELP1 (proline, glutamic acid and leucine rich protein 1) is a nuclear receptor coregulator that is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. Here, we show that PELP1 modulates expression of metastasis-influencing microRNAs (miRs) to promote cancer metastasis. Whole genome miR array analysis using PELP1-overexpressing and PELP1-underexpressing model cells revealed that miR-200 and miR-141 levels inversely correlated with PELP1 expression. Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2. PELP1 knockdown significantly reduced tumor growth and metastasis compared with parental cells in an orthotopic xenograft tumor model. Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1-overexpressing cells reversed PELP1 target gene expression, decreased PELP1-driven migration/invasion in vitro and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis. Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier histone deacetylase 2 (HDAC2) as revealed by chromatin immunoprecipitation, small interfering RNA and HDAC inhibitor assays. Taken together, our results suggest that PELP1 regulates tumor metastasis by controlling the expression and functions of the tumor metastasis suppressors miR-200a and miR-141.

Emerging significance of estrogen cancer coregulator signaling in breast cancer.

Estrogen receptor (ERα) plays an important role in the initiation and progression of breast cancer. Many breast cancer patients respond positively to hormonal therapy; however, initial or acquired resistance to endocrine therapies frequently occurs and tumors recur as metastases. Emerging evidence suggests that ERα action is complex and involves functional interactions with coregulators. The levels of ERα coregulator proteins are tightly regulated under normal conditions and ERα-coregulator proteins have the potential to be differentially expressed in malignant tumors and have altered functions leading to tumor progression and metastases. In this review, we summarize recent findings that relate to ERα coregulators and discuss implications of ERα coregulator deregulation in breast cancer metastasis.

Extranuclear signaling by estrogen: role in breast cancer progression and metastasis.

The estrogen receptor (ERa) is implicated in the progression of breast cancer. Hormonal therapies which block ER functions or local and systemic estrogen production are currently used to treat ERa positive breast cancer. Hormonal therapy shows beneficial effects, however, initial or acquired resistance to endocrine therapies frequently occurs, and tumors recur as metastasis. Emerging evidence suggests in addition to exerting its well-studied nuclear functions, ERa also participates in extranuclear signaling that involve growth factor signaling components, adaptor molecules and the stimulation of cytosolic kinases. ERa extranuclear pathways have the potential to activate gene transcription, modulate cytoskeleton, and promote tumor cell proliferation, survival, and metastasis. Cytoplasmic/membrane ERa is detected in a subset of breast tumors and expression of extranuclear components ERa is deregulated in tumors. The extranuclear actions of ER are emerging as important targets for tumorigenic and metastatic control. Inhibition of ERa extranuclear actions has the potential to prevent breast tumor progression and may be useful in preventing ERa positive metastasis. In this review, we summarize the results of recent research into the role of ERa mediated extranuclear actions in breast tumorigenesis and metastasis.

Emerging significance of ER-coregulator PELP1/MNAR in cancer.

The estrogen receptors ERalpha and ERbeta have been implicated in the progression of a wide variety of cancers. The actions of ER are regulated by ER coregulator proteins, including proline-, glutamic acid- and leucine-rich-protein-1 (PELP1/MNAR). PELP1 has been shown to participate in both genomic and nongenomic functions of ER. The expression and localization of PELP1/MNAR are deregulated in a wide variety of tumors and have been implicated in the development of hormonal resistance in cancer cell lines. Emerging data suggest that PELP1/MNAR interacts with many proteins and activates several oncogenes, including Src kinase, phosphotidyl inositol 3 kinase (PI3K), and signal transducers and activators of transcription 3 (STAT3). These new results suggest that PELP1/MNAR may act as an oncogene as well as cooperating with other oncogenes. Thus, PELP1/MNAR may contribute to the tumorigenic potential of cancer cells by serving as a scaffolding protein that couples various signaling complexes with ER.

Novel estrogen receptor coregulators and signaling molecules in human diseases.

The steroid hormone estrogen and signaling from its receptors are increasingly recognized as critical mediators of a variety of organ-specific biological processes. Recent advances in the identification and functional characterization of novel estrogen receptor interacting proteins clearly show the complexity of hormonal signaling regulation, but may also contribute to our understanding of the roles of estrogen signaling in normal physiology and the pathobiology of human disease.

Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R(1) in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis.

Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R(1) (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K(d) approximately 1nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R(1) revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R(1) cDNA, subsequently killing the cells. Recruitment of BT-R(1) by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.

Growth factor regulation of the molecular chaperone calnexin.

Heregulin-beta1 (HRG) is a regulatory polypeptide having several distinct biological effects in mammary epithelial cells. To address the hypothesis that HRG selectively regulates gene expression, we performed differential display screening using cells grown in the presence or absence of HRG. One cDNA clone upregulated by HRG was identical to human calnexin, a protein with molecular chaperone function. This is the first demonstration of the regulation of calnexin mRNA and protein expression by a physiologically relevant polypeptide factor in human breast cancer cells. HRG stimulation also caused a rapid redistribution of calnexin from vesicle-like structures in the cell cytoplasm to the perinuclear area and to the cell membrane. Furthermore, HRG induced colocalization and physical interaction of calnexin with the HER2 growth factor receptor. Finally, calnexin protein levels were increased in progressive stages of human breast cancer. These findings suggest that stimulation of calnexin expression by HRG may constitute a mechanism of protein redistribution and facilitate downstream signaling events in growth-factor-activated cells.

Molecular cloning and characterization of PELP1, a novel human coregulator of estrogen receptor alpha.

Nuclear hormone receptors (NRs) are transcription factors whose activity is regulated by ligands and by coactivators or corepressors. We report the characterization of a new NR coregulator: proline-, glutamic acid-, leucine-rich protein 1 (PELP1), a novel human protein that comprises 1,282 amino acids and is localized on chromosome 17. The primary structure of PELP1 consists of several motifs present in most transcriptional regulators including nine NR-interacting boxes (LXXLL motifs), a zinc finger, and glutamic acid- and proline-rich regions. We demonstrate that PELP1 is a coactivator of estrogen receptor alpha (ERalpha). PELP1 enhances 17beta-estradiol-dependent transcriptional activation from the estrogen response element in a dose-dependent manner. PELP1 interacts with ERalpha and also with general transcriptional coactivators p300 and cAMP response element-binding protein-binding protein. PELP1 was differentially expressed in various human and murine tissues with the highest expression levels in the testes, mammary glands, and brain. We also provide evidence supporting the developmental regulation of PELP1 expression in murine mammary glands, the detectable expression of PELP1 in human mammary cancer cell lines, and the enhanced expression of PELP1 in human breast tumors. These findings suggest that PELP1 is a novel coregulator of ERalpha and may have a role in breast cancer tumorigenesis.

Tiam1 overexpression potentiates heregulin-induced lymphoid enhancer factor-1/beta -catenin nuclear signaling in breast cancer cells by modulating the intercellular stability.

Heregulin-beta1 (HRG) promotes motility, scattering, and invasiveness of breast cancer cells. Tiam1, a newly identified guanine nucleotide exchange factor, has been shown to inhibit or promote cell migration in a cell type-dependent manner. In this study, we identified Tiam1 as a target of HRG signaling. HRG stimulation of breast cancer epithelial cells induced the phosphorylation and redistribution of Tiam1 to the membrane ruffles and the loosening of intercellular junctions. In addition, HRG-mediated scattering of breast epithelial cells was accompanied by stimulation of tyrosine phosphorylation and redistribution of beta-catenin from the cell junctions to the cytosol and, finally, entry into the nucleus. Decompaction of breast cancer epithelial cells by HRG was accompanied by a transient physical association of the tyrosine-phosphorylated beta-catenin with the activated human epidermal growth factor receptor 2 and subsequent nuclear translocation of beta-catenin, as well as beta-catenin-dependent transactivation of T-cell factor.lymphoid enhancer factor-1. All of these HRG-induced phenotypic changes were regulated in a phosphatidylinositol-3 kinase-sensitive manner. HRG-induced cellular ruffles, loss of intercellular adhesiveness, and increased cell migration could be mimicked by overexpression of a fully functional Tiam1 construct. Furthermore, ectopic expression of Tiam1 or of an active beta-catenin mutant led to potentiation of the beta-catenin-dependent T-cell factor.lymphoid enhancer factor-1 transactivation and invasiveness of HRG-treated cells. We also found preliminary evidence suggesting a close correlation between the status of Tiam1 expression and invasiveness of human breast tumor cells with the degree of progression of breast tumors. Together, these findings suggest that HRG regulate Tiam1 activation and lymphoid enhancer factor/beta-catenin nuclear signaling via phosphatidylinositol-3 kinase in breast cancer cells.

Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicity of breast cancer cells.

Etk/Bmx, a member of the Tec family of nonreceptor protein-tyrosine kinases, is characterized by an N-terminal pleckstrin homology domain and has been shown to be a downstream effector of phosphatidylinositol 3-kinase. P21-activated kinase 1 (Pak1), another well characterized effector of phosphatidylinositol 3-kinase, has been implicated in the progression of breast cancer cells. In this study, we characterized the role of Etk in mammary development and tumorigenesis and explored the functional interactions between Etk and Pak1. We report that Etk expression is developmentally regulated in the mammary gland. Using transient transfection, coimmunoprecipitation and glutathione S-transferase-pull down assays, we showed that Etk directly associates with Pak1 via its N-terminal pleckstrin homology domain and also phosphorylates Pak1 on tyrosine residues. The expression of wild-type Etk in a non-invasive human breast cancer MCF-7 cells significantly increased proliferation and anchorage-independent growth of epithelial cancer cells. Conversely, expression of kinase-inactive mutant Etk-KQ suppressed the proliferation, anchorage-independent growth, and tumorigenicity of human breast cancer MDA-MB435 cells. These results indicate that Pak1 is a target of Etk and that Etk controls the proliferation as well as the anchorage-independent and tumorigenic growth of mammary epithelial cancer cells.

Butyric acid induces apoptosis by up-regulating Bax expression via stimulation of the c-Jun N-terminal kinase/activation protein-1 pathway in human colon cancer cells.

BACKGROUND & AIMS: The colonic epithelial cells near the top of the crypt have been shown to undergo apoptosis. Because butyric acid (BA) is the major short-chain fatty acid produced by fermentation of dietary fiber in the large bowel, it may be an important regulator of apoptosis in colorectal cancer. We investigated which signaling pathway is triggered by BA to undergo apoptosis in human colorectal cancer cells. METHODS: Human DiFi and FET colorectal cells were treated with BA to undergo apoptosis and were assayed for activation of c-Jun N-terminal kinase (JNK), transcription factor activation protein 1 (AP1) and NF-kappaB, and the proapoptotic molecule Bax. The contribution of specific pathways was assessed by examining the effects of dominant-negative mutants of JNK/AP1 or NF-kappaB on BA-induced Bax expression and apoptosis. RESULTS: BA-mediated DNA fragmentation and Bax induction were preceded by early stimulation of JNK, and the DNA-binding activities of AP1 and NF-kappaB. BA-induced enhancement of DNA fragmentation and stimulation of Bax promoter activity were blocked by the expression of dominant-negative mutants of JNK1 or AP1 but not NF-kappaB. CONCLUSIONS: These findings suggest that apoptosis triggered by BA involves transcriptional stimulation of the Bax gene via activation of the JNK/AP1 pathway in colonic epithelial cells.

Transcriptional repression of oestrogen receptor by metastasis-associated protein 1 corepressor.

Activation of the heregulin/HER2 pathway in oestrogen receptor (ER)-positive breast-cancer cells leads to suppression of oestrogen-receptor element (ERE)-driven transcription and disruption of oestradiol responsiveness, and thus contributes to progression of tumours to more invasive phenotypes. Here we report the identification of metastatic-associated protein 1 (MTA1), a component of histone deacetylase (HDAC) and nucleosome-remodelling complexes, as a gene product induced by heregulin-beta1 (HRG). Stimulation of cells with HRG is accompanied by suppression of histone acetylation and enhancement of deacetylase activity. MTA1 is also a potent corepressor of ERE transcription, as it blocks the ability of oestradiol to stimulate ER-mediated transcription. The histone-deacetylase inhibitor trichostatin A blocks MTA1-mediated repression of ERE transcription. Furthermore, MTA1 directly interacts with histone deacetylase-1 and -2 and with the activation domain of ER-alpha. Overexpression of MTA1 in breast-cancer cells is accompanied by enhancement of the ability of cells to invade and to grow in an anchorage-independent manner. HRG also promotes interaction of MTA1 with endogenous ER and association of MTA1 or HDAC with ERE-responsive target-gene promoters in vivo. These results identify ER-mediated transcription as a nuclear target of MTA1 and indicate that HDAC complexes associated with the MTA1 corepressor may mediate ER transcriptional repression by HRG.

Regulation of elongation factor-1alpha expression by growth factors and anti-receptor blocking antibodies.

The epidermal growth factor (EGF) family and its receptors regulate normal and cancerous epithelial cell proliferation, a process that could be suppressed by anti-receptor blocking antibodies. Polypeptide elongation factor-1alpha (EF-1alpha) is a multifunctional protein whose levels are positively correlated with the proliferative state of cells. To identify genes, whose expression may be modulated by anti-receptor blocking antibodies, we performed a differential display screening and isolated differentially expressed cDNAs. Isolates from one clone were 100% identical to human EF-1alpha. Both EGF and heregulin-beta1 (HRG) induced EF-1alpha promoter activity and mRNA and protein expression. Growth factor-mediated EF-1alpha expression was effectively blocked by pretreatment with humanized anti-EGF receptor antibody C225 or anti-human epidermal growth factor receptor-2 (HER2) antibody herceptin. Mutants and pharmacological inhibitors of p38(MAPK) and MEK, but not phosphatidylinositol 3-kinase, suppressed both constitutive and HRG-induced stimulation of EF-1alpha promoter activity in MCF-7 cells. Deletion analysis of the promoter suggested the requirement of the -393 to -204 region for growth factor-mediated transcription of EF-1alpha. Fine mapping and point mutation studies revealed a role of the SP1 site in the observed HRG-mediated regulation of the EF-1alpha promoter. In addition, we also provide new evidence to suggest that HRG stimulation of the EF-1alpha promoter involves increased physical interactions with acetylated histone H3 and histone H4. These results suggest that regulation of EF-1alpha expression by extracellular signals that function through human EGF receptor family members that are widely deregulated in human cancers and that growth factor regulation of EF-1alpha expression involve histone acetylation.

Evidence of Rab3A expression, regulation of vesicle trafficking, and cellular secretion in response to heregulin in mammary epithelial cells.

Heregulin beta1 (HRG), a combinatorial ligand for human growth factor receptors 3 and 4, is a regulatory polypeptide that promotes the differentiation of mammary epithelial cells into secretory lobuloalveoli. Emerging evidence suggests that the processes of secretory pathways, such as biogenesis and trafficking of vesicles in neurons and adipose cells, are regulated by the Rab family of low-molecular-weight GTPases. In this study, we identified Rab3A as a gene product induced by HRG. Full-length Rab3A was cloned from a mammary gland cDNA library. We demonstrated that HRG stimulation of human breast cancer cells and normal breast epithelial cells induces the expression of Rab3A protein and mRNA in a cycloheximide-independent manner. HRG-mediated induction of Rab3A expression was blocked by an inhibitor of phosphatidylinositol 3-kinase but not by inhibitors of mitogen-activated protein kinases p38(MAPK) and p42/44(MAPK). Human breast epithelial cells also express other components of regulated vesicular traffic, such as rabphilin 3A, Doc2, and syntaxin. Rab3A was predominantly localized in the cytosol, and HRG stimulation of the epithelial cells also raised the level of membrane-bound Rab3A. HRG treatment induced a profound alteration in the cell morphology in which cells displayed neuron-like membrane extensions that contained Rab3A-coated, vesicle-like structures. In addition, HRG also promoted the secretion of cellular proteins from the mammary epithelial cells. The ability of HRG to modify exocytosis was verified by using a growth hormone transient-transfection system. Analysis of mouse mammary gland development revealed the expression of Rab3A in mammary epithelial cells. Furthermore, expression of the HRG transgene in Harderian tumors in mice also enhanced the expression of Rab3A. These observations provide new evidence of the existence of a Rab3A pathway in mammary epithelial cells and suggest that it may play a role in vesicle trafficking and secretion of proteins from epithelial cells in response to stimulation by the HRG expressed within the mammary mesenchyma.

Vascular endothelial growth factor up-regulation via p21-activated kinase-1 signaling regulates heregulin-beta1-mediated angiogenesis.

Heregulin-beta1 promotes the activation of p21-activated kinase 1 (Pak1) and the motility and invasiveness of breast cancer cells. In this study, we identified vascular endothelial growth factor (VEGF) as a gene product induced by heregulin-beta1. The stimulation by heregulin-beta1 of breast cancer epithelial cells induced the expression of the VEGF mRNA and protein and its promoter activity. Heregulin-beta1 also stimulated angiogenesis in a VEGF-dependent manner. Herceptin, an anti-HER2 antibody inhibited heregulin-beta1-mediated stimulation of both VEGF expression in epithelial cells and angiogenesis in endothelial cells. Because the activation of Pak1 and VEGF expression are positively regulated by heregulin-beta1, we hypothesized that Pak1 regulates VEGF expression, and hence explored the role of Pak1 in angiogenesis. We provide new evidence to implicate Pak1 signaling in VEGF expression. Overexpression of a kinase-dead K299R Pak1 leads to suppression of VEGF promoter activity, as well as VEGF mRNA expression and secretion of VEGF protein. Conversely, kinase-active T423E Pak1 promotes the expression and secretion of VEGF. Furthermore, expression of the heregulin-beta1 transgene, HRG, in harderian tumors in mice enhances the activation of Pak1 as well as expression of VEGF and angiogenic marker CD34 antigen. These results suggest that heregulin-beta1 regulates angiogenesis via up-regulation of VEGF expression and that Pak1 plays an important role in controlling VEGF expression and, consequently, VEGF secretion and function.

Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells.

Stimulation of growth factor signaling has been implicated in the development of invasive phenotypes and the activation of p21-activated kinase (Pak1) in human breast cancer cells (Adam, L., Vadlamudi, R., Kondapaka, S. B., Chernoff, J., Mendelsohn, J., and Kumar, R. (1998) J. Biol. Chem. 273, 28238-28246; Adam, L., Vadlamudi, R., Mandal, M., Chernoff, J., and Kumar, R. (2000) J. Biol. Chem. 275, 12041-12050). To study the role of Pak1 in the regulation of motility and growth of breast epithelial cells, we developed human epithelial MCF-7 clones that overexpressed the kinase-active T423E Pak1 mutant under an inducible tetracycline promoter or that stably expressed the kinase-active H83L,H86L Pak1 mutant, which is deficient in small GTPase binding sites. The expression of both T423E and H83L,H86L Pak1 mutants in breast epithelial cells was accompanied by increased cell motility without any apparent effect on the growth rate of cells. The T423E Pak1 mutant was primarily localized to filopodia, and the H83L,H86L Pak1 mutant was primarily localized to ruffles. Cells expressing T423E Pak1 exhibited a regulatable stimulation of mitogen-activated protein kinase and Jun N-terminal kinase activities. The expression of kinase-active Pak1 mutants significantly stimulated anchorage-independent growth of cells in soft agar in a preferential mitogen-activated protein kinase-sensitive manner. In addition, regulatable expression of kinase-active Pak1 resulted in an abnormal organization of mitotic spindles characterized by appearance of multiple spindle orientations. We also provide evidence to suggest a close correlation between the status of Pak1 kinase activity and base-line invasiveness of human breast cancer cells and breast tumor grades. This study is the first demonstration of Pak1 regulation of anchorage-independent growth, potential Pak1 regulation of invasiveness, and abnormal organization of mitotic spindles of human epithelial breast cancer cells.

Apoptosis in mammary gland and cancer.

Homeostasis in normal tissue is regulated by a balance between proliferative activity and cell loss by apoptosis. Apoptosis is a physiological mechanism of cell loss that depends on both pre-existing proteins and de novo protein synthesis, and the process of apoptosis is integral to normal mammary gland development and in many diseases, including breast cancer. The mammary gland is one of the few organ systems in mammals that completes its morphologic development postnatally during two discrete physiologic states, puberty and pregnancy. The susceptibility of the mammary gland to tumorigenesis is influenced by its normal development, particularly during stages of puberty and pregnancy that are characterized by marked alterations in breast cell proliferation and differentiation. Numerous epidemiologic studies have suggested that specific details in the development of the mammary gland play a critical role in breast cancer risk. Mammary gland development is characterized by dynamic changes in the expression profiles of Bcl-2 family members. The expression of Bcl-2 family proteins in breast cancer is also influenced by estradiol and by progestin. Since the ratio of proapoptotic to antiapoptotic proteins determines apoptosis or cell survival, hormone levels may have important implications in the therapeutic prevention of breast cancer.