Regulation of estrogen receptor alpha by the tumor suppressor p53 in breast cancer cells

Estrogen receptor (ER) and the tumor suppressor p53 are key prognostic indicators in breast cancer. Estrogen signaling through its receptor (ER) controls proliferation of normal as well as transformed mammary epithelial cells, and the presence of ER is established as a marker of good prognosis and response to therapy. The p53 tumor suppressor gene is often referred to as the "cellular gatekeeper" due to its extensive control of cell proliferation and apoptosis. Loss of functional p53 is a negative prognostic indicator and is correlated with lack of response to antiestrogens, reduced disease-free interval and increased chance of disease recurrence. Clinical studies have demonstrated that tumors with mutated p53 tend to be ER negative, while ER positive tumors tend to have wild type p53. ^ Recent studies from our lab indicate that p53 genotype correlates with estrogen receptor expression in mammary tumors in vivo. We therefore hypothesized that p53 regulates ER expression in mammary cancer cells by recruitment of specific cofactors to the ER promoter. To test this, MCF-7 cells were treated with doxorubicin or ionizing radiation, both of which stimulated significant increases in p53 expression, as expected, but also increased ER expression in a p53-dependent manner. Furthermore, in cells treated with siRNA targeting p53, both p53 and ER protein levels were significantly reduced. P53 was also demonstrated to transcriptionally regulate the ER promoter in luciferase assays and chromatin immunoprecipitation assays showed that p53 was recruited to the ER promoter along with CARM1, CBP, c-Jun and Sp1 and that this multifactor complex was formed in a p53-dependent manner. The regulation of ER by p53 has therapeutic implications, as the treatment of breast cancer cells with doxorubicin sensitized these cells to tamoxifen treatment. Furthermore, response to tamoxifen as well as to estrogen was dependent on p53 expression in ER positive human breast cancer cells. Taken together, these data demonstrate that p53 regulates ER expression through transcriptional control of the ER promoter, accounting for their concordant expression in human breast cancer and identifying potentially beneficial therapeutic strategies for the treatment of ER positive breast cancers. ^

Respiratory syncytial virus infection in hematopoietic stem cell transplant patients

Respiratory Syncytial Virus (RSV) is a major cause of respiratory tract infections in immunocompromised patients such as children less than 2 years, premature infants with congenital heart disease and chronic lung disease, elderly patients and patients who have undergone hematopoietic stem cell transplant (HSCT). HSCT patients are at high risk of RSV infection, at increased risk of developing pneumonia, and RSV-related mortality. Immunodeficiency can be a major risk factor for severe infection & mortality. Therapy of RSV infection with Ribavirin, Palivizumab and Immunoglobulin has shown to reduce the risk of progression to LRI and mortality, especially if initiated early in the disease. Data on RSV infection in HSCT patients is limited, especially at various levels of immunodeficiency. 323 RSV infections in HSCT patients have been identified between 1/1995 and 8/2009 at University of Texas M D Anderson Cancer Center (UTMDACC). In this proposed study, we attempted to analyze a de-identified database of these cases and describe the epidemiologic characteristics of RSV infection in HSCT patients, the course of the infection, rate of development of pneumonia and RSV-related mortality in HSCT patients at UTMDACC.^ Key words: RSV infections, HSCT patients ^

PROSTATE CANCER STEM CELLS: DRUG TOLERANCE, EXPERIMENTAL RECONSTITUTION AND CASTRATION RESISTANCE

Prostate cancer (PCa) is one of the leading malignancies affecting men in the Western world. Although tremendous effort has been made towards understanding PCa development and developing clinical treatments in the past decades, the exact mechanisms of PCa are still not clearly understood. Emerging evidence has postulated that a population of stem cell-like cells inside a tumor, termed ‘cancer stem cells (CSCs)’, may be the cells responsible for tumor initiation, progression, recurrence, metastasis and therapy resistance. Like CSC studies in other cancer types, it has been reported that PCa also contains CSCs. However, there remain several unresolved questions that need to be clarified. First, the relationship between prostate CSCs (PCSCs) and therapy resistance (chemo- and radio-) is not known. Herein, we have found that not all CSCs are drug-tolerant, and not all drug-tolerant cells are CSCs. Second, whether primary human PCa (HPCa) actually contain PCSCs remains unclear, due to the well-known fact that we have yet to establish a reliable assay system that can reproducibly and faithfully reconstitute tumor regeneration from single HPCa cells. Herein, after utilizing more than 114 HPCa samples we have provided evidence that immortalized bone marrow-derived stromal cells (Hs5) can help dissociated HPCa cells generate undifferentiated tumors in immunodeficient NOD/SCID-IL2Rγ-/- mice, and the undifferentiated PCa cells seem to have a survival advantage to generate tumors. Third, the evolution of PCa from androgen dependent to the lethally castration resistant (CRPC) stage remains enigmatic, and the cells responsible for CRPC development have not been identified. Herein, we have found a putative cell population, ALDH+CD44+α2β1+ PCa cells that may represent a cell-of-origin for CRPC. Taken together, our work has improved our understanding of PCSC properties, possibly highlighting a potential therapeutic target for CRPC.

Survival analysis of circulating tumor cells in triple-negative breast cancer

Background. Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females, accounting for 23% (1.38 million) of the total new cancer cases and 14% (458,400) of the total cancer deaths in 2008. [1] Triple-negative breast cancer (TNBC) is an aggressive phenotype comprising 10–20% of all breast cancers (BCs). [2-4] TNBCs show absence of estrogen, progesterone and HER2/neu receptors on the tumor cells. Because of the absence of these receptors, TNBCs are not candidates for targeted therapies. Circulating tumor cells (CTCs) are observed in blood of breast cancer patients even at early stages (Stage I & II) of the disease. Immunological and molecular analysis can be used to detect the presence of tumor cells in the blood (Circulating tumor cells; CTCs) of many breast cancer patients. These cells may explain relapses in early stage breast cancer patients even after adequate local control. CTC detection may be useful in identifying patients at risk for disease progression, and therapies targeting CTCs may improve outcome in patients harboring them. Methods . In this study we evaluated 80 patients with TNBC who are enrolled in a larger prospective study conducted at M D Anderson Cancer Center in order to determine whether the presence of circulating tumor cells is a significant prognostic factor in relapse free and overall survival . Patients with metastatic disease at the time of presentation were excluded from the study. CTCs were assessed using CellSearch System™ (Veridex, Raritan, NJ). CTCs were defined as nucleated cells lacking the presence of CD45 but expressing cytokeratins 8, 18 or 19. The distribution of patient and tumor characteristics was analyzed using chi square test and Fisher's exact test. Log rank test and Cox regression analysis was applied to establish the association of circulating tumor cells with relapse free and overall survival. Results. The median age of the study participants was 53years. The median duration of follow-up was 40 months. Eighty-eight percent (88%) of patients were newly diagnosed (without a previous history of breast cancer), and (60%) of patients were chemo naïve (had not received chemotherapy at the time of their blood draw for CTC analysis). Tumor characteristics such as stage (P=0.40), tumor size (P=69), sentinel nodal involvement (P=0.87), axillary lymph node involvement (P=0.13), adjuvant therapy (P=0.83), and high histological grade of tumor (P=0.26) did not predict the presence of CTCs. However, CTCs predicted worse relapse free survival (1 or more CTCs log rank P value = 0.04, at 2 or more CTCs P = 0.02 and at 3 or more CTCs P < 0.0001) and overall survival (at 1 or more CTCs log rank P value = 0.08, at 2 or more CTCs P = 0.01 and at 3 or more CTCs P = 0.0001. Conclusions. The number of circulating tumor cells predicted worse relapse free survival and overall survival in TNBC patients.^

INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER

INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER Midan Ai, Ph.D. Supervisory Professor: Zhen Fan, M.D. Breast tumor kinase (Brk) is a nonreceptor protein-tyrosine kinase that is highly expressed in approximately two thirds of breast cancers but is not detectable or is expressed at very low levels in normal mammary epithelium. Brk plays important roles in promoting proliferation, survival, invasion, and metastasis of breast cancer cells, but the mechanism(s) of which remain largely unknown. Recent studies showed that Brk is frequently co-overexpressed with human epidermal growth factor receptor-2 (HER2) and is physically associated with HER2 in breast cancer. The mechanism needs to be determined. In my studies, I found that high expression of HER2 is correlated with high expression of Brk in breast cancer cell lines. Silencing HER2 expression via RNA interference in HER2 over-expressed breast cancer cells resulted in Brk protein decrease and overexpression of HER2 in HER2 low-expressed breast cancer cells up-regulated Brk expression. The mechanism study indicated that overexpression of HER2 increased Brk protein stability. Brk was degraded through a Ca2+-dependent protease pathway involving calpain and HER2 stimulated Brk expression via inhibiting calpain activity. Calpastatin is a calpain endogenous inhibitor and the calpain-calpastatin system has been implicated in a number of cell physiological functions. HER2 restrained calpain activation via up-regulating calpastatin expression and HER2 downstream signaling, MAPK pathway, was involved in the regulation. Furthermore, silencing of Brk expression by RNA interference in HER2-overexpressing breast cancer cells decreased HER2-mediated cell proliferation, survival, invasion/metastasis potential and increased cell sensitivity to HER2 kinase inhibitor, lapatinib, treatment, indicating that Brk plays important roles in regulating and mediating the oncogenic functions of HER2. The Stat3 pathway played important roles in Brk mediated cell survival and invasion/metastasis in the context of HER2-overexpressing breast cancer cells. However, transgenic mice with inducible expression of constitutively active Brk (CA) in the mammary epithelium failed to develop malignant change in the mammary glands after Brk induction for 15 months which indicated that expression of Brk protein alone was not sufficiently to induce spontaneous breast tumor. Bitransgenic mice with co-expression of HER2/neu and inducible expression of Brk in the mammary epithelium developed multifocal mammary tumors, but there were no significant difference in the tumor occurring time, tumor size, tumor weight and tumor multiplicity between the mouse group with co-expression of Brk and HER2/neu and the mouse group with HER2/neu expression only.

The Role of Type I Insulin-Like Growth Factor Receptor Signaling in Breast Cancer Brain Metastasis

Brain metastasis is a common cause of mortality in cancer patients. Approximately 20-30% of breast cancer patients acquire brain metastasis, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF- IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that the IGF-IR signaling axis is constitutively active in brain-seeking sublines of breast cancer cells, driving an increase in in vitro metastatic properties. We demonstrate that IGF-IR signaling is activated in an autocrine manner as a result of IGFBP3 overexpression in brain-seeking cells. Transient and stable knockdown of IGF-IR results in a downregulation of IGF-IR downstream signaling through phospho-AKT, as well as decreased in vitro migration and invasion of MDA- MB-231Br brain-seeking cells. Using an in vivo experimental brain metastasis model, we show that IGF-IR ablation attenuates the establishment of brain metastases and prolongs survival. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.

Mechanisms of HER2/{\it neu\/} proto-oncogene overexpression in breast cancer cells

Overexpression and amplification of HER2/neu have been documented in many primary tumors, most notably in breast. Not only do approximately 30% of breast cancer patients carry tumors that overexpress the gene, but those that do generally have shorter overall and disease-free survival times than patients with tumors expressing low levels of HER2/neu. Thus, overexpression of HER2/neu plays an important role in the pathogenesis of breast cancer. We have examined the mechanisms that result in HER2/neu overexpression in breast cancer by using, as a model system, established breast cancer cell lines that express much higher levels of HER2/neu mRNA than normal breast tissue while maintaining a near normal HER2/neu gene copy number. Nuclear run-on experiments indicate that the breast cancer cell lines MDA-MB453, BT483, and BT474 have an increased HER2/neu gene transcription rate. By using HER2/neu promoter-CAT constructs, we have found that the enhanced HER2/neu transcription rate in MDA-MB453 cells is due to activation of the gene in trans, while the enhanced transcription rate in BT483 cells is due to activation of the gene in either trans or cis. In BT474 cells, transcriptional upregulation is primarily due to gene amplification. Since the levels of increased transcription are not as high as the levels of HER2/neu mRNA in any of these three lines, post-transcriptional deregulation that increases HER2/neu expression must also be functioning in these cells. The half-life of HER2/neu mRNA was measured and found to be equivalent in these lines as in a control. Thus, the post-transcriptional deregulation is not increased stability of the HER2/neu transcript.^ Much work has been performed in characterizing the altered trans-acting factor involved in increased HER2/neu transcription in MDA-MB453 cells. Using promoter deletion constructs linked to a reporter gene, the region responsive to this factor was localized in the rat neu promoter. When human HER2/neu promoter constructs were used, the homologous sequence in the human promoter was identified. Furthermore, a number of protein/DNA complexes are detected when these promoter regions are used in gel mobility shift assays. UV-crosslinking experiments indicate DNA-binding proteins of roughly 110 kDa, 70 kDa, and 35 kDa are capable of interacting with the human promoter element. ^

The effect of protein tyrosine phosphatase SHP1 on tumorigenicity of the MDA -MB231 breast cancer cells

SHP1 is a cytosolic protein tyrosine phosphatase that contains two SH2 domains. It is highly expressed in hematopoietic cells and expressed in normal epithelium at lower levels. While SHP1 in hematopoietic cells is thought to be a negative regulator of cellular signaling by associating with and dephosphorylating various receptors and their downstream effectors after they become activated, its precise function in epithelium remains to be understood. The potential involvement of SHP1 in human tumorigenesis has been hypothesized from the findings that SHP1 can interact with, dephosphorylate, and regulate the activity of several protein tyrosine kinases (PTKs) implicated in human cancer. These PTKs include epidermal growth factor receptor (EGFR) and Src. Such speculation is also supported by the report that SHP1 is overexpressed in human ovarian cancers. ^ Here we report, for the first time, that the levels of SHP1 expression and activity are altered in human breast cancer cells in comparison with normal breast epithelium. In particular, SHP1 expression is nearly lost in the breast cancer cell lines MDA-MB231 and MDA-MB435. After the re-introduction of SHP1 both in wild type (wt) and enzymatically inactive (dn) forms, into the MDA-MB231 cells, we observed no changes in cellular proliferation. However, the overexpression of wt SHP1 led to increased anchorage-independent growth in the MDA-MB231 cells. SHP1 phosphatase activity is essential for such an increase since the overexpression of dn SHP1 had no effect. Enhanced turnorigenicity in nude mice was also observed in the MDA-MB231 cells overexpressing wt SHP1, but not dn SHP1, suggesting the crucial function of SHP1 enzymatic activity in this process. Our observations in this study indicate that SHP1 promotes tumorigenesis by a mechanism or mechanisms apart from enchancing angiogenesis. In addition, we have found no evidence that the overexpression of SHP1 could affect metastatic potential in the MDA-MB231 cells. ^ In the MDA-MB231 cells stably transfected with either wt or dn SHP1 the peak level of EGFR tyrosine phosphorylation induced by EGF, as well as the sensitivity to EGF stimulation, was not altered. However, the overexpression of wt SHP1 led to a slight increase in the kinetics of EGFR dephosphorylation, whereas the overexpression of dn SHP1 led to slightly delayed kinetics of EGFR dephosphorylation. The overexpression of either the wt or dn SHP1 did not lead to any significant increase in Src kinase activity. ^ In NIH3T3 cells, the transient overexpression of SHP1 led to no significant changes in MAP kinase (ERK2) activation by EGF or Akt activation by PDGF. In 3T3H4 cells, the transient overexpression of SHP1 led to no significant changes in MAP kinase (ERK2) activation by heregulin. The transient overexpression of wt SHP1 in the MDA-MB231 cells caused an apparent increase, ranging from 10% to 20%, in the G0/G1 population of the cells with a corresponding decrease in the S phase population. ^ In order to understand the mechanisms by which SHP1 exerts its positive effect on the tumorigenic potential of the MDA-MB231 cells, we employed two-dimensional electrophoresis in an attempt to identify cellular protein(s) with significantly altered tyrosine phosphorylation level upon wt SHP1 overexpression. The overexpression of wt SHP1 but not dn SHP1, leads increased tyrosine phosphorylation of a protein with a molecular weight of approximately 40 kDa and a pI between 5.9 to 6.6. ^

The role of erbB2 receptor in human breast cancer metastasis

Overexpression of c-erbB-2 gene-encoded p185 has been correlated with lymph node metastasis and poor prognosis in breast cancer patients. To investigate whether overexpression of c-erbB-2 can enhance metastatic potential of human breast cancer cells, we compared the metastatic phenotypes of the parental MDA-MB-435 cells and the c-erbB-2 gene transfected 435.eB cells. In vivo experimental metastasis assays demonstrated that mice injected erbB2-overexpressing 435.eB transfectants formed significantly more metastatic tumors than the mice injected with parental and control cells. The changes in metastatic potential in vivo were accompanied by increased invasiveness in vitro . The transfectants and the parental cells all had similar growth rates and transformation potential. These findings suggest that c- erbB-2 gene can enhance the intrinsic metastatic potentials of MDA-MB-435 cells without increasing their transformation abilities. ^ Homophilic adhesion may affect invasive and metastatic potential of tumor cells. We found that Heregulin-β1 (HRG-β1), a growth factor that activates receptor kinases erbB3 and erbB4, can enhance aggregation of MCF-7 and SKBR3 human breast cancer cells. While investigating the downstream signals involved in HRG-β1-increased cell aggregation, we observed that HRG-β1 increased the kinase activities of extracellular signal-regulated protein kinase (ERK) and PI3K in these cells. By using different kinase inhibitors, we found that the HRG-β1-activated MEK1-ERK pathway has no demonstrable role in the induction of cell aggregation, whereas HRG-β1-activated PI3K is required for enhancing breast cancer cell aggregation. These results have provided one mechanism by which HRG-β1-activated signaling of erbB receptors may affect invasive/metastatic properties of breast cancer cells. ^ To identify the structural motifs within the erbB2 receptor that are required for erbB2 increased metastatic potential in breast cancer cells, we injected different forms of mutated erbB2 expressing MDA-MB-435 cell line transfectants with or without the EGF-like domain of heregulin-β1 protein (HRG/egf) into ICR-SCID mice to test the metastatic survival rate. The results show that an intact kinase domain of erbB2 receptor is required for erbB2 enhanced metastatic potential in these cells. The C-terminal tyrosine 1248 residue of erbB2 may also play a role in enhancing metastatic potential. Moreover, the results suggest that HRG/egf promote the metastatic potential of human breast cancer cells in vivo. ^

Activation of matrix metalloproteinase -9 (MMP-9) by heregulin in human breast cancer cells involves multiple signaling pathways

Matrix metalloproteinase-9 (MMP-9) plays an important role in tumor invasion and angiogenesis. Secretion of MMP-9 has been reported in various cancer types including lung cancer, brain cancer, colon cancer, and breast cancer. Heregulin is a growth factor that regulates growth and differentiation of normal breast cells as well as mammary tumor cells. To study the role of heregulin in breast cancer metastasis, we tested whether heregulin may regulate MMP-9 secretion. By screening a panel of breast cancer cell line for their ability to respond to heregulin and produce MMP-9, we have found that MMP-9 secretion can be induced by heregulin-β1 in two breast cancer cell lines, SKBr3 and MCF-7. In both cell lines, increase of MMP-9 activity as shown by zymography was accompanied by increased protein level as well as mRNA level of MMP-9. Using a reporter luciferase assay, we have identified that proximal −670bp promoter of MMP-9 had similar activity to a 2.2kb MMP-9 promoter in response to heregulin stimulation. Heregulin treatment of SKBr3 and MCF-7 activated multiple signaling pathways inside cells. These include the Erk pathway, the p38 kinase pathway, PKC pathway, and PI-3K pathway. To examine which pathways are involved in MMP-9 activation by heregulin, we have used a panel of chemical inhibitors to specifically inhibit each one of these pathways. Ro-31-8220 (PKC inhibitor) and SB203580 (p38 kinase inhibitor) completely blocked heregulin activation of MMP-9. On the other hand, PD098059 (MEK-1 inhibitor) partially blocked MMP-9 activation, whereas PI-3K inhibitor, wortmannin, had no effect. Therefore, at least three signaling pathways are involved in activation of MMP-9 by heregulin. Since MMP-9 is tightly associated with metastatic potential, our study also suggests that heregulin may enhance breast tumor metastasis through induction of MMP-9 expression. ^

The antiapoptotic effect of overexpression c -erBb2 in breast cancer

Overexpression of the receptor tyrosine kinase p185ErbB2 confers taxol resistance in breast cancers and activation of p34Cdc2 is required for taxol-induced apoptosis and cytotoxicity. Here, we investigated the underlying mechanisms and found that overexpression of p185 ErbB2 inhibits taxol-induced apoptosis through two branches to inhibit activation of p34Cdc2. ^ Overexpression of p185ErbB2 in MDA-MB-435 cells by transfection transcriptionally upregulated p21Cip1, which associates with p34Cdc2, inhibits taxol-mediated p34Cdc2 activation, delays cell entrance to G2/M phase, and thereby inhibits taxol-induced apoptosis. In p21Cip1 antisense-transfected MDA-MB-435 cells or in p21−/− MEF cells, p185ErbB2 was unable to inhibit taxol-induced apoptosis. Therefore, p21Cip1 participates in the regulation of a G2/M checkpoint that contributes to resistance to taxol-induced apoptosis in p185ErbB2-overexpressing breast cancer cells. ^ Direct phosphorylation on Tyrosine-15 of p34Cdc2 by p185 ErbB2 receptor tyrosine kinase inhibits p34Cdc2 activation. The wild-type p185ErbB2 but not the kinase-defective mutant, when overexpressed in breast cancer cells, can phosphorylate p34Cdc2 on tyrosine (Tyr)15, an inhibitory phosphorylation site of p34 Cdc2. The kinase domain of the ErbB2 receptor was sufficient for binding to p34Cdc2 and directly phosphorylating the recombinant Cdc2. Phosphospecific Cdc2-Tyr15 immunoblot analyses, immunocomplex kinase assays, and phospho-amino acid analyses revealed that p185ErbB2 specifically phosphorylates Cdc2 on Tyr15. Phosphorylation of Cdc2-Tyr15 by ErbB2 is modulated during cell cycle and corresponded with delayed cell entry into G2/M phase. The kinase-defective p185ErbB2, which incapable of phosphorylating Cdc2-Tyr15, failed to inhibit taxol-induced activation and apoptosis, whereas the wild-type and the constitutive-active p185ErbB2 did. Increased Cdc2-Tyr15 phosphorylation was found in Erb132-overexpressing tumors from breast cancer patients. Thus, direct phosphorylation of Cdc2-Tyr15 by p185 ErbB2 RTK in breast cancer cells inhibits taxol-induced p34 Cdc2 activation and apoptosis, thereby conferring taxol resistance. ^

Estradiol and insulin cross -talk in breast cancer

Approximately 33% of clinical breast carcinomas require estrogens to proliferate. Epidemiological data show that insulin resistance and diabetes mellitus is 2–3 times more prevalent in women with breast cancer than those with benign breast lesions, suggesting a clinical link between insulin and estradiol. Insulin and estradiol have a synergistic effect on the growth of MCF7 breast cancer cells, and long-term estradiol treatment upregulates the expression of the key insulin signaling protein IRS-1. The goal of this study was to further define the mechanism(s) of cross-talk between insulin and estradiol in regulating the growth of breast cancer. Using MCF7 cells, acute treatment with insulin or estradiol alone was found to stimulate two activities associated with growth: Erk MAP kinase and PI 3-kinase. However, combined acute treatment had an antagonistic effect on both activities. Acute estradiol treatment inhibited the insulin-stimulated tyrosine phosphorylation of IRS-1 while increasing its serine phosphorylation; the serine phosphorylation was attenuated by the PI 3-kinase inhibitor wortmannin. The acute antagonism observed with combined estradiol and insulin are not consistent with the long-term synergistic effect on growth. In contrast, chronic estradiol treatment enhanced the insulin-sensitivity of breast cancer cells as measured by increases in total cellular insulin-stimulated tyrosine phosphorylation of IRS-1 and activation of PI 3-kinase. Estradiol stimulation of gene transcription was found to require PI 3-kinase activity but not MAP kinase activity. Insulin alone had no effect on ER transcriptional activity, but chronic treatment in combination with estradiol resulted in synergism of ER transcription. The synergistic effect of insulin and estradiol on MCF7 cell growth was also found to require PI 3-kinase but not MAP kinase activity. Therefore, chronic estradiol treatment increases insulin stimulation of PI 3-kinase, and PI 3-kinase is required for estradiol stimulation of gene transcription alone and in combined synergy with insulin. These data demonstrate that PI 3-kinase is the locus for the cross-talk between insulin and estradiol which results in enhanced breast cancer growth with long-term exposure to both hormones. This may have important clinical implications for women with high risk for breast cancer and/or diabetes mellitus. ^

Significance of tissue transglutaminase expression in multidrug resistant MCF -7 human breast cancer cells

Tissue transglutaminase (tTGase) is an enzyme that catalyzes the posttranslational modification of proteins via Ca2+-dependent cross-linking reactions. In this study, we extended our earlier observation that tTGase is highly expressed in MCF-7 human breast carcinoma cells selected for the multidrug resistance phenotype (MCF-7/DOX). To directly assess the involvement of tTGase in drug resistance, parental MCF-7 (MCF-7/WT) cells were transfected with cDNAs encoding either a catalytically active (wildtype) or inactive (mutant) tTGase protein. Expression of wildtype tTGase led to spontaneous apoptosis in MCF-7/WT cells, while the mutant tTGase was tolerated by the cells but did not confer resistance to doxorubicin. Analysis of calcium by a spectrofluorometric technique revealed that MCF-7/DOX cells exhibit a defective mechanism in intracellular calcium mobilization, which may play a role in preventing the in situ activation of tTGase and thus allowing the cells to grow despite expressing this enzyme. An elevation in intracellular calcium by treatment with the calcium ionophore A23187 induced rapid and substantial apoptosis in MCF-7/DOX cells as determined by morphological and biochemical criteria. Pretreatment of MCF-7/DOX cells with a tTGase-specific inhibitor (monodansylcadaverine) suppressed A12387-induced apoptosis, suggesting the possible involvement of tTGase-catalyzed protein cross-linking activity. A23187-induced apoptosis in MCF-7/DOX cells was further characterized by PARP cleavage and activation of downstream caspases (-3, -6, and -7). Another interesting aspect of tTGase/A23187-induced apoptosis in MCF-7/DOX cells was that these cells failed to show any prototypic changes associated with the mitochondrial (altered membrane potential, cytochrome c release, caspase-9 activation), receptor-induced (Bid cleavage), or endoplasmic reticulum-stressed (caspase-12 activation) apoptotic pathways. In summary, our data demonstrate that, despite being highly resistant to conventional chemotherapeutic drugs, MCF-7/DOX cells are highly sensitive to apoptosis induced by increased intracellular calcium. We conclude that tTGase does not play a direct role in doxorubicin resistance in MCF-7/DOX cells, but may play a role in enhancing the sensitivity of these cells to undergo apoptosis. ^

Breast cancer -specific activation of topoisomerase IIalpha and its application in the tumor -targeting gene therapy

To ensure the success of systemic gene therapy, it is critical to enhance the tumor specificity and activity of the promoter. In the current study, we identified the breast cancer-specific activity of the topoisomerase IIα promoter. We further showed that cdk2 and cyclin A activate topoisomerase IIα promoter in a breast cancer-specific manner. An element containing an inverted CCAAT box (ICB) was shown to respond this signaling. When the ICB-harboring topoisomerase IIα minimal promoter was linked with an enhancer sequence from the cytomegalovirus immediate early gene promoter (CMV promoter), this composite promoter, CT90, exhibited activity comparable to or higher than the CMV promoter in breast cancer cells in vitro and in vivo, yet expresses much lower activity in normal cell lines and normal organs than the CMV promoter. A CT90-driven construct expressing BikDD, a potent pro-apoptotic gene, was shown to selectively kill breast cancer cells in vitro and to suppress mammary tumor development in an animal model of intravenously administrated, liposome-delivered gene therapy. Expression of BikDD was readily detectable in the tumors but not in the normal organs of CT90-BikDD-treated animals. Finally, we demonstrated that CT90-BikDD treatment potentially enhanced the sensitivity of breast cancer cells to chemotherapeutic agents, especially doxorubicin and taxol. The results indicate that liposomal CT90-BikDD is a novel and effective systemic breast cancer-targeting gene therapy, and its combination with chemotherapy may further improve the current adjuvant therapy for breast cancer. ^

Heregulin -beta 1 directly associates with RhoA via the immunoglobulin -like domain thereby modulating breast cancer metastasis-related properties

Heregulins constitute a family of growth factors belonging to the epidermal growth factor (EGF) family. Breast cancers that overexpress specific members of the EGF receptor family (EGFR, ErbB2, ErbB3, ErbB4) have increased metastatic potential, and Heregulin-β1 (HRGβ1), a ligand for ErbB3 and ErbB4, has also been shown to induce metastasis-related properties in breast cancer cells in vitro. The secreted form of the HRGβ1 is composed of five distinct structural domains, including the N-terminal domain, an immunoglobulin-like domain (IgG-like), a glycosylation domain, an EGF-like domain, and a β1-specific domain. Of these, the EGF-like domain is well characterized for its function in metastasis-related properties as well as its structure. However, the contributions of the other HRGβ1 domains in breast cancer metastasis remains unclear. ^ To investigate this, HRGβ1 proteins with targeted domain deletions were purified and subjected to assays for metastasis-related properties, including aggregation, invasion, activation of EGFR family members, and motility of breast cancer cells. These assays showed that retaining the EGF-like domain of HRGβ1 is important for activation of EGFRs. Interestingly, the HRGβ1 protein lacking the IgG-like domain (NGEB) led to a decrease in breast cancer cell motility, indicating the IgG-like domain modulates cell motility, an important step in cancer metastasis. ^ To understand the underlying mechanisms, I performed protein sequence and structural analysis of HRGβ1 and identified that the IgG-like domain of HRGβ1 shares sequence homology and three-dimensional structural similarity with the IgG-like domain of TRIO. TRIO is a cytoplasmic protein that directly associates with RhoA, a GTPase involved in cell reorganization and cell motility. Therefore, I hypothesized that HRGβ1 may translocate inside the breast cancer cells through receptor mediated endocytosis and bind to RhoA via its IgG-like domain. I show wild type HRGβ1 but not NGEB binds RhoA in vitro and in vivo, leading to RhoA activation. Inhibition of HRG-β1 internalization via endocytosis disrupted HRGβ1 binding to RhoA. Additionally, breast cancer cell motility induced by HRG-β1 is reduced after treatment with inhibitors to both endocytosis and RhoA function, similar to levels seen with NGEB treatment. ^ Thus, in addition to the well-known role of HRGβ1 as an extracellular stimulator of the EGFR family members, HRGβ1 also functions within the cell as a binding partner and activator of RhoA to modulate cancer cell motility. ^