MicroRNA (miRNA) in cancer.

In recent years, there has been a tremendous and growing interest among researchers to investigate the role of mircoRNA (miRNA) in normal cellular as well as in disease processes. miRNAs are a family of small non-coding RNAs which were reported to regulate the expression of various oncogenes or tumor suppressor genes. The expression profiling of miRNAs has already entered into cancer clinics as diagnostic and prognostic biomarkers to assess tumor initiation, progression and response to treatment in cancer patients. This review summarizes: (i) the current understanding of interactions between miRNAs and their target genes, (ii) recent advances in the regulatory mechanisms that control the expression of genes related to carcinogenesis, and (iii) the role of miRNAs in cancer diagnosis and therapy.

Functional role of miR-10b in tamoxifen resistance of ER-positive breast cancer cells through down-regulation of HDAC4.

BACKGROUND: For breast cancer patients diagnosed with estrogen receptor (ER)-positive tumors, treatment with tamoxifen is the gold standard. A significant number of patients, however, develop resistance to tamoxifen, and management of such tamoxifen-resistant patients is a major clinical challenge. With an eye to identify novel targets for the treatment of tamoxifen-resistant tumors, we observed that tamoxifen-resistant cells derived from ER-positive MCF-7 cells (MCF7TR) exhibit an increased expression of microRNA-10b (miR-10b). A role of miR-10b in drug-resistance of breast cancer cells has never been investigated, although its is very well known to influence invasion and metastasis. METHODS: To dileneate a role of miR-10b in tamoxifen-resistance, we over-expressed miR-10b in MCF-7 cells and down-regulated its levels in MCF7TR cells. The mechanistic role of HDAC4 in miR-10b-mediated tamoxifen resistance was studied using HDAC4 cDNA and HDAC4-specific siRNA in appropriate models. RESULTS: Over-expression of miR-10b in ER-positive MCF-7 and T47D cells led to increased resistance to tamoxifen and an attenuation of tamoxifen-mediated inhibition of migration, whereas down-regulation of miR-10b in MCF7TR cells resulted in increased sensitivity to tamoxifen. Luciferase assays identified HDAC4 as a direct target of miR-10b. In MCF7TR cells, we observed down-regulation of HDAC4 by miR-10b. HDAC4-specific siRNA-mediated inactivation of HDAC4 in MCF-7 cells led to acquisition of tamoxifen resistance, and, moreover, reduction of HDAC4 in MCF7TR cells by HDAC4-specific siRNA transfection resulted in further enhancement of tamoxifen-resistance. CONCLUSIONS: We propose miR-10b-HDAC4 nexus as one of the molecular mechanism of tamoxifen resistance which can potentially be expolited as a novel targeted therapeutic approach for the clinical management of tamoxifen-resistant breast cancers.

Protein kinase Cδ and caspase-3 modulate TRAIL-induced apoptosis in breast tumor cells.

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL-induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL-induced apoptosis was significantly inhibited in PKCδ overexpressing MCF-7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase-8 activation, a first step in TRAIL-induced apoptosis, thus preventing TRAIL-induced apoptosis. Inhibition of PKCδ using rottlerin or PKCδ siRNA reverses the inhibitory effect of PKCδ on caspase-8 activation leading to TRAIL-induced apoptosis. To determine if caspase-3-induced PKCδ cleavage reverses its inhibition on caspase-8, we developed stable cell lines that either expresses wild-type PKCδ (MCF-7/cas-3/PKCδ) or caspase-3 cleavage-resistant PKCδ mutant (MCF-7/cas-3/PKCδ mut) utilizing MCF-7 cells expressing caspase-3. Cells that overexpress caspase-3 cleavage-resistant PKCδ mutant (MCF-7/cas-3/PKCδmut) significantly inhibited TRAIL-induced apoptosis when compared to wild-type PKCδ (MCF-7/cas-3/PKCδ) expressing cells. In MCF-7/cas-3/PKCδmut cells, TRAIL-induced caspase-8 activation was blocked leading to inhibition of apoptosis when compared to wild-type PKCδ (MCF-7/cas-3/PKCδ) expressing cells. Together, these results strongly suggest that overexpression of PKCδ inhibits caspase-8 activation leading to inhibition of TRAIL-induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase-3 sensitizes cells to TRAIL-induced apoptosis. Clinically, PKCδ overexpressing tumors can be treated with a combination of PKCδ inhibitor(s) and TRAIL as a new treatment strategy.

Stem Cells: Current Status and Therapeutic Implications.

Cancer stem cells (CSCs) are a class of pluripotent cells that have been observed in most types of cancers. Evolving evidence suggests that CSCs, has the ability to self-renew and initiate tumors, may be responsible for promoting therapeutic resistance, tumor recurrence and metastasis. Tumor heterogeneity is originating from CSCs and its progenitors are recognized as major difficulty in efficaciously treating cancer patients. Therefore, understanding the biological mechanisms by which CSCs survive chemo- and-radiation therapy has the potential to identify new therapeutic strategies in the future. In this review, we summarized recent advances in CSC biology and their environment, and discuss about the potential therapies to prevent therapeutic resistance.

Novel strategies to target chemoresistant triple-negative breast cancer.

Previous studies from our group and others have shown that current drug treatment(s) strategies eliminate bulk of tumor cells (non-CSCs) but it had a minimal effect on cancer stem cells (CSCs) leading to resistance and tumor recurrence. We studied the effects of CFM-4.16 (CARP-1 functional mimetic) and/or cisplatin on four Triple-negative breast cancer (TNBC) MDA-MB-468, MDA-MB-231, CRL-2335 and BR-1126, two cisplatin resistant CisR/MDA-231 and CisR/MDA-468 and cancer stem cells (CSCs) from resistant cell lines. TNBC cells treated with CFM-4.16 plus cisplatin inhibited the expression of FZD8, LRP6 and c-Myc and significantly enhanced cell death in all the cell lines by ~70%-80% compared with the control(s). When Cisplatin resistant CisR/MDA-231 and CisR/MDA-468 were treated with CFM-4.16 plus cisplatin, they also showed a reduction in FZD8 and LRP6 and increased apoptosis compared to control group. Similarly, CFM-4.16 plus cisplatin treatment reduced mammospheres formation abilities of CSCs by 80-90% compared to control group, increased PARP cleavage and apoptosis. Data shows CFM-4.16 plus cisplatin treatment significantly increased apoptosis/cell death in parental, cisplatin resistant and CSCs. Taken together the data suggests that FZD8-mediated Wnt-signaling plays a major role in mediating CSCs growth and resistance to chemotherapy and its inhibition enhances the chemotherapeutic response in TNBC.

Inhibition of the MAP kinase activity suppresses estrogen-induced breast tumor growth both in vitro and in vivo.

Elevated expression of mitogen-activated protein kinase (Erk/MAPK) has been noted in a significant percentage of primary human breast cancers. To directly assess the importance of Erk/MAPK activation in estrogen (E2)-induced tumor progression, we blocked E2-signaling with MEK-inhibitor CI-1040 and/or tamoxifen (Tam). Our data show that both MEK-inhibitor CI-1040 and Tam blocked E2-induced MAPK phosphorylation and cell proliferation in MCF-7 breast cancer cells in vitro. However, in vivo studies show that anti-tumor efficacy of combining the CI-1040 and Tam was similar to single agent(s). Furthermore, sequential treatment with Tam followed by CI-1040 or CI-1040 followed by Tam did not significantly reduce E2-induced tumor growth. This suggests that the combination of CI-1040 and Tam may not be synergistic in inhibiting E2-induced tumor growth. However, these findings also indicate that MAPK plays a critical role in E2-induced tumor growth, and that this could be a potential therapeutic target to combat hormonally regulated growth in ER-positive tumors.

Activation function-1 domain of estrogen receptor regulates the agonistic and antagonistic actions of tamoxifen.

The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.

Myc mediates cancer stem-like cells and EMT changes in triple negative breast cancers cells.

Women with triple negative breast cancer (TNBC) have poor prognosis compared to other breast cancer subtypes. There were several reports indicating racial disparity in breast cancer outcomes between African American (AA) and European American (EA) women. For example, the mortality rates of AA breast cancer patients were three times higher than of EA patients, even though, the incidence is lower in AA women. Our in vitro studies indicate that cancer stem-like cells (CSCs) derived from AA TNBC cell lines have significantly higher self-renewal potential (mammosphere formation) than CSCs derived from EA cell lines. TNBC tumors express high levels of Myc compared to luminal A or HER2 expressing breast cancers. We studied the effects of c-Myc overexpression on CSCs and chemotherapy in AA, and EA derived TNBC cell line(s). Overexpression of c-Myc in AA derived MDA-MB-468 (Myc/MDA-468) cells resulted in a significant increase in CSCs and with minimal changes in epithelial-to-mesenchymal transition (EMT) compared to the control group. In contrast, overexpression of c-Myc in EA derived MDA-MB-231(Myc/MDA-231) cells led to increased epithelial-to-mesenchymal transition (EMT), with a minimal increase in CSCs compared to the control group. Myc/MDA-468 cells were resistant to standard chemotherapeutic treatments such as iniparib (PARP inhibitor) plus cisplatin, / iniparib, cisplatin, paclitaxel and docetaxel. However, Myc/MDA-231 cells, which showed EMT changes responded to iniparib with cisplatin, but were resistant to other drugs, such as iniparib, cisplatin, paclitaxel and docetaxel. Collectively, our results indicate that intrinsic differences in the tumor biology may contribute to the breast cancer disparities.

Upregulation of PKC-delta contributes to antiestrogen resistance in mammary tumor cells.

Acquired resistance to tamoxifen (Tam) in breast cancer patients is a serious therapeutic problem. We have previously reported that protein kinase C-delta (PKC-delta) plays a major role in estrogen (E2)-mediated cell proliferation. To determine if PKC-delta is one of the major alternate signaling pathways that supports cell growth in the presence of Tam, we determined the levels of PKC isoforms in four different models of antiestrogen-resistant cells. Three out of four antiestrogen resistance cell lines (Tam/MCF-7, ICI/MCF-7 and HER-2/MCF-7) expressed significantly high levels of both total and activated PKC-delta levels compared to sensitive cells. Estrogen receptor (ER) alpha content and function are maintained in all the antiestrogen-resistant cell lines. Overexpressing active PKC-delta in Tam-sensitive MCF-7 cells (PKC-delta/MCF-7) led to Tam resistance both in vitro and in vivo. Inhibition of PKC-delta by rottlerin (a relatively specific inhibitor of PKC-delta) or siRNA significantly inhibited estrogen- and Tam-induced growth in antiestrogen-resistant cells. PKC-delta levels are significantly higher in Tam-resistant tumors compared to Tam-sensitive tumors in xenograft model (P<0.05). Taken together, these data suggest that PKC-delta plays a major role in antiestrogen resistance in breast tumor cells and thus provides a new target for treatment.

MAP kinase/estrogen receptor cross-talk enhances estrogen-mediated signaling and tumor growth but does not confer tamoxifen resistance.

The estrogen receptor alpha (ERalpha) signaling plays an essential role in breast cancer progression and endocrine therapy. Mitogen-activated protein kinase (MAPK/Erk1/2) has been implicated in ligand-independent activation of ER, resulting in the cross-talk between growth factor and ER mediated signaling. In this study, we examined the effect of the cross-talk on estradiol (E(2))-mediated signaling, tumor growth and its effect on anti-estrogen therapy. Our findings demonstrate that expression of constitutively activated mitogen activated kinase kinase (MEK1), an immediate upstream activator of MAPK in estrogen receptor positive MCF-7 breast cancer cells (MEK/MCF-7), showed an increase in ERalpha-driven transcriptional activation. In MEK/MCF-7 cells maximal transactivation levels were achieved in response to treatment with much lower E(2) concentrations (10(-10) M E(2)) when compared to MCF-7 control cells (10(-8) M E(2)). Furthermore, we have seen an increased association between ERalpha and its nuclear coactivators AIB1 or TIF-2, in MEK/MCF-7 cells relative to those seen in MCF-7 control cells. In addition, in vivo studies show that MEK/MCF-7 cell tumors are approximately threefold larger than those of MCF-7 cell, in the presence of E(2). Immunohistochemical staining demonstrates that progesterone receptor (PR) and pS2, two E(2)-regulated gene products, are significantly increased in MEK/MCF-7 cell tumors compared to those of MCF-7 control tumors, suggesting that activation of ERalpha by MAPK enhances the expression of E(2)-regulated genes and accelerates tumor growth. Remarkably, the antiestrogens tamoxifen and ICI 182,780, were shown both in vitro and in vivo studies to efficiently antagonize the stimulatory effects of E(2) on ER regulated transactivation and tumor growth in MEK/MCF-7 as well as MCF-7 cell lines. Taken together, these data suggest that MAPK/ER cross-talk enhances ERalpha-mediated signaling and accelerates E(2)-dependent tumor growth without diminishing sensitivity to the inhibitory effects of anti-estrogens.

Apoptosis signaling by the novel compound 3-Cl-AHPC involves increased EGFR proteolysis and accompanying decreased phosphatidylinositol 3-kinase and AKT kinase activities.

The threonine and serine protein kinase AKT plays a major role in inhibiting apoptosis in a number of malignant cell types including prostate and breast carcinoma. Activation of AKT is a complex process involving translocation to the plasma membrane and phosphorylation of serine and threonine amino-acid residues. We now report that the novel compound 4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC), induces apoptosis in breast and prostate carcinoma cells and inhibits AKT activity in these cells. Overexpression of a constitutively activated AKT inhibits 3-Cl-AHPC-mediated apoptosis. Decrease in AKT activity occurs through 3-Cl-AHPC inhibition of phosphatidylinositol 3 kinase (PI3-K) activity. 3-Cl-AHPC inhibits PI3-K activity by enhancing epidermal growth factor receptor (EGFR) proteolysis and thus inhibiting EGFR association with the p85 subunit of PI3-K. 3-Cl-AHPC-mediated decrease in PI3-K activity results in the reduced synthesis of phosphatidylinositol 3,4 bisphosphate and phosphatidylinositol 3,4,5 triphosphate with the subsequent inhibition of integrin-linked kinase activity and serine-473 phosphorylation of AKT. Overexpression of EGFR results in increased AKT activity and inhibition of 3-Cl-AHPC-mediated decrease in AKT activation, AKT activity and 3-Cl-AHPC-mediated apoptosis. Inhibition of AKT activity by this compound results in the inability of AKT to phosphorylate and inactivate the proapoptotic forkhead transcription factor.

Distinct mechanisms mediate the initial and sustained phases of cell migration in epidermal growth factor receptor-overexpressing cells.

Elevated levels of epidermal growth factor receptor (EGFR) are predictive of increased invasion and metastasis in many human cancers. In the present study, we have shown that two distinct pathways regulate cell migration in EGFR-overexpressing invasive cells such as MDA 468 breast cancer cells: mitogen-activated protein kinase (MAPK or ERK 1 and 2) pathways play a major role in early stages to cell migration; and protein kinase C delta isoforms (PKC-delta) play a significant role in later stages of sustained cell migration. Inhibition of MAPK activity with MAP kinase kinase (MEK) inhibitor PD98059 blocks early stages of cell migration (up to 4 h); however, cells revert back to enhanced cell migration after 4 h. While inhibition of PKC-delta activity with rottlerin or dominant-negative PKC-delta expression blocks sustained cell migration after 4 h and up to 12 h, the combination of MAPK and PKC inhibitors completely blocked transforming growth factor alpha (TGF-alpha)-induced cell migration in EGFR-overexpressing breast cancer cells. However, inhibition of MAPK activity completely blocked cell migration in low EGFR-expressing non-invasive breast cancer cells such as MCF-7 cells. Forced overexpression of EGFR in MCF-7 cells (EGFR/MCF-7 cells) resulted in cell migration patterns seen in MDA 468 cells, that is, MAPK pathways play a major role in early stages to cell migration, and PKC-delta plays a major role in later stages of sustained cell migration. The above data demonstrate that EGFR-overexpressing invasive cells have the ability to compensate the loss of MAPK-mediated signaling through activation of PKC-delta signaling for cell migration, which plays a major role in invasion and metastasis. In addition, data suggest that inhibition of MAPK and PKC-delta signaling pathways should abrogate cell migration and invasion in EGFR-overexpressing human breast cancer cells.

Anti-estrogen­resistant breast cancer cells are sensitive to cisplatin plus TRAIL treatment.

Breast cancer patients who are positive for estrogen receptor (ER) are usually treated with anti-estrogen drugs, such as tamoxifen (Tam). However, a great majority of such patients eventually develop resistance to Tam. In this study, MCF-7 cells (with de novo and/or acquired resistance to Tam) as well as T47D cells (acquired resistance to Tam) models were used to investigate the effect of treatment with cisplatin plus tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The results in the two cell types treated with cisplatin plus TRAIL showed significantly increased cell death compared to that in the untreated control cells. A similar treatment had a minimal effect on normal breast cells, the increased cell death appeared to be caused by the activation of caspases and, inhibition of the activity of caspases (using relatively specific inhibitors) reduced the cell death caused by cisplatin plus TRAIL treatment. Taken together, the results suggested that cisplatin plus TRAIL treatment has the potential to provide a novel treatment strategy to improve the treatment outcome in anti-estrogen­resistant breast cancer patients.

Identification and Testing of Novel CARP-1 Functional Mimetic Compounds as Inhibitors of Non-Small Cell Lung and Triple Negative Breast Cancers.

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

Tumor-initiating cells and FZD8 play a major role in drug resistance in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) studies have shown that neoadjuvant chemotherapy before surgery was effective in the minority of women, whereas the majority who had residual tumor had a relatively poor outcome. To identify the mechanism by which residual cancer cells survive chemotherapy, we initially conducted gene expression profiling using the CRL2335 TNBC cell line derived from a squamous breast carcinoma before and after treatment with cisplatin plus TRAIL. We found a significant increase in the expression of FZD8, one of Wnt receptors, and its downstream targets LEF1 and TCF in residual CRL2335 tumor cells after treatment with cisplatin plus TRAIL. Increased FZD8 levels were further confirmed in other TNBC cell lines. Inhibition of FZD8 by siRNA in CRL2335 cells in the presence of cisplatin plus TRAIL reduced ß-catenin and survivin levels and increased apoptosis compared with scrambled siRNA-treated cells. In vivo data show that cisplatin plus TRAIL treatment significantly reduces tumor volume in NOD/SCID mice. However, we found that cisplatin plus TRAIL treatment predominantly eliminated non-tumor-initiating cells, as shown by whole-body fluorescent imaging of mice injected with mammosphere-forming CRL2335 cells stably transfected with DsRed. This led to TIC enrichment in residual tumors, as confirmed by immunostaining for TIC markers. Moreover, an increase in FZD8 expression was observed in residual tumors treated with cisplatin and TRAIL. Taken together, our findings suggest that FZD8-mediated Wnt signaling may play a major role in mediating resistance to chemotherapy, making it a potential target to enhance chemotherapeutic efficacy in patients with TNBCs.

Cisplatin and TRAIL enhance breast cancer stem cell death.

Triple negative breast cancer (TNBC) has increased recurrence and poor survival, despite a high response rate to neoadjuvant chemotherapy. The aim of this study was to determine whether current drug treatment(s) eliminates bulk of tumor cells, but it has a minimal effect on cancer stem cells (CSCs) leading to tumor recurrence. We studied the effects of PARP inhibitors (AZD2281 and BSI-201), paclitaxel, docetaxel, cisplatin and cisplatin plus TRAIL on CSCs derived from CRL-2335 and MDA-MB-468 TNBC cells in vitro. The in vitro data indicate that cisplatin plus TRIAL treatment was most effective in eliminating CSCs compared to PARP inhibitors, cisplatin, paclitaxel and docetaxel. Treatment with cisplatin plus TRAIL also inhibits Wnt-1 signaling and its downstream target, ß-catenin, phospho ß-catenin, cyclin D1, increased apoptosis, reduced proliferation and mammosphere formation. Inhibition of Wnt-1 by siRNA significantly reduced the ability of CSCs to form mammospheres compared to control. However, maximum effect was seen in cisplatin plus TRAIL-treated cells. Taken together the data suggest that cisplatin plus TRAIL treatment has the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients.

Enhanced anticancer effect of the combination of cisplatin and TRAIL in triple-negative breast tumor cells.

Women with triple-negative breast cancer (TNBC) have a worse prognosis compared with other breast cancer subtypes. Hormonal or Herceptin-based therapies were found to be ineffective because of the loss of target receptors, such as ER, PR, and HER-2 amplification. Conventional chemo- and/ or radiation therapy also seems to have limited efficacy in TNBC patients. We studied the effects of cisplatin plus TRAIL on 1 normal and 2 TNBC cells in vitro. The in vitro studies indicate that cisplatin plus TRAIL significantly enhanced cell death in TNBC cell lines CRL2335 and MDA-MB-468 by approximately 60%-70% compared with approximately 10%-15% in CRL8799 normal breast cell line. Treatment with cisplatin/TRAIL also inhibited the expression of EGFR, p63, survivin, Bcl-2, and Bcl-xL in TNBC cells. Specific inhibition of EGFR and/or p63 protein in TNBC cells by small interfering RNA (siRNA) does not increase TRAIL-induced apoptosis. However, inhibition of survivin by siRNA enhances TRAIL-induced apoptosis. These observations suggested the possibility that survivin played an important role in cisplatin plus TRAIL-induced apoptosis in TNBC cells. In vivo experiments, treatment of mice with cisplatin plus TRAIL resulted in a significant inhibition of CRL2335 xenograft tumors compared with untreated control tumors. Taken together the data suggest that cisplatin plus TRAIL treatment have the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients.

Mechanism of 17-beta-estradiol-induced Erk1/2 activation in breast cancer cells. A role for HER2 AND PKC-delta.

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

Role of MAP kinase in tumor progression and invasion.

Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is a frequent event in tumorigenesis. MAPKs have been implicated in cell migration, proteinase-induction, regulation of apoptosis, and angiogenesis, events that are essential for successful completion of metastasis. In this review, we discuss the potential role that MAPKs play in metastasis by regulating cell migration, proteinase-induction and apoptosis.