Harikrishna Nakshatri

Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth.

Breast cancers often progress from a hormone-dependent, nonmetastatic, antiestrogen-sensitive phenotype to a hormone-independent, antiestrogen- and chemotherapy-resistant phenotype with highly invasive and metastatic growth properties. This progression is usually accompanied by altered function of the estrogen receptor (ER) or outgrowth of ER-negative cancer cells. To understand the molecular mechanisms responsible for metastatic growth of ER-negative breast cancers, the activities of the transcription factor NF-kappaB (which modulates the expression of genes involved in cell proliferation, differentiation, apoptosis, and metastasis) were compared in ER-positive (MCF-7 and T47-D) and ER-negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines. NF-kappaB, which is usually maintained in an inactive state by protein-protein interaction with inhibitor IkappaBs, was found to be constitutively active in ER-negative breast cancer cell lines. Constitutive DNA binding of NF-kappaB was also observed with extracts from ER-negative, poorly differentiated primary breast tumors. Progression of the rat mammary carcinoma cell line RM22-F5 from an ER-positive, nonmalignant phenotype (E phenotype) to an ER-negative, malignant phenotype (F phenotype) was also accompanied by constitutive activation of NF-kappaB. Analysis of individual subunits of NF-kappaB revealed that all ER-negative cell lines, including RM22-F5 cells of F phenotype, contain a unique 37-kDa protein which is antigenically related to the RelA subunit. Cell-type-specific differences in IkappaB alpha, -beta, and -gamma were also observed. In transient-transfection experiments, constitutive activity of an NF-kappaB-dependent promoter was observed in MDA-MB-231 and RM22-F5 cells of F phenotype, and this activity was efficiently repressed by cotransfected ER. Since ER inhibits the constitutive as well as inducible activation function of NF-kappaB in a dose-dependent manner, we propose that breast cancers that lack functional ER overexpress NF-kappaB-regulated genes. Furthermore, since recent data indicate that NF-kappaB protects cells from tumor necrosis factor alpha-, ionizing radiation-, and chemotherapeutic agent daunorubicin-mediated apoptosis, our results provide an explanation for chemotherapeutic resistance in ER-negative breast cancers.

NF-κB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: Potential involvement of ZEB-1 and ZEB-2

The transcription factor nuclear factor kappa B (NF-κB) is constitutively active in both cancer cells and stromal cells of breast cancer; however, the precise role of activated NF-κB in cancer progression is not known. Using parental MCF10A cells and a variant that expresses the myoepithelial marker p63 stably overexpressing the constitutively active p65 subunit of NF-κB (MCF10A/p65), we show that NF-κB suppresses the expression of epithelial specific genes E-cadherin and desmoplakin and induces the expression of the mesenchymal specific gene vimentin. P65 also suppressed the expression of p63 and the putative breast epithelial progenitor marker cytokeratin 5/6. MCF10A/p65 cells were phenotypically similar to cells undergoing epithelial to mesenchymal transition (EMT). MCF10A/p65 cells failed to form characteristic acini in three-dimensional Matrigel. Analysis of parental and MCF10A/p65 cells for genes previously shown to be involved in EMT revealed elevated expression of ZEB-1 and ZEB-2 in MCF10A/p65 cells compared to parental cells. In transient transfection assays, p65 increased ZEB-1 promoter activity. Furthermore, MCF10A cells overexpressing ZEB-1 showed reduced E-cadherin and p63 expression and displayed an EMT phenotype. The siRNA against ZEB-1 or ZEB-2 reduced the number of viable MCF10A/p65 but not parental cells, suggesting the dependence of MCF10A/p65 cells to ZEB-1 and ZEB-2 for cell cycle progression or survival. MCF10A cells chronically exposed to tumor necrosis factor alpha (TNFα), a potent NF-κB inducer, also exhibited the EMT-like phenotype and ZEB-1/ZEB-2 induction, both of which were reversed following TNFα withdrawal.

Cutting Edge: IL-17F, a Novel Cytokine Selectively Expressed in Activated T Cells and Monocytes, Regulates Angiogenesis and Endothelial Cell Cytokine Production

A novel secreted cytokine, termed IL-17F, was cloned using nested RACE PCR. This cytokine bears homology to IL-17. IL-17F was expressed only in activated CD4+ T cells and activated monocytes. Recombinant human IL-17F did not stimulate the proliferation of hematopoietic progenitors or the migration of mature leukocytes. However, it markedly inhibited the angiogenesis of human endothelial cells and induced endothelial cells to produce IL-2, TGF-β, and monocyte chemoattractant protein-1.

Paclitaxel sensitivity of breast cancer cells with constitutively active NF-κB is enhanced by IκBα super-repressor and parthenolide

The transcription factor nuclear factor-κB (NF-κB) regulates genes important for tumor invasion, metastasis and chemoresistance. Normally, NF-κB remains sequestered in an inactive state by cytoplasmic inhibitor-of-κB (IκB) proteins. NF-κB translocates to nucleus and activates gene expression upon exposure of cells to growth factors and cytokines. We and others have shown previously that NF-κB is constitutively active in a subset of breast cancers. In this study, we show that constitutive activation of NF-κB leads to overexpression of the anti-apoptotic genes c-inhibitor of apoptosis 2 (c-IAP2) and manganese superoxide dismutase (Mn-SOD) in breast cancer cells. Furthermore, expression of the anti-apoptotic tumor necrosis factor receptor associated factor 1 (TRAF1) and defender-against cell death (DAD-1) is regulated by NF-κB in certain breast cancer cells. We also demonstrate that NF-κB-inducible genes protect cancer cells against paclitaxel as MDA-MB-231 breast cancer cells modified to overexpress IκBα required lower concentrations of paclitaxel to arrest at the G2/M phase of the cell cycle and undergo apoptosis when compared to parental cells. The effect of NF-κB on paclitaxel-sensitivity appears to be specific to cancer cells because normal fibroblasts derived from embryos lacking p65 subunit of NF-κB and wild type littermate embryos were insensitive to paclitaxel-induced G2/M cell cycle arrest. Parthenolide, an active ingredient of herbal remedies such as feverfew (tanacetum parthenium), mimicked the effects of IκBα by inhibiting NF-κB DNA binding activity and Mn-SOD expression, and increasing paclitaxel-induced apoptosis of breast cancer cells. These results suggest that active ingredients of herbs with anti-inflammatory properties may be useful in increasing the sensitivity of cancers with constitutively active NF-κB to chemotherapeutic drugs.

FOXA1 expression in breast cancer- : Correlation with luminal subtype A and survival

Purpose: FOXA1, a forkhead family transcription factor, is essential for optimum expression of ∼50% of estrogen receptor α (ERα):estrogen responsive genes. FOXA1 is expressed in breast cancer cells. It segregates with genes that characterize the luminal subtypes in DNA microarray analyses. The utility of FOXA1 as a possible independent prognostic factor has not been determined in breast cancers. Materials and Methods: A tissue microarray comprising tumors from 438 patients with 15.4 years median follow-up was analyzed for FOXA1 expression by immunohistochemistry. Interpretable FOXA1 expression obtained in 404 patients was analyzed along with other prognostic factors like tumor grade, size, nodal status, ER, progesterone receptor (PR), and HER2/neu. Results: FOXA1 expression (score >3) was seen in 300 of 404 breast cancers and it correlated with ER (P = 0.000001), PR (P = 0.00001), and luminal A subtype (P = 0.000001). Loss of expression was noted with worsening tumor grade (P = 0.001). Univariate analysis showed nodal status (P = 0.0000012), tumor size (P = 0.00001), FOXA1 (P = 0.0004), and ER (P = 0.012) to be predictors of breast cancer–specific survival. Multivariate analysis showed only nodal status (P = 0.001) and tumor size (P = 0.039) to be significant prognostic factors, whereas FOXA1 (P = 0.060) and ER (P = 0.131) were not significant. In luminal subtype A patient subgroup, FOXA1 expression was associated with better cancer-specific survival (P = 0.024) and in ER-positive subgroup, it was better predictor of cancer-specific survival (P = 0.009) than PR (P = 0.213). Conclusion: FOXA1 expression correlates with luminal subtype A breast cancer and it is significant predictor of cancer-specific survival in patients with ER-positive tumors. Prognostic ability of FOXA1 in these low-risk breast cancers may prove to be useful in clinical treatment decisions.

Nuclear Factor-κB Is Constitutively Activated in Prostate Cancer In vitro and Is Overexpressed in Prostatic Intraepithelial Neoplasia and Adenocarcinoma of the Prostate

Purpose: The transcription factor nuclear factor-κB (NF-κB) promotes the production of angiogenic, antiapoptotic, and prometastatic factors that are involved in carcinogenesis. Experimental Design: Electromobility gel shift assays were used to evaluate NF-κB DNA binding in vitro. The functional relevance of NF-κB DNA binding was assessed by both cDNA array analyses and proliferation assays of prostate cancer cells with and without exposure to an NF-κB inhibitor, parthenolide. Immunohistochemistry staining for the p65 NF-κB subunit was used to determine the frequency and location of NF-κB in 97 prostatectomy specimens. The amount of staining was quantified on a 0–3+ scale. Results: An electromobility gel shift assay confirmed the presence of NFκB DNA binding in all four prostate cancer cell lines tested. The binding was inhibited by parthenolide, and this agent also decreased multiple gene transcripts under the control of NF-κB and inhibited proliferation of prostate cancer cells. The staining results revealed overexpression of p65 in the prostatic intraepithelial neoplasia and cancer compared with the benign epithelium. Specifically, there was a predominance of 1+ and 2+ with no 3+ staining in benign epithelium, whereas there was only 2+ and 3+ staining (30 and 70%, respectively) in the cancerous areas. These differences were statistically different. There was no correlation with tumor grade or stage. Conclusions: NF-κB is constitutively activated in prostate cancer and functionally relevant in vitro. Immunohistochemistry of human prostatectomy specimens demonstrated overexpression of the active subunit of NF-κB, p65, and that this occurs at an early stage in the genesis of prostate cancer. This work supports the rationale for targeting NF-κB for the prevention and/or treatment of prostate cancer.

Prognostic impact of ALDH1 in breast cancer: a story of stem cells and tumor microenvironment

The concept of cancer cells being hierarchically organized and arising from their own progenitor stem cells will have important implications on cancer therapy. If this hypothesis were to be true then the paucity of estrogen receptors in stem cells as well as their inherent drug resistance mechanisms pose a challenge to current targeted therapies. In this study, we sought to examine the prognostic relevance of ALDH1, a putative cancer stem cell marker, by immunohistochemistry. The four cohorts analyzed included an adjuvantly treated series of 245 invasive cancers, a neoadjuvantly treated series of 34 cases, and two series of 58 and 40 triple negative cases, respectively. Both tumor cell and stromal expression for ALDH1 was evaluated, where possible. Tumor cell ALDH1 expression significantly correlated only with basal-like and HER2 tumor types in the adjuvant series and tumor grade in the neoadjuvant cohort. No significant enrichment for ALDH1 positive cells was observed in the postneoadjuvant therapy specimens compared to pretreatment samples. On the other hand, high degree of stromal expression was significantly associated with best disease-free survival as well as a trend for overall survival. The association of stromal expression was confirmed in an independent cohort of triple negative cases. The novel finding is that tumor microenvironment may play a significant role in determining the prognostic impact of stem/progenitor cells in human breast tumors.

Antitumor agent parthenolide reverses resistance of breast cancer cells to tumor necrosis factor-related apoptosis-inducing ligand through sustained activation of c-Jun N-terminal kinase.

The antitumor activity of the sesquiterpene lactone parthenolide, an active ingredient of medicinal plants, is believed to be due to the inhibition of DNA binding of transcription factors NF-κB and STAT-3, reduction in MAP kinase activity and the generation of reactive oxygen. In this report, we show that parthenolide activates c-Jun N-terminal kinase (JNK), which is independent of inhibition of NF-κB DNA binding and generation of reactive oxygen species. Parthenolide reversed resistance of breast cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Cancer cells treated with a combination of TRAIL and parthenolide underwent massive typical apoptosis and atypical apoptosis involving the loss of plasma membrane integrity. JNK activity is necessary for the parthenolide-induced sensitization to TRAIL because a dominant-negative JNK or the JNK inhibitor SP600125 reduced TRAIL plus parthenolide-induced apoptosis. Parthenolide induced phosphorylation of Bid and increased TRAIL-dependent cleavage of Bid without affecting caspase 8 activities. Cytochrome c but not Smac/DIABLO was released from the mitochondria in cells treated with parthenolide alone. Parthenolide through JNK increased the TRAIL-mediated degradation of the antiapoptotic protein X-linked inhibitor of apoptosis (XIAP). Enhanced XIAP cleavage correlated with increased and prolonged caspase 3 activity and PARP cleavage, suggesting that the sensitization to TRAIL involves ‘feed forward’ activation of caspase 3. These results identify a new antitumor activity of parthenolide, which can be exploited to reverse resistance of cancer cells to TRAIL, particularly those with elevated XIAP levels.

Identification of signal transduction pathways involved in constitutive NF-κB activation in breast cancer cells

Nuclear factor-κB (NF-κB) is usually maintained in an inactive form in the cytoplasm through its association with inhibitor of kappaB (IκB) proteins, and is activated upon stimulation of cells with a variety of signals. However, constitutive activation of NF-κB is observed in a number of cancers including breast cancer. The signaling pathways that are involved in constitutive NF-κB activation remain largely unknown. Using breast cancer cell lines derived from transgenic mice that overexpress specific oncogene/growth factors in the mammary gland, we show that heregulin but not her2/neu, c-Myc or v-Ha-ras plays a major role in constitutive NF-κB activation. Her2/neu potentiated tumor necrosis factor alpha (TNFα)-inducible NF-κB activation whereas c-Myc potentiated 12-o-tetracecanyolphorbol-13-acetate (TPA)-induced NF-κB activation. Heregulin-mediated NF-κB activation correlated with phosphorylation of epidermal growth factor receptor (EGFR) and ErbB3 but not her2/neu. Tryphostin AG1517, which inhibits heregulin-mediated phosphorylation of EGFR, her2/neu and ErbB3 reduced NF-κB activation. In contrast, emodin, which blocks phosphorylation of her2/neu by heregulin, failed to reduce NF-κB activation. These results suggest that heregulin induces NF-κB independent of her2/neu. PI3 kinase/AKT, protein kinase A (PKA) and IκB kinase appear to be downstream signaling molecules involved in NF-κB activation as specific inhibitors of these kinases but not inhibitors of ERK/MAP kinase or protein kinase C reduced heregulin-mediated NF-κB activation. Based on these results, we propose that heregulin increases the expression of pro-invasive, pro-metastatic and anti-apoptotic genes in cancer cells through autocrine activation of NF-κB, which leads to invasive and drug-resistant growth of breast cancer.

Parthenolide and sulindac cooperate to mediate growth suppression and inhibit the nuclear factor-κB pathway in pancreatic carcinoma cells

Activation of the transcription factor nuclear factor-κB (NF-κB) has been implicated in pancreatic tumorigenesis. We evaluated the effect of a novel NF-κB inhibitor, parthenolide, a sesquiterpene lactone isolated from the herb feverfew, in three human pancreatic tumor cell lines (BxPC-3, PANC-1, and MIA PaCa-2). Parthenolide inhibited pancreatic cancer cell growth in a dose-dependent manner with substantial growth inhibition observed between 5 and 10 μmol/L parthenolide in all three cell lines. Parthenolide treatment also dose-dependently increased the amount of the NF-κB inhibitory protein, IκB-α, and decreased NF-κB DNA binding activity. We have previously shown that nonsteroidal anti-inflammatory drugs (NSAID) suppress the growth of pancreatic cancer cells. To determine whether inhibition of the NF-κB pathway by parthenolide could sensitize pancreatic cancer cells to NSAID inhibition, BxPC-3, PANC-1, and MIA PaCa-2 cells were treated with parthenolide and the NSAID sulindac, either alone or in combination. Treatment with the combination of parthenolide and sulindac inhibited cell growth synergistically in MIA PaCa-2 and BxPC-3 cells and additively in PANC-1 cells. In addition, treatment with the parthenolide/sulindac combination lowered the threshold for apoptosis. Increased levels of IκB-α protein were detected, especially in MIA PaCa-2 cells, after treatment with parthenolide and sulindac compared with each agent alone. Similarly, decreased NF-κB DNA binding and transcriptional activities were detected in cells treated with the combination compared with the single agents, demonstrating cooperative targeting of the NF-κB pathway. These data provide preclinical support for a combined chemotherapeutic approach with NF-κB inhibitors and NSAIDs for the treatment of pancreatic adenocarcinoma.