Aurora-A Mitotic Kinase Induces Endocrine Resistance through Down-Regulation of ERα Expression in Initially ERα+ Breast Cancer Cells

Development of endocrine resistance during tumor progression represents a major challenge in the management of estrogen receptor alpha (ERα) positive breast tumors and is an area under intense investigation. Although the underlying mechanisms are still poorly understood, many studies point towards the ‘cross-talk’ between ERα and MAPK signaling pathways as a key oncogenic axis responsible for the development of estrogen-independent growth of breast cancer cells that are initially ERα+ and hormone sensitive. In this study we employed a metastatic breast cancer xenograft model harboring constitutive activation of Raf-1 oncogenic signaling to investigate the mechanistic linkage between aberrant MAPK activity and development of endocrine resistance through abrogation of the ERα signaling axis. We demonstrate for the first time the causal role of the Aurora-A mitotic kinase in the development of endocrine resistance through activation of SMAD5 nuclear signaling and down-regulation of ERα expression in initially ERα+ breast cancer cells. This contribution is highly significant for the treatment of endocrine refractory breast carcinomas, because it may lead to the development of novel molecular therapies targeting the Aurora-A/SMAD5 oncogenic axis. We postulate such therapy to result in the selective eradication of endocrine resistant ERα^low/− cancer cells from the bulk tumor with consequent benefits for breast cancer patients.     

Inhibition of β2-Microglobulin/Hemochromatosis Enhances Radiation Sensitivity by Induction of Iron Overload in Prostate Cancer Cells

Background

Bone metastasis is the most lethal form of several cancers. The β2-microglobulin (β2-M)/hemochromatosis (HFE) complex plays an important role in cancer development and bone metastasis. We demonstrated previously that overexpression of β2-M in prostate, breast, lung and renal cancer leads to increased bone metastasis in mouse models. Therefore, we hypothesized that β2-M is a rational target to treat prostate cancer bone metastasis.

Results

In this study, we demonstrate the role of β2-M and its binding partner, HFE, in modulating radiation sensitivity and chemo-sensitivity of prostate cancer. By genetic deletion of β2-M or HFE or using an anti-β2-M antibody (Ab), we demonstrate that prostate cancer cells are sensitive to radiation in vitro and in vivo. Inhibition of β2-M or HFE sensitized prostate cancer cells to radiation by increasing iron and reactive oxygen species and decreasing DNA repair and stress response proteins. Using xenograft mouse model, we demonstrate that anti-β2-M Ab sensitizes prostate cancer cells to radiation treatment. Additionally, anti-β2-M Ab was able to prevent tumor growth in an immunocompetent spontaneous prostate cancer mouse model. Since bone metastasis is lethal, we used a bone xenograft model to test the ability of anti-β2-M Ab and radiation to block tumor growth in the bone. Combination treatment significantly prevented tumor growth in the bone xenograft model by inhibiting β2-M and inducing iron overload. In addition to radiation sensitive effects, inhibition of β2-M sensitized prostate cancer cells to chemotherapeutic agents.

Conclusion

Since prostate cancer bone metastatic patients have high β2-M in the tumor tissue and in the secreted form, targeting β2-M with anti-β2-M Ab is a promising therapeutic agent. Additionally, inhibition of β2-M sensitizes cancer cells to clinically used therapies such as radiation by inducing iron overload and decreasing DNA repair enzymes.     

Tamoxifen resistance and metastasis of human breast cancer cells were mediated by the membrane-associated estrogen receptor ER-α36 signaling in vitro

The drug resistance and tumor metastasis have been the main obstacles for the longer-term therapeutic effects of tamoxifen (TAM) on estrogen receptor-positive (ER⁺) breast cancer, but the mechanisms underlying the TAM resistance are still unclear. Here, we demonstrated that the membrane-associated estrogen receptor ER-α36 signaling, but not the G protein-coupled estrogen receptor 1 (GPER1) signaling, might be involved in the TAM resistance and metastasis of breast cancer cells. In this study, a model of ER⁺ breast cancer cell MCF-7 that involves the up-regulated expression of ER-α36 and unchanged expression of ER-α66 and GPER1 was established via the removal of insulin from the cell culture medium. The mechanism of TAM resistance in the ER⁺ breast cancer cell line MCF-7 was investigated, and the results showed that the stimulating effect of insulin on susceptibility of MCF-7 to TAM was mediated by ER-α36 and that the expression level of ER-α36 in TAM-resistant MCF-7 cells was also significantly increased. Both TAM and estradiol (E2) could promote the migration of triple negative (ER-α66^?/PR^?/HER2^?) and ER-α36⁺/GPER1⁺ breast cancer cells MDA-MB-231. The migration of MDA-MB-231 cells was inhibited by the down-regulated intracellular expression of ER-α36 by transient transfection of specific small interfering RNA, whereas no effect of GPER1 down-regulation was observed. Meanwhile, the effect of TAM on the migration of ER-α36-down-regulated MDA-MB-231 cells was also reduced. Furthermore, it was found that TAM enhanced the distribution of integrin β1 on the cell surface but did not affect the expression of integrin β1 in MDA-MB-231 cells. Collectively, these data suggested that ER-α36 signaling might play critical roles in acquired and de novo TAM resistance and metastasis of breast cancer, and ER-α36 might present a potential biomarker of TAM resistance in the clinical diagnosis and treatment of ER⁺ breast cancer.     

3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

Herceptin failure is a major clinical problem in breast cancer. A subset of breast cancer patients with high HER-2/neu levels eventually experience metastatic disease progression when treated with Herceptin as a single agent. Mechanistic details of development of this aggressive disease are not clear. Therefore, there is a dire need to better understand the mechanisms by which drug resistance develops and to design new combined treatments that benefit patients with aggressive breast cancer and have minimal toxicity. We hypothesized that 3, 3′-diindolylmethane (DIM), a non-toxic agent can be combined with Herceptin to treat breast cancers with high levels of HER-2/neu. Here, we evaluated the effects of Herceptin alone and in combination with DIM on cell viability, apoptosis and clonogenic assays in SKBR3 (HER-2/neu-expressing) and MDA-MB-468 (HER-2/neu negative) breast cancer cells. We found that DIM could enhance the effectiveness of Herceptin by significantly reducing cell viability, which was associated with apoptosis-induction and significant inhibition of colony formation, compared with single agent treatment. These results were consistent with the down-regulation of Akt and NF-kB p65. Mechanistic investigations revealed a significant upregulation of miR-200 and reduction of FoxM1 expression in DIM and Herceptin-treated breast cancer cells. We, therefore, transfected cells with pre-miR-200 or silenced FoxM1 in these cells for understanding the molecular mechanism involved. These results provide experimental evidence, for the first time, that DIM plus Herceptin therapy could be translated to the clinic as a therapeutic modality to improve treatment outcome of patients with breast cancer, particularly for the patients whose tumors express high levels of HER-2/neu.     

MicroRNA-3646 Contributes to Docetaxel Resistance in Human Breast Cancer Cells by GSK-3β/β-Catenin Signaling Pathway

Acquisition of resistance to docetaxel (Doc) is one of the most important problems in treatment of breast cancer patients, but the underlying mechanisms are still not fully understood. In present study, Doc-resistant MDA-MB-231 and MCF-7 breast cancer cell lines (MDA-MB-231/Doc and MCF-7/Doc) were successfully established in vitro by gradually increasing Doc concentration on the basis of parental MDA-MB-231 and MCF-7 cell lines (MDA-MB-231/S and MCF-7/S). The potential miRNAs relevant to the Doc resistance were screened by miRNA microarray. We selected 5 upregulated miRNAs (has-miR-3646, has-miR-3658, has-miR-4438, has-miR-1246, and has-miR-574-3p) from the results of microarray for RT-qPCR validation. The results showed that expression level of miR-3646 in MDA-MB-231/Doc cells was significantly higher than in MDA-MB-231/S cells. Compared to MCF-7/S cells, miR-3646 expression was up-regulated in MCF-7/Doc cells. Further studies revealed that transfection of miR-3646 mimics into MDA-MB-231/S or MCF-7/S cells remarkably increased their drug resistance, in contrast, transfection of miR-3646 inhibitors into MDA-MB-231/Doc or MCF-7/Doc cells resulted in significant reduction of the drug resistance. By the pathway enrichment analyses for miR-3646, we found that GSK-3β/β-catenin signaling pathway was a significant pathway, in which GSK-3β was an essential member. RT-qPCR and Western blot results demonstrated that miR-3646 could regulate GSK-3β mRNA and protein expressions. Furthermore, a marked increase of both nuclear and cytoplasmic β-catenin expressions (with phosphorylated-β-catenin decrease) was observed in MDA-MB-231/Doc cells compared with MDA-MB-231/S cells, and their expression were positively related to miR-3646 and negatively correlated with GSK-3β. Taken together, our results suggest that miR-3646-mediated Doc resistance of breast cancer cells maybe, at least in part, through suppressing expression of GSK-3β and resultantly activating GSK-3β/β-catenin signaling pathway.     

Mitochondrial Bioenergetics of Metastatic Breast Cancer Cells in Response to Dynamic Changes in Oxygen Tension: Effects of HIF-1α

Solid tumors are characterized by regions of low oxygen tension (OT), which play a central role in tumor progression and resistance to therapy. Low OT affects mitochondrial function and for the cells to survive, mitochondria must functionally adapt to low OT to maintain the cellular bioenergetics. In this study, a novel experimental approach was developed to examine the real-time bioenergetic changes in breast cancer cells (BCCs) during adaptation to OT (from 20% to <1% oxygen) using sensitive extracellular flux technology. Oxygen was gradually removed from the medium, and the bioenergetics of metastatic BCCs (MDA-MB-231 and MCF10CA clones) was compared with non-tumorigenic (MCF10A) cells. BCCs, but not MCF10A, rapidly responded to low OT by stabilizing HIF-1α and increasing HIF-1α responsive gene expression and glucose uptake. BCCs also increased extracellular acidification rate (ECAR), which was markedly lower in MCF10A. Interestingly, BCCs exhibited a biphasic response in basal respiration as the OT was reduced from 20% to <1%. The initial stimulation of oxygen consumption is found to be due to increased mitochondrial respiration. This effect was HIF-1α-dependent, as silencing HIF-1α abolished the biphasic response. During hypoxia and reoxygenation, BCCs also maintained oxygen consumption rates at specific OT; however, HIF-1α silenced BCC were less responsive to changes in OT. Our results suggest that HIF-1α provides a high degree of bioenergetic flexibility under different OT which may confer an adaptive advantage for BCC survival in the tumor microenvironment and during invasion and metastasis. This study thus provides direct evidence for the cross-talk between HIF-1α and mitochondria during adaptation to low OT by BCCs and may be useful in identifying novel therapeutic agents that target the bioenergetics of BCCs in response to low OT.     

Chaperonin CCT-Mediated AIB1 Folding Promotes the Growth of ERα-Positive Breast Cancer Cells on Hard Substrates

Clinical observations have revealed a strong association between estrogen receptor alpha (ERα)-positive tumors and the development of bone metastases, however, the mechanism underlying this association remains unknown. We cultured MCF-7 (ERα-positive) on different rigidity substrates. Compared with cells grown on more rigid substrates (100 kPa), cells grown on soft substrates (10 kPa) exhibited reduced spreading ability, a lower ratio of cells in the S and G2/M cell cycle phases, and a decreased proliferation rate. Using stable isotope labeling by amino acids (SILAC), we further compared the whole proteome of MCF-7 cells grown on substrates of different rigidity (10 and 100 kPa), and found that the expression of eight members of chaperonin CCT increased by at least 2-fold in the harder substrate. CCT folding activity was increased in the hard substrate compared with the soft substrates. Amplified in breast cancer 1 (AIB1), was identified in CCT immunoprecipitates. CCT folding ability of AIB1 increased on 100-kPa substrate compared with 10- and 30-kPa substrates. Moreover, using mammalian two-hybrid protein-protein interaction assays, we found that the polyglutamine repeat sequence of the AIB1 protein was essential for interaction between CCTζ and AIB1. CCTζ-mediated AIB1 folding affects the cell area spreading, growth rate, and cell cycle. The expressions of the c-myc, cyclin D1, and PgR genes were higher on hard substrates than on soft substrate in both MCF-7 and T47D cells. ERα and AIB1 could up-regulate the mRNA and protein expression levels of the c-myc, cyclin D1, and PgR genes, and that 17 β-estradiol could enhance this effects. Conversely, 4-hydroxytamoxifen, could inhibit these effects. Taken together, our studies demonstrate that some ERα-positive breast cancer cells preferentially grow on more rigid substrates. CCT-mediated AIB1 folding appears to be involved in the rigidity response of breast cancer cells, which provides novel insight into the mechanisms of bone metastasis.     

Protein Kinase C�� Supports Survival of MDA-MB-231 Breast Cancer Cells by Suppressing the ERK1/2 Pathway

Mechanisms that mediate apoptosis resistance are attractive therapeutic targets for cancer. Protein kinase Cδ (PKCδ) is considered a pro-apoptotic factor in many cell types. In breast cancer, however, it has shown both pro-survival and pro-apoptotic effects. Here, we report for the first time that down-regulation of PKCδ per se leads to apoptosis of MDA-MB-231 cells. Inhibition of MEK1/2 by either PD98059 or U0126 suppressed the induction of apoptosis of PKCδ-depleted MDA-MB-231 cells but did not support survival of MCF-7 or MDA-MB-468 cells. Basal ERK1/2 phosphorylation was substantially higher in MDA-MB-231 cells than in the other cell lines. PKCδ depletion led to even higher ERK1/2 phosphorylation levels and also to lower expression levels of the ERK1/2 phosphatase MKP3. Depletion of MKP3 led to apoptosis and higher levels of ERK1/2 phosphorylation, suggesting that this may be a mechanism mediating the effect of PKCδ down-regulation. However, PKCδ silencing also induced increased MEK1/2 phosphorylation, indicating that PKCδ regulates ERK1/2 phosphorylation both upstream and downstream. Moreover, PKCδ silencing led to increased levels of the E3 ubiquitin ligase Nedd4, which is a potential regulator of MKP3, because down-regulation led to increased MKP3 levels. Our results highlight PKCδ as a potential target for therapy of breast cancers with high activity of the ERK1/2 pathway.     

Advances in the understanding of the structure and function of ER-α36,a novel variant of human estrogen receptor-alpha

Estrogen receptors (ERs) belong to the nuclear receptor superfamily, whose members include ER-α66, ER-α36, ER-α46 and ER-β. Each receptor performs specific functions through binding with a specific ligand, such as estrogen. Recently, ER-α36, a novel variant of human estrogen receptor-alpha (ER-α), was identified and cloned. ER-α36 inhibits, in a dominant-negative manner, the transactivation of both the wild-type ER-α (ER-α66) and ER-β. As a predominantly membrane-based ER, ER-α36 mediates nongenomic estrogen signaling and is involved in the resistance of breast cancer to endocrine therapy, i.e., tamoxifen. This review summarizes recent studies on the structure and function of ER-α36 and the relationship of ER-α36 with cancer, with special emphasis on its function in the resistance of breast cancer to endocrine therapy.     

The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells

BACKGROUND: MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood. METHODS: We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apoptosis analysis (Annexin V detection), and induction of senescence (β-galactosidase analysis). RESULTS: In a screen for MDM2 splice variants in MCF-7 breast cancer cells, extended with data from healthy leukocytes, we found P2-MDM2-10 and MDM2-Δ5 to be the splice variants expressed at highest levels. Contrasting MDM2 full-length protein, we found normal tissue expression levels of P2-MDM2-10 and MDM2-Δ5 to be highest in individuals harboring the promoter SNP309TT genotype. While we detected no protein product coded for by MDM2-Δ5, the P2-MDM2-10 variant generated a protein markedly more stable than MDM2-FL. Both splice variants were significantly upregulated in stressed cells (P = 4.3 × 10^?4 and P = 7.1 × 10^?4, respectively). Notably, chemotherapy treatment and overexpression of P2-MDM2-10 or MDM2-Δ5 both lead to increased mRNA levels of the endogenous MDM2-FL (P = .039 and P = .070, respectively) but also the proapoptotic gene PUMA (P = .010 and P = .033, respectively), accompanied by induction of apoptosis and repression of senescence. CONCLUSION: We found P2-MDM2-10 and MDM2-Δ5 to have distinct biological functions in breast cancer cells. GENERAL SIGNIFICANCE: Alternative splicing may influence the oncogenic effects of the MDM2 gene.     

Prospective Validation of Immunological Infiltrate for Prediction of Response to Neoadjuvant Chemotherapy in HER2-Negative Breast Cancer – A Substudy of the Neoadjuvant GeparQuinto Trial

Introduction

We have recently described an increased lymphocytic infiltration rate in breast carcinoma tissue is a significant response predictor for anthracycline/taxane-based neoadjuvant chemotherapy (NACT). The aim of this study was to prospectively validate the tumor-associated lymphocyte infiltrate as predictive marker for response to anthracycline/taxane-based NACT.

Patients and Methods

The immunological infiltrate was prospectively evaluated in a total of 313 core biopsies from HER2 negative patients of the multicenter PREDICT study, a substudy of the neoadjuvant GeparQuinto study. Intratumoral lymphocytes (iTuLy), stromal lymphocytes (strLy) as well as lymphocyte-predominant breast cancer (LPBC) were evaluated by histopathological assessment. Pathological complete response (pCR) rates were analyzed and compared between the defined subgroups using the exact test of Fisher.

Results

Patients with lymphocyte-predominant breast cancer (LPBC) had a significantly increased pCR rate of 36.6%, compared to non-LPBC patients (14.3%, p<0.001). LPBC and stromal lymphocytes were significantly independent predictors for pCR in multivariate analysis (LPBC: OR 2.7, p = 0.003, strLy: OR 1.2, p = 0.01). The amount of intratumoral lymphocytes was significantly predictive for pCR in univariate (OR 1.2, p = 0.01) but not in multivariate logistic regression analysis (OR 1.2, p = 0.11).

Conclusion

Confirming previous investigations of our group, we have prospectively validated in an independent cohort that an increased immunological infiltrate in breast tumor tissue is predictive for response to anthracycline/taxane-based NACT. Patients with LPBC and increased stromal lymphocyte infiltration have significantly increased pCR rates. The lymphocytic infiltrate is a promising additional parameter for histopathological evaluation of breast cancer core biopsies.