Target For Breast Cancer Therapy Research Articles

Interplay Between HGF/SF-Met-Ras Signaling, Tumor Metabolism and Blood Flow as a Potential Target for Breast Cancer Therapy.

High glucose uptake and increase blood flow is a characteristic of most metastatic tumors. Activation of Ras signaling increases glycolytic flux into lactate, de novo nucleic acid synthesis and uncoupling of ATP synthase from the proton gradient. Met tyrosine kinase receptor signaling upon activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF), increases glycolysis, oxidative phosporylation, oxygen consumption, and tumor blood volume. Ras is a key factor in Met signaling. Using the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS), we investigated interplay between HGF/SF-Met-Ras signaling, metabolism, and tumor blood-flow regulation. In vitro, HGF/SF-activated Met increased Ras activity, Erk phosphorylation, cell motility and glucose uptake, but did not affect ATP. FTS inhibited basal and HGF/SF-induced signaling and cell motility, while further increasing glucose uptake and inhibiting ATP production. In vivo, HGF/SF rapidly increased tumor blood volume. FTS did not affect basal blood-flow but abolished the HGF/SF effect. Our results further demonstrate the complex interplay between growth-factor-receptor signaling and cellular and tumor metabolism, as reflected in blood flow. Inhibition of Ras signaling does not affect glucose consumption or basal tumor blood flow but dramatically decreases ATP synthesis and the HGF/SF induced increase in tumor blood volume. These findings demonstrate that the HGF/SF-Met-Ras pathway critically influences tumor-cell metabolism and tumor blood-flow regulation. This pathway could potentially be used to individualize tumor therapy based on functional molecular imaging, and for combined signaling/anti-metabolic targeted therapy.

Upregulated microRNA-301a in breast cancer promotes tumor metastasis by targeting PTEN and activating Wnt/β-catenin signaling

MicroRNAs (miRNAs) are strongly implicated in many cancers, including breast cancer. Recently, microRNA-301a (miR-301a) has been proved to play a substantial role in gastric cancer, but its functions in the context of breast cancer remain unknown. Here we report that miR-301a was markedly upregulated in primary tumor samples from patients with distant metastases and pro-metastatic breast cancer cell lines. Gain-of-function and loss-of-function studies showed that ectopic overexpression of miR-301a promoted breast cancer cell migration, invasion and metastasis both in vitro and in vivo. Notably, Wnt/β-catenin signaling was hyperactivated in metastatic breast cancer cells that express miR-301a, and mediated miR-301a-induced invasion and metastasis. Furthermore, miR-301a directly targeted and suppressed PTEN, one negative regulator of the Wnt/β-catenin signaling cascade. These results demonstrate that miR-301a maintains constitutively activated Wnt/β-catenin signaling by directly targeting PTEN, which promotes breast cancer invasion and metastasis. Taken together, our findings reveal a new regulatory mechanism of miR-301a and suggest that miR-301a might be a potential target in breast cancer therapy.

Inhibition of Leptin and Leptin Receptor Gene Expression by Silibinin-Curcumin Combination

Leptin and its receptor are involved in breast carcinogenesis as mitogenic factors. Therefore, they could be considered as targets for breast cancer therapy. Expression of the leptin receptor gene could be modulated by leptin secretion. Silibinin and curcumin are herbal compounds with anti-cancer activity against breast cancer. The aim of this study was to assess their potential to inhibit of expression of the leptin gene and its receptor and leptin secretion. Cytotoxic effects of the two agents on combination on T47D breast cancer cells was investigated by MTT assay test after 24h treatment. With different concentrations the levels of leptin, leptin receptor genes expression were measured by reverse-transcription real-time PCR. Amount of secreted leptin in the culture medium was determined by ELISA. Data were statistically analyzed by one-way ANOVA test. The silibinin and curcumin combination inhibited growth of T47D cells in a dose dependent manner. There were also significant difference between control and treated cells in leptin expression and the quantity of secreted leptin with a relative decrease in leptin receptor expression. In conclusion, these herbal compounds inhibit the expression and secretion of leptin and it could probably be used as drug candidates for breast cancer therapy through leptin targeting in the future.

Abstract C184: Thyroid hormone receptors: Future targets for breast cancer therapy?.

Abstract Introduction: Thyroid receptors (TR) have been documented in breast cancer tissue. Given their role in cell metabolism and proliferation, they may serve as future therapeutic targets. Prior to targeting TRs for therapy, their prevalence and degree of expression must be elucidated. In this study, the expression and sub-cellular location of three TR isoforms (TRA1, TRA2, TRB1) in breast cancer tissue is documented and correlated to standard pathological and clinical prognostic markers. Methods: 131 archived breast tumors were selected sequentially starting from January 2007, to allow for collection of 5-year overall survival (OS) and disease-free survival (DFS) data. The tumors were assessed retrospectively for TRA1, TRA2 and TRB1 expression by immunohistochemistry, using commercially available antibodies. Nuclear versus cytoplasmic location was noted. The Allred score of each tumor was assessed in triplicate; an average score ≥5 was considered significant in the statistical analysis. TR expression was correlated with pathological markers (ER/PR and Her2/neu positivity, tumor size, grade, nodal involvement, lymphovascular invasion, mitotic count) and clinical factors (age, OS, DFS) using correlation analyses and binary regression models. Results: The age of patients was 65±15 (mean± standard deviation) years. The majority had T1c (31%) or T2 (56%) disease, and 39% had lymph node involvement. 60% of tumors were ER positive and 13% were Her2/neu positive. TRs were expressed to some extent in 130 of 131 assessed breast tumors; TRA1 was highly expressed (Allred score ≥5) in 81% of tumors, while TRA2 and TRB1 were highly expressed in 51% and 29% of tumors, respectively. TRA1 and TRA2 were expressed in the nucleus, while 95% of TRB1 receptors were expressed in the cytoplasm. Cytoplasmic TRB1 expression was associated with a mitotic count <10 [OR 0.4 (95%CI 0.20-0.93), p=0.03], but not with other variables. High TRA2 expression was associated with ER [OR 5.5 (95%CI 2.2-13.7), p<0.01] and PR [OR 5.3 (95%CI 2.4-11.8), p<0.01] positivity, and also 5-year DFS [OR 2.6 (95%CI 1.1-6.1), p=0.03]. Conversely, low TRA expression was associated with Her2/neu positivity [OR 3.2, (95%CI 1.0-9.5), p=0.04], high tumor grade [OR 6.0, (95%CI 2.3-15.2), p<0.01] and a mitotic count >10 [OR 2.8, (95%CI 1.4-5.8), p=0.05]. There was no significant association between TR expression and tumor size, lymph node involvement or OS. Conclusion: Thyroid receptors are widely expressed in breast cancer and they are known to influence cellular proliferation. High TRA2 expression is statistically significant in predicting favorable pathologic markers and even 5-year DFS. As with ER, its favorable prognostic profile should not exclude TRA2 as a therapeutic target; it may have the potential to expand our armamentarium of hormone directed therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C184. Citation Format: Katarzyna J. Jerzak, Anita Bane, Bindi Dhesy-Thind. Thyroid hormone receptors: Future targets for breast cancer therapy?. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C184.

Abstract A049: The sfRon kinase as a potential novel target for breast cancer therapy

Abstract Background: Our recent data revealed striking predominance of an alternatively transcribed form of the Ron receptor tyrosine kinase, known as short-form Ron (sfRon), in breast cancer. sfRon is the major active Ron isoform in breast tumors from patients, and has remarkable effects on metastasis. sfRon lacks the extracellular ligand-binding domain, but organizes into a constitutively-active receptor. Our in vitro studies revealed that overexpression of sfRon in breast cancer cell lines promotes aggressive phenotypes including an epithelial-mesenchymal transition and invasion through basement membranes. We observed that sfRon strongly signals through the PI3K pathway and that interaction between sfRon and PI3K was required for sfRon function. In vivo studies revealed striking results that expression of sfRon was sufficient to convert slow growing non-metastatic tumors into fast growing tumors that spontaneously metastasized from the mammary gland to liver, spleen and bones. An increased understanding of how sfRon contributes to tumor progression and metastasis is critical. Such data carries additional importance because sfRon does not require ligand binding for activity, appears to function in a manner distinct from full length Ron, and may not be targetable using standard approaches such as antibody inhibitors (e.g. RON8, which is now being tested in clinical trials). Instead, our data suggest that inhibition of Ron kinase activity, which would block both Ron and sfRon, would be preferable and that inhibition of downstream PI3K might also be effective in breast tumors expressing sfRon. Results: Through collaborations with various pharmaceutical companies we have obtained several compounds that specifically target Ron/sfRon kinase (e.g. ASLAN002) or PI3 kinase (e.g. BKM120). Our in vitro data demonstrate that ASLAN002 inhibits the activity of sfRon kinase in MCF7 breast cancer cells. The drug also completely blocked PI3K signaling downstream of sfRon. Our studies also showed BKM120 to be a potent inhibitor of PI3K pathway in sfRon-expressing breast cancer cells, effective in doses as low as 250 nM. We have also shown that, while the RON8 antibody inhibits activity of full length Ron kinase, it is not effective against sfRon activity. Based on these promising in vitro results, we extended our work to in vivo assays. We used NOD/SCID mice to orthotopically transplant sfRon-expressing MCF7 cells. After tumors reached 200mm3 we initiated treatment every other day with vehicle control, RON8 antibody, ASLAN002, BKM120, or ASLAN002+BKM120 combination therapy. Our data showed that although the RON8 antibody designed to target Ron is not effective against tumors expressing sfRon, monotherapy with ASLAN002 or BKM120 significantly inhibited tumor growth. However, the most effective treatment regimen was ASLAN002+BKM120 combination therapy. Most importantly, we also observed reduced formation of metastasis in mice treated with ASLAN002, BKM120, or the combination therapy compared to vehicle controls (% of spontaneous metastasis: 44%, 44%, and 33% vs 75%, respectively). Conclusion: Our experimental approach supports the novel concept to use Ron and PI3K pathway inhibitors as potential new therapeutic strategies in breast cancer. These data could have significant impact on future clinical trials for both Ron and PI3K inhibitors, which has high potential to impact patients in the short-term. Citation Format: Magdalena Bieniasz, Najme Faham, Alana L. Welm. The sfRon kinase as a potential novel target for breast cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A049.

Effects of a combined treatment with tamoxifen and estrogen receptor β agonists on human breast cancer cell lines

Coexpression of estrogen receptors (ER) α and β is present in about half of all breast cancer cases. Whereas ERα is a well-established target for endocrine therapy with the selective estrogen receptor modulator tamoxifen, the applicability of ERβ as target in breast cancer therapy is unclear. In this study, we examined the effects of two synthetic ERβ agonists alone and in combination with tamoxifen on ERα/β-positive breast cancer cells. We treated MCF-7 and T-47D breast cancer cells with the ERβ agonists ERB-041 and WAY-200070 and measured the effects on cell growth. In addition, transcriptome analyses were performed by means of Affymetrix GeneChip arrays. When given alone, ERβ agonists ERB-041 and WAY-200070 did not affect the growth of MCF-7 or T-47D cells. In contrast, addition of these drugs to tamoxifen increased its growth-inhibitory effect on both cell lines. This effect was more pronounced under serum-free conditions, but was also observed in the presence of serum in T-47D cells. Transcriptome analyses revealed a set of genes regulated after addition of ERβ agonists including S100A8 and CD177. The observed enhanced growth-inhibitory effects of a combination of tamoxifen and ERβ agonists in vitro encourage further studies to test its possible use in the clinical setting.

DNA Homologous Recombination Factor SFR1 Physically and Functionally Interacts with Estrogen Receptor Alpha

Estrogen receptor alpha (ERα), a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER’s ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER’s transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

Application of Radiolabeled Antibodies in Targeting Therapy of Breast Cancer

Breast cancer continues to be the second-leading cause of cancer in world. Early detection, improved surgical techniques, and more effective therapy methods have improved quality of life and survival for patients, particularly those with less advanced diseases. Therefore, tremendous amounts of time and efforts are dedicated to search toward earlier detection and more efficient treatment of breast cancer. Advances in molecular cancer biology achieved an increased understanding of the biologic factors that contribute to breast cancer pathogenesis and progression. This understanding has already led to more effective treatment. Among different therapy modalities, nuclear medicine provides an important role to the clinical management of breast cancer. In this review, the history of radioimmunotherapy of breast cancer as a targeted molecular method is reviewed.

Molecular Mechanisms of Hormone Resistance of Breast Cancer

More than 70% malignant mammary tumors contain steroid hormone receptors; this suggests the possibility of hormone therapy in the majority of patients with breast cancer (BC). The main cause of inefficiency of hormone therapy in BC is hormone resistance (tumor resistance to hormonal cytostatics). Here we discuss the main mechanisms of hormone resistance of BC and the mechanisms underlying the formation of hormone resistance of the tumors are analyzed at the molecular level. The data on the signal pathways of estrogen receptors (ER), the key regulators of BC cell proliferation, are presented. The most important factors of BC hormone resistance are: high activity/expression of receptor tyrosine kinases; high activity of proteins regulating cell defense mechanisms (Akt PI3K, mTOR); changes in the activities of cell cycle regulator proteins (Myc, c-Fos, Cyclin D1). Our experiments have demonstrated that estrogen-independent BC cell growth is supported by VEGF/VEGFR2 and EGF/EGFR mitogenic signal pathways. Our data indicate that NF-kappaB transcription factor is directly involved in the regulation of hormone-resistant BC cell growth and survival, while NF-kappaB suppression determines cell sensitivity to apoptotic activity of antitumor compounds. On the whole, the results indicate good prospects of using EGFR, HER-2/neu, mTOR, VEGFR, PI3K/Akt molecular pathways as targets for BC therapy, including therapy for BC resistant forms.

Curcumin and Silibinin Inhibit Telomerase Expression in T47D Human Breast Cancer Cells

Telomerase has been considered as an attractive molecular target for breast cancer therapy. The main objective of this work is to assess the inhibitory effects of silibinin and curcumin, two herbal substances, on telomerase gene expression in breast cancer cells. For determination of cell viability tetrazolium-based assays were conducted after 24, 48, and 72 h exposure times and expression of human telomerase reverse transcriptase gene was measured with real-time PCR. Each compound exerted cytotoxic effects on T47D cells and inhibited telomerase gene expression, both in a time-and dose-dependent manner. The mixture of curcumin and silibinin showed relatively more inhibitory effect on growth of T47D cells and hTERT gene expression as compared with either agent alone. These findings suggest that cell viability along with hTERT gene expression in breast cancer cells could be reduced by curcumin and silibinin.

Oncogenic Variant RON160 Expression in Breast Cancer and its Potential as a Therapeutic Target by Small Molecule Tyrosine Kinase Inhibitor

Aberrant expression of the RON receptor tyrosine kinase contributes to breast cancer malignancy. Although clinical trials of RON targeting are underway, the intriguing issue is the diversity of RON expression as evident by cancer cells expressing different variants including oncogenic RON160. The current study determines aberrant RON160 expression in breast cancer and its potential as a target for breast cancer therapy. Using mouse monoclonal antibody Zt/h12 in immunohistochemical staining of breast cancer tissue microarray, we observed that RON160 was expressed in high frequency in primary invasive ductal (77.2%, 61/79 cases), lobular (42.5%, 34/80 cases), and lymph node-involved (63.9%, 26/36 cases) breast cancer samples. Moreover, RON160 overexpression was predominantly observed in invasive ductal (26.6%, 21/79 cases) and lymph node-involved (33.3%, 12/36) cases. Among a panel of breast cancer cell lines analyzed, Du4475 cells naturally expressed RON160. Silencing RON160 expression by siRNA reduced Du4475 cell viability. Inhibition of RON160 signaling by tyrosine kinase inhibitor PHA665752 also suppressed Du4475 cell anchorage-independent growth and induced apoptotic cell death. Studies in vivo revealed that PHA665752 inhibited 3T3- RON160 and Du4475 cell-mediated tumor growth in mouse mammary fat pad. A 60% reduction in tumor volume compared to controls was achieved after a 13-day treatment. We conclude from these studies that RON160 is highly expressed in breast cancer and its signaling is integrated into cellular signaling network for tumor cell growth and survival. Experimental treatment by PHA665752 in Du4475 breast cancer xenograft model highlights the significance of RON160 as a drug target in molecular-targeted breast cancer therapy.

Abstract 920: KW-2450, a novel IGF-1R/IR inhibitor, enhances the antitumor effect of lapatinib, letrozole or 4-hydroxy-tamoxifen in breast cancer cells.

Abstract [Background] Both the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptors (IR) have been found to be promising targets for breast cancer therapy because their activation has been associated with breast cancer development, progression and drug resistance. KW-2450, a potent inhibitor of both IGF-1R and IR, is currently in clinical development in combination with lapatinib and letrozole for HER2-positive advanced or metastatic breast cancer. To support the rationale for evaluating this combination, we examined the combined effects of KW-2450 and lapatinib, a HER2/EGFR inhibitor, in a HER2/IGF-1R-expressing breast cancer cell line, and the combined effects of KW-2450 and either letrozole or 4-hydroxy-tamoxifen in a hormone-dependent breast cancer cell line. [Results] KW-2450 and lapatinib showed a strong synergistic effect against the HER2/IGF-1R double-positive cell line, MDA-MB-361, probably caused by enhanced caspase-3/7 activation, compared to the treatment with lapatinib alone. In this setting, KW-2450 completely inhibited the phosphorylation of IGF-1R/IR, whereas lapatinib mostly inhibited the phosphorylation of HER2. Interestingly, neither KW-2450 nor lapatinib inhibited Akt phosphorylation, however, the combined treatment showed a remarkable inhibition of Akt phosphorylation. In fact, KW-2450 showed a potent combined anti-tumor effect in vivo. Furthermore, a microarray analysis identified other biomarkers, such as Ki67, survivin and TIMP3, whose expression levels were changed by the combined treatment with KW-2450 and lapatinib. In an aromatase-dependent cell line, MCF-7-Ac1, the combination of KW-2450 and letrozole showed a strong synergistic effect. Moreover, the combination of KW-2450 and 4-hydroxy-tamoxifen showed a synergistic effect in an estrogen-dependent cell line, MCF-7. [Conclusions] Our results suggest that the combination of KW-2450 and lapatinib in HER2/IGF-1R double-positive breast cancers and that of KW-2450 and anti-hormone agents in hormone-dependent breast cancers are highly effective, exhibiting a synergistic anti-cancer activity. Therefore, KW-2450 may be a promising platform for carrying out combination therapy for breast cancer. Citation Format: Hiroshi Umehara, Fumito Koizumi, Yoshimi Maekawa, Makiko Shimizu, Hiroaki Nakamura, Toshio Ota, Shinji Nara, Takeshi Takahashi, Yutaka Kanda, Norihiko Shiraishi, Shiro Akinaga, Yukimasa Shiotsu, Shiro Soga. KW-2450, a novel IGF-1R/IR inhibitor, enhances the antitumor effect of lapatinib, letrozole or 4-hydroxy-tamoxifen in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 920. doi:10.1158/1538-7445.AM2013-920

Abstract 872: Novel potent and orally active STAT3 inhibitors for cancer therapies.

Abstract Signal transducers and activators of transcription (STATs) are proteins which have dual roles as cytoplasmic signaling proteins and transcription factors that activate sets of genes including those contribute to tumorigenesis. Among the STATs family members, STAT3 has been shown to play an important role in breast carcinogenesis and is often overexpressed in primary breast tumors. Increased STAT3 transcriptional activity is reported to correlate with ER-negative phenotype in breast cancer cell lines and in primary human invasive ductal breast carcinomas. Therefore, STAT3 is a promising target for breast cancer therapies. Current strategies of inhibiting STAT3 activity did not sufficiently block STAT3 activation in cancer cells due to their limited potency, poor solubility or bioavailability. Despite several STAT3 inhibitors have been advanced into early clinical trials, there is an urgent need to develop orally active, potent STAT3 inhibitors as cancer therapeutics. We have designed, synthesized and screened a series of novel STAT3 inhibitors utilizing fragment-based drug design strategy. A number of small molecules with novel scaffolds have been discovered with low-micromolar to nanomolar potency in inhibiting the proliferation of various human breast cancer and pancreatic cancer cells. The identified compounds HJC0152, HJC0123, HJC0125, and HJC0416 significantly suppress STAT3 promoter activity and STAT3 phosphorylation (pY705), reduce total STAT3 protein expression, induce cell growth arrest, and early apoptosis in ER-negative breast cancer cell line MDA-MB-231. These novel compounds have demonstrated a better efficacy in suppressing the growth of MDA-MB-231 breast xenograft tumors via intraperitoneal and oral administrative routes, with remarkably improved aqueous solubility and less toxicity than the lead compounds. In conclusion, we have developed a new class of orally active STAT3 inhibitors with superior drug-like properties, for further evaluation in cancer therapeutic and preventive settings. This work was supported by The University of Texas MD Anderson Cancer Center DFI Funding Program. Citation Format: Zhengduo Yang, Haijun Chen, Lili Chu, Yusong Zhang, Chunyong Ding, Huiling Liu, Jia Zhou, Qiang Shen. Novel potent and orally active STAT3 inhibitors for cancer therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 872. doi:10.1158/1538-7445.AM2013-872

Abstract 1403: Early tumor regression after endocrine and kinase inhibitor therapy in an experimental model of breast cancer.

Abstract More than 70% of breast cancers express estrogen and progesterone receptors (ER and PR), and are thus susceptible to adjuvant endocrine therapy. Treatments for these patients are mainly focused on blocking ER function at multiple levels. In the last few years, evidence has linked progestin to breast cancer pathogenesis. According to that, PR would be a valid target for breast cancer therapy. In addition, up to 40% of hormone-receptor positive breast tumors have PI3K activating mutations, and then PI3K/AKT inhibition is becoming a new approach for breast cancer treatment. The aim of this work was to study early stages of tumor regression in an experimental model of breast cancer. We tested the effect of an antiprogestin and a PI3K inhibitor for 48 hs, alone or in combination. We used the medroxyprogesterone acetate (MPA)-induced breast cancer model. Tumors of this model express high levels of ER and PR. Initially, they are MPA-dependent, but after a few passages MPA-independent variants appear spontaneously. These variants may respond to an endocrine therapy or may progress to a hormonal resistant phenotype. We have previously demonstrated that the MPA-independent variant C4-HI regresses after 48 hs administration of the antiprogestin Mifepristone (MFP, 12 mg/kg/day) or the PI3K inhibitor Wortmannin (WN, 1 mg/kg/day), displaying an increase in ductal differentiation. We now explored the mechanisms involved in these inhibitory effects. Proliferation of tumor cells, determined as mitotic and Ki67 indexes, is reduced (p<0.01) similarly by MFP and WN respect to vehicle. Consistently, we detected a downregulation in the expression of cell cycle proteins c-MYC and Cyclin D1 determined by western blot and immunohistochemistry. However, after 48hs only WN increased necrosis, apoptosis (p<0.01) and the BAX/BCL-XL ratio. As expected, only WN decreased pAKT and pS6 levels. Furthermore, when given in combination, MFP and WN improved all the above mentioned parameters. In an antiprogestin resistant tumor variant, PI3K inhibition still suppressed tumor growth. We detected that after 7 days of treatment WN-treated C4-2-HI tumors were smaller than vehicle-treated tumors (p<0.001). However, no evident signs of regression were histologically observed. Ongoing experiments are being conducted to evaluate histologically tissue response at earlier time points (48hs) after WN therapy. Taken together, these results suggest that inhibiting PI3K pathway could represent a useful tool for the treatment of hormone receptor-positive tumors, even of those that have progressed to a hormonal resistant phenotype. Understanding the early mechanisms involved in tumor regression may improve the design and monitoring of cancer therapies. Citation Format: Maria Laura Polo, Marina Riggio, Claudia Lanari, Virginia Novaro. Early tumor regression after endocrine and kinase inhibitor therapy in an experimental model of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1403. doi:10.1158/1538-7445.AM2013-1403

Abstract 1401: Overexpression of HER4 isoforms in transgenic mice reveal isoform-specific role in mammary gland development and carcinogenesis.

Abstract Human epidermal growth factor receptor 4 (HER4/ERBB4) belongs to the family of four receptor tyrosine kinases (HER1-4). While HER1, HER2 and HER3 are often overexpressed in breast cancer and predict poor prognosis, the role of HER4 in cancer is still unclear. Some studies suggest a tumor suppressor role of HER4, while contradictory reports suggest its oncogenic potential. HER4 gene produces multiple isoforms as a result of alternative splicing, in contrast to other members of this receptor family. We have used a gain-of-function MMTV-HER4 model isoforms, to evaluate potential developmental and carcinogenic roles of HER4-CYT1 and CYT2. Mammary gland ductal morphogenesis was significantly suppressed by expressing one of the transgene isoforms, strongly suggesting a role of HER4 in normal mammary ductal morphogenesis in addition to its role in lactation described previously. Interestingly, sustained expression of one HER4 transgene isoform resulted in formation of mammary tumor lesions, whereas no significant findings were observed in the control group. Formation of tumor lesions in these transgenic mice strongly suggests an oncogenic function of a HER4 isoform. These novel findings may help improve the rational design of targeted breast cancer therapy. Supported by NIH grant RO1 CA80065. Citation Format: Vikram B. Wali, Maureen Gilmore-Hebert, Klaus Elenius, David F. Stern. Overexpression of HER4 isoforms in transgenic mice reveal isoform-specific role in mammary gland development and carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1401. doi:10.1158/1538-7445.AM2013-1401

The CXCR4 Antagonist AMD3465 Regulates Oncogenic Signaling and Invasiveness In Vitro and Prevents Breast Cancer Growth and Metastasis In Vivo

CXCR4, the receptor for stromal-derived factor-1, is reportedly involved in breast carcinogenesis. However, the mechanisms through which CXCR4 contributes to breast cancer cell growth and metastases are poorly understood. In this study, we examined the putative in vitro and in vivo anti-cancer effects of the specific CXCR4 inhibitor AMD3465. Here, we report that AMD3465 triggers a reduction in breast cancer cell invasiveness in vitro, and promotes marked changes in oncogenic signaling proteins including a reduction in STAT3, JAK2, AKT, and CXCR4 phosphorylation and the reduced expression of GSK3 and cMYC. Using three breast cancer cell lines as murine syngeneic immunocompetent breast cancer models, we found that AMD3465 inhibited breast tumor formation and reduced tumor cell metastases to the lung and liver. Furthermore, treatment with AMD3465 significantly reduced the infiltration of myeloid CD11b positive cells at the aforementioned metastatic sites as well as the spleen implying this agent could regulate the formation of the tumor microenvironment and conceivably the premetastatic niche. In conclusion, our studies suggest that AMD3465 inhibits breast cancer growth and metastases by acting on tumor cells as well as immune cells that constitute the tumor microenvironment. This process appears to be regulated, at least in part, through the modulation of oncogenic signaling that includes the STAT3 pathway. Thus, CXCR4 could be a novel target for breast cancer therapy.

Novel Approaches for Molecular Targeted Therapy of Breast Cancer: Interfering with PI3K/AKT/mTOR Signaling

Breast cancer is one of the most prevalent and devastating malignant diseases in women worldwide. Fortunately, while breast cancer incidence is still increasing, its death rate is declining. This is mainly due to early diagnosis and potent therapies such as blockade of estrogen receptor- or of ErbB2 (HER2-neu) membrane receptor signaling.In recent years, the PI3K/AKT/mTOR pathway, which transmits signals from the cell membrane into the nucleus and activates multiple oncogenic programs, has been found to play a crucial role in the regulation of breast cancer cell growth. This pathway is densely interconnected with a multitude of other important regulatory systems for glucose-,lipid- and amino acid-metabolism, for energy balance, and for autophagy. It has been found that PI3K/AKT/mTOR signaling modulates estrogen receptor function. Using transverse and feedback regulatory loops the PI3K/AKT/mTOR cascade can communicate with concurrent and with upstream systems. Thus, PI3K/AKT/mTOR is a crucial element within a complicated signaling network. This pathway is hyperactive in more than 70% of breast tumors. Hence, the protein kinases located along this route represent very attractive and promising drug targets for breast cancer therapy. Currently, numerous small molecular drugs that inhibit PI3K, AKT and/or mTOR are being developed in preclinical and clinical models of breast cancer. Some of these compounds are highly selective blocking only one particular kinase complex, whereas others interfere with two (mTORC1+mTORC2) or even three effectors (PI3K+mTORC1+mTORC2) of the pathway. Due to the many interactions with other regulatory systems, silencing of the pathway can cause unexpected results. Therefore, detailed preclinical and clinical evaluation of these compounds as single drugs and in combination is required to achieve optimal results with maximal clinical benefit and acceptable toxicity. Also, reliable biomarkers for the identification of patient subsets that will maximally benefit from PI3K/AKT/mTOR inhibition need to be developed. Thus, selective silencing of PI3K/AKT/mTOR signaling represents a promising approach for breast cancer and might prove useful when combined with other drugs. Here we review the current preclinical and clinical data and compare the potential benefits of multi- versus single-targeting PI3K/AKT/mTOR drugs.

Trials and tribulations of cytotoxic and targeted breast cancer therapy: a clinical perspective on the next phase of progress

Interview by Sophia Maprayil and Alexandra Hemsley, Commissioning EditorsChris Twelves is a medical oncologist and leads the Section of Oncology and Clinical Research at Cancer Research UK’s Clinical Centre at St James’s Hospital, Leeds. His particular interest lies in new drug development and clinical pharmacology; his clinical practice to date has been in the field of colorectal and breast cancer. After training in London he was Senior Lecturer, then Reader, in Medical Oncology in Glasgow at the Beatson Oncology Centre before taking up his current post as Professor of Clinical Cancer Pharmacology and Oncology at the University of Leeds and St James’s Institute of Oncology. In this role, Twelves leads his team to perform hypothesis-driven studies, prioritizing therapeutics developed locally or through the Cancer Research UK New Agents Committee, of which he has been a member.Twelves also heads the Experimental Cancer Medicine Centre in Leeds; previously he was Chair of the New Drug Development Group of the European Organisation for Research and Treatment of Cancer. He has published over 150 papers, in addition to several books, and recently presented his group’s findings from a Phase III study into the breast cancer drug eribulin.

The level of phosphorylated Akt predominantly reflects the expressive status of CerbB2 in invasive breast cancer.

Although some evidence has been documented on EGFR/PI3K mediation of Akt activation in breast cancers, ILK and DNA-PK have not been investigated so far. The aim of this study was to analyze the expression of phosphorylated Akt (pAkt) in breast cancer, with respect to its upstream regulators. The immunostaining of pAkt (Ser473) in 70 invasive breast cancers revealed that status of CerbB2 could play a major role in Akt phosphorylation, while ILK was also involved in the stimulated level of pAkt. The results would provide an important clue for the activation of Akt and potential targeted therapy in breast cancer.

Silencing of Atp6v1c1 Prevents Breast Cancer Growth and Bone Metastasis

Previous studies have shown that Atp6v1c1, a regulator of the assembly of the V0 and V1 domains of the V-ATPase complex, is up-regulated in metastatic oral tumors. Despite these studies, the function of Atp6v1c1 in tumor growth and metastasis is still unknown. Atp6v1c1's expression in metastatic oral squamous cell carcinoma indicates that Atp6v1c1 has an important function in cancer growth and metastasis. We hypothesized that elevated expression of Atp6v1c1 is essential to cancer growth and metastasis and that Atp6v1c1 promotes cancer growth and metastasis through activation of V-ATPase activity. To test this hypothesis, a Lentivirus-mediated RNAi knockdown approach was used to study the function of Atp6v1c1 in mouse 4T1 mammary tumor cell proliferation and migration in vitro and cancer growth and metastasis in vivo. Our data revealed that silencing of Atp6v1c1 in 4T1 cancer cells inhibited lysosomal acidification and severely impaired 4T1 cell growth, migration, and invasion through Matrigel in vitro. We also show that Atp6v1c1 knockdown with Lenti-c1s3, a lentivirus targeting Atp6v1c1 for shRNA mediated knockdown, can significantly inhibit 4T1 xenograft tumor growth, metastasis, and osteolytic lesions in vivo. Our study demonstrates that Atp6v1c1 may promote breast cancer growth and bone metastasis through regulation of lysosomal V-ATPase activity, indicating that Atp6v1c1 may be a viable target for breast cancer therapy and silencing of Atp6v1c1 may be an innovative therapeutic approach for the treatment and prevention of breast cancer growth and metastasis.