Antitumor Effects In Breast Cancer Research Articles

Arsenic Trioxide Overcomes Rapamycin-Induced Feedback Activation of AKT and ERK Signaling to Enhance the Anti-Tumor Effects in Breast Cancer

Inhibitors of the mammalian target of rapamycin (mTORi) have clinical activity; however, the benefits of mTOR inhibition by rapamycin and rapamycin-derivatives (rapalogs) may be limited by a feedback mechanism that results in AKT activation. Increased AKT activity resulting from mTOR inhibition can be a result of increased signaling via the mTOR complex, TORC2. Previously, we published that arsenic trioxide (ATO) inhibits AKT activity and in some cases, decreases AKT protein expression. Therefore, we propose that combining ATO and rapamycin may circumvent the AKT feedback loop and increase the anti-tumor effects. Using a panel of breast cancer cell lines, we find that ATO, at clinically-achievable doses, can enhance the inhibitory activity of the mTORi temsirolimus. In all cell lines, temsirolimus treatment resulted in AKT activation, which was decreased by concomitant ATO treatment only in those cell lines where ATO enhanced growth inhibition. Treatment with rapalog also results in activated ERK signaling, which is decreased with ATO co-treatment in all cell lines tested. We next tested the toxicity and efficacy of rapamycin plus ATO combination therapy in a MDA-MB-468 breast cancer xenograft model. The drug combination was well-tolerated, and rapamycin did not increase ATO-induced liver enzyme levels. In addition, combination of these drugs was significantly more effective at inhibiting tumor growth compared to individual drug treatments, which corresponded with diminished phospho-Akt and phospho-ERK levels when compared with rapamycin-treated tumors. Therefore, we propose that combining ATO and mTORi may overcome the feedback loop by decreasing activation of the MAPK and AKT signaling pathways.

Beta-naphthoflavone (DB06732) mediates estrogen receptor-positive breast cancer cell cycle arrest through AhR-dependent regulation of PI3K/AKT and MAPK/ERK signaling

Beta-naphthoflavone (BNF, DB06732) is an agonist of aryl hydrocarbon receptor (AhR) and a putative chemotherapeutic agent that has antitumor activity against mammary carcinomas in vivo. However, the mechanism by which BNF exerts this antitumor effect remains unclear. Thus, we explored mechanisms of BNF's antitumor effects in human breast cancer cells. This study showed that BNF suppressed cell proliferation and induced cell cycle arrest in the G0/G1 phase with downregulation of cyclin D1/D3 and CDK4 and upregulation of p21(Cip1/Waf1), leading to a senescence-like phenotype in estrogen receptor (ER)-positive MCF-7 cells, but not in ER-negative MDA-MB-231 cells. In addition, BNF inhibited PI3K/AKT signaling, and the PI3K inhibitor, LY294,002, exhibited the same inhibitory effects on cyclinD1/D3, CDK4 and the cell cycle as BNF. Interestingly, BNF activated mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK) signaling, and more notably, MEK inhibitor PD98059 significantly blocked the BNF-induced cell cycle arrest and upregulation of p21(Cip1/Waf1). Furthermore, specific ERα and AhR siRNA studies indicate that ERα is required in BNF-induced p21(Cip1/Waf1) expression, and BNF-mediated cell cycle arrest and modulation of AKT and ERK signaling is AhR-dependent. Taken together, AhR-dependent inhibition of the PI3K/AKT pathway, activation of MAPK/ERK and modulation of ERα is a novel mechanism underlying BNF-mediated antitumor effects in breast cancer, which may represent a promising strategy to be exploited in future clinical trials.

Incorporation of Lapatinib Into Lipoprotein-Like Nanoparticles with Enhanced Water Solubility and Anti-Tumor Effect in Breast Cancer

The poor water solubility of many active compounds is a serious deterrent to their use as commercial drugs. Lapatinib is a dual inhibitor of the EGF receptor and EGF receptor 2 approved by the US FDA to treat advanced breast cancer. This study prepares lapatinib-incorporated lipoprotein-like nanoparticles (LTNPs) to enhance the water solubility and elevate the anti-tumor effect of lapatinib. Bovine albumin was used to bind with lapatinib, and egg yolk lecithin was used to stabilize the conjugation of bovine albumin and lapatinib. The characteristics of LTNPs were evaluated by several experiments. Cell uptake and toxicity were performed on BT-474 cells. In vivo anti-tumor effect was performed on BT-474 xenograft-bearing mice. LTNPs contained a lipid corona and a core of lapatinib and albumin. LTNPs could be effectively taken up by BT-474 cells and induced apoptosis. An in vivo study demonstrated that LTNPs could passively distribute into a tumor via the enhanced permeability and retention effect and induce anti-tumor activity in breast cancer. The authors present a convenient nanoformulation with improved anti-tumor effect, which is a promising candidate for clinical trials.

Androgen receptor-induced tumor suppressor, KLLN, inhibits breast cancer growth and transcriptionally activates p53/p73-mediated apoptosis in breast carcinomas

Androgen receptor (AR) expression by immunohistochemistry correlates with better prognosis and survival among breast cancer patients. We and others have shown that AR inhibits proliferation and induces apoptosis in breast cancer cells. However, the mechanism of AR's anti-tumor effect in breast cancer is still not fully understood. Our recent study indicates that AR upregulates expression of tumor suppressor gene PTEN by promoter activation in breast cancer. KLLN, encoding KLLN protein, is a newly identified gene, which shares a bidirectional promoter with PTEN and is transcribed in the opposite direction. So far, the function of KLLN has never been studied in tumorigenesis. Here, we define KLLN as a tumor suppressor in breast carcinomas, which inhibits tumor growth and invasiveness. After analyzing 188 normal breast and 1247 malignant breast cancer tissues, we observed the loss of KLLN in multiple breast cancer subtypes and this decreased KLLN expression associates with tumor progression and increasing histological grade in invasive carcinomas. We characterize KLLN, for the first time, as a transcription factor, directly promoting the expression of TP53 and TP73, with consequent elevated apoptosis and cell cycle arrest in breast cancer cells. We demonstrate, in vitro and in murine xenograph models, that both KLLN and PTEN are AR-target genes, mediating androgen-induced growth inhibition and apoptosis in breast cancer cells. Our observations suggest that KLLN might be used as a potential prognostic marker and novel therapy target for breast carcinomas.

Abstract P5-10-10: The miR-34a is down-regulated in breast cancer and breast stem cells and a potential to eradicating breast cancer via a systemic delivery of a VISA –miR-34a nanoparticle system

Abstract Breast cancer is the most common disease in women around the world and the current treatment strategies are not potent enough for the patients, especially those who are the triple-negative type of molecular classifications. Therefore, novel and more effective treatments are pressingly needed. Of the current methods, target therapy, which not only retains cancer-specific expression but also limits toxicity, is a new strategy for treatment of cancers. In this study, it was to investigate miR-34a expression in breast caner specimen and its relationship with patient's clinical status, and develop targeted miR-34a delivery system as a potential method for breast cancer therapy. miR-34a expression was investigated by qRT-PCR and related to clinicopathologic significance and found to be down-regulated in breast cancer as compared with normal adjacent tissues and involved in breast cancer stem cell through down-regulation of CD44, ZEB1, and Bmi1. We designed targeted miR-34a expression using T-VISA system (hTERT promoter driven VP16-Gal4-WPRE Integrated Systemic Amplifier) liposomed-based nanoparticles and evaluated the antitumor effect in breast cancer cells in vitro and in orthotopic animal model as well as its systemic toxicity. The T-VISA-miR-34a system robust targeted to breast cancer cells and breast cancer stem cells and prolonged duration expression of miR-34a. Furthermore, a systemic delivery of a VISA –miR-34a nanoparticle system targeted efficiently expression of miR-34a to tumors and could significantly inhibit tumor growth and prolong mouse survival in multiple living imaging xenograft and syngeneic models of orthotopic breast cancer and without toxicity in intact mice. Our study demonstrated that miR-34a expression was down-regulated in breast cancer and breast cancer stem cells through down-regulation of CD44, ZEB1, and Bmi1. The T-VISA-miR-34a nanoparticle system showed robust antitumor effects in breast cancer and could be a potential therapeutic approach to eradicating breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-10.

Abstract 4396: Combination of intratumoral regulated IL-12 gene delivery and systemic chemotherapy with Palifosfamide (IPM-Tris) enhances anti-tumor effects in breast cancer (4T1) model

Abstract A major obstacle for the development of effective immunotherapy is the ability of tumors to escape detection and/or destruction by the immune system. This study explored pairing a potent immunomodulatory cytokine (IL-12) with a bifunctional alkylating agent currently in Phase III clinical trials for soft tissue sarcoma (Palifosfamide, IPM-Tris). In this study intratumorally administered adenoviral vector, Ad-RTS-mIL-12, expressing IL-12 under the control of Rheoswitch Therapeutic System®, (RTS®), a novel inducible promoter system was used. Expression of IL-12 is regulated by oral administration of a small molecule activator ligand, INXN-1001 (AL). The in vitro transduction of the human fibrosarcoma cell line, HT1080, with Ad-RTS-hIL12 + AL at a MOI of 400 pfu, produced >6000-fold induction of human IL-12 above background, while IPM-Tris (1-3 uM) was cytotoxic in a variety of cancer cell lines. The effect of Ad-RTS-mIL12 + AL in combination with IPM-Tris was evaluated in a subcutaneous 4T1 syngeneic BALB/c mouse mammary tumor model at low doses, which were chosen to have minimal effect on tumor growth rate. Indeed, IPM-Tris alone (40 - 120 mg/m2) had little effect on tumor growth rate (< 30%) compared to vehicle. In contrast, a single intratumoral injection of Ad-RTS-mIL12 (1x1010 vp) and oral administration of AL (15, 30, 75 or 150 mg/m2) once daily for 5 days led to significant AL dose-related tumor growth inhibition (28, 29, 62, 59%, respectively; p<0.05) with the Ad-RTS-mIL12 having no effect relative to control by Day 35. Combined treatment with Ad-RTS-mIL12 + AL (30 mg/m2) and IPM-Tris (40 or 120 mg/m*2 administered IP QD for 3 days) significantly inhibited tumor growth inhibition (∼71-90%, p<0.001) and increased median survival rate by 8-14 days when compared to the single agents alone, without overt toxicity as assessed by change in body weight. These results indicate that local intratumoral immunotherapy combined with low dose chemotherapy with palifosfamide offers a distinct advantage over either therapeutic modality alone. These results are supportive of our hypothesis that cytotoxic agents at low doses may prime the immune system to enhance immunotherapy, which could potentially be translated into a safe clinical regimen for the treatment of metastatic breast cancer. Additional studies are ongoing to explore in depth the immunologic mechanism(s) that account for the more potent immunotherapeutic effects of IL-12 when combined with IPM-Tris. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4396. doi:1538-7445.AM2012-4396

Abstract 4855: KLLN, which shares a bidirectional promoter with PTEN, is an AR-regulated tumor suppressor, and induces p53-dependent and -independent apoptosis in breast cancer

Abstract 70% of breast cancer patients are diagnosed after menopause where the ovaries cease androgen production. Maintaining functional androgen receptor (AR) has been reported to correlate with good prognosis and better survival among breast cancer patients. We and others have shown that AR inhibits proliferation and induces apoptosis in breast cancer cells. However, the mechanism of AR's anti-tumor effect in breast cancer is still not fully understood. KLLN is a newly identified gene whose function has never been studied in tumorigenesis. KLLN and tumor suppressor gene PTEN, both located in 10q23.31, share the same promoter region but are transcribed in opposite directions. To perform in silico comparisons of KLLN expression between normal breast and breast cancer tissues, we selected 56 normal breast and 1170 tumor tissues from patients without exposure to any treatment using publicly available whole genome transcriptome datasets. We found that KLLN is significantly downregulated in breast carcinomas and that loss of KLLN expression is associated with increasing histological grade in invasive carcinomas. By using MCF-7 and MDA-MB-453 breast cancer cell lines as our models, we show that AR activates this bi-directional promoter, resulting in the upregulation of both KLLN and PTEN expression. For the first time, we identified KLLN as an AR-activated tumor suppressor, inhibiting breast cancer cell proliferation and suppressing tumor cell invasiveness in both cell lines. In MCF-7 cells, overexpression of KLLN directly activates TP53 expression as a transcription activator, accompanied by significant increase of PARP cleavage and apoptosis. We also found that KLLN could promote TP73 expression in both MCF-7 (p53-wildtype) and MDA-MB-453 (p53-null) cells at the transcriptional level, resulting in p53-independnt apoptosis. Therefore, we discovered a new pathway, which mediates AR-induced growth inhibition and apoptosis in breast cancer cells. Our study also indicates KLLN as a potential prognostic marker and novel therapy target for breast carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4855. doi:1538-7445.AM2012-4855

P1-01-04: Immunological Effects of Bisphosphonates on gdT Cells in Breast Cancer.

Abstract Background: Human γδT cells play an important role in the first-line of defence. Accumulating data demonstrate that γδ T cells recognize nonpeptide antigens such as IPP and HMBPP and activated γδT cells have potent cytotoxic activity against tumor cells. Nitrogen-containing bisphosphonates like zoledronic acid inhibit farnesyl diphosphate synthetase, resulting in the accumulation of upstream metabolites such as IPP. Previous studies on the adoptive transfer of ex-vivo amplified γδ T cells demonstrated the safety and feasibility of γδT cell-based immunotherapy for metastatic cancer. According to ABCSG-12 clinical trial, zoldedoric acid improved DFS of patients with early-stage breast cancer under adjuvant endocrine therapy. Whereas a precise mechanism remained unclear, it is likely that γδT cells are involved in this anti-tumor effect in breast cancer. Materials and Methods: Peripheral blood mononuclear cells derived from 63 (54 untreated non-metastatic and 9 metastatic) patients with breast cancer were examined for the baseline population of γδT cells and their proliferative responses to zoledronic acid in vitro. Tumor-infiltrating lymphocytes were isolated from 20 tumor biopsies and 2 pleural effusions. Peripheral blood γδT cells were stimulated with 5 μM zoledronic acid and cultured in the presence of 100 IU/ml IL-2 for 10 days. Amplified γδ T cells were analyzed for cytokine production and cytotoxic activity. Results: The proportion of γδ (TCRVδ2 -bearing) T cells among peripheral blood CD3+ cells was 4.2 ± 5.0%. Although it was equivalent to the previously reported value of 4.3 ± 2.2% in healthy volunteers, 14% (9/63) of patients exhibited an increase in the number of peripheral blood γδT cells at baseline. The level of TCRVδ2 T cells with potent anti-tumor activity was significantly higher than that of TCRVδ1 T cells (4.2± 5.0% vs. 1.5 ± 1.8%, p<0.001), whereas they were present in tumor-infiltrating cells at essentially the same levels (4.4± 9.6% vs. 3.7 ± 9.7%, p=0.434). Percentage of TCR Vδ2+ CD3+ cells among amplified γδ T cells in vitro and cytokine (IFN-γ and TNF-α) production by them were significantly proportional to the initial level of TCRVδ2+/CD3+ ratio in peripheral blood. Upon treatment with zoledronic acid, breast cancer cell lines became susceptible to γδ T cells. Conclusions: γδ (TCR Vδ2-bearing) T cells increased in some patients with breast cancer and the TCRVδ2+/CD3+ ratio at baseline was correlated with the efficiency of proliferation and cytotoxic activity when treated with zoledronic acid. Zoledronic acid efficiently sensitized breast tumor cells to be lyzed by γδ T cells. The present results suggest that γδ T cells stimulated with zoledronic acid can be utilized for immunotherapy against breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-04.

Zoledronic acid for adjuvant use in patients with breast cancer

Zoledronic acid, a nitrogen-containing bisphosphonate, is firmly established in the management of metastatic bone disease. It inhibits farnesyl diphosphonate synthase within the mevalonate pathway and, through this mechanism, is a potent inhibitor of osteoclast-mediated bone resorption. In addition, there are preclinical data suggesting that farnesyl diphosphonate synthase inhibition by zoledronic acid has anti-tumor effects in breast cancer. Adjuvant therapies for early breast cancer are associated with substantial decreases in bone mineral density. Results from three clinical trials, ABCSG-12, Z-FAST and ZO-FAST, indicate that the addition of twice-yearly zoledronic acid to standard adjuvant endocrine therapy in premenopausal and postmenopausal patients with hormone receptor-positive breast cancer prevents cancer treatment-induced bone loss. Moreover, it is becoming evident that it may also exert anticancer effects in an estrogen-deprived state in the adjuvant and neoadjuvant setting. However, long-term side effects need to be taken into consideration for treatment decisions.

HLA-matched allo-SCT after reduced intensity conditioning with fludarabine/CY in patients with metastatic breast cancer

Fifteen patients with chemosensitive metastatic breast cancer (MBC) underwent reduced intensity (RIST) allo-SCT between 1999 and 2006. The purpose of this single-center study was to evaluate the feasibility, safety and efficacy of this therapeutic approach. The pretransplant conditioning regimen consisted of fludarabine (25 mg/m(2) at days -5 to -1) and CY (60 mg/kg at days -2, -1). Stem cells were from HLA-matched sibling donors. The treatment-related mortality was 2/15 (13%). Median PFS and OS were 144 days (43-509 days) and 303 days (122-1376 days), respectively. The 1-year PFS was 20%, and the 1-year and 2-year OS was 40 and 20%, respectively. No objective tumor responses were observed, but the relatively long PFS does suggest a graft-vs-tumor effect. Although RIST using this CY/fludarabine regimen is feasible, the efficacy in this set of patients was limited. Future clinical trials should be performed to improve the knowledge of mechanisms of antitumor effects in breast cancer.

Curcumin as a Possible Lead Compound against Hormone‐Independent, Multidrug‐Resistant Breast Cancer

We examine the possible evidence that the phytochemical curcumin may overcome resistance to hormonal and cytotoxic agents in breast cancer. We present our observations on MCF-7R, a multidrug-resistant (MDR) variant of the MCF-7 breast cancer cell line. In contrast to MCF-7, MCF-7R lacks aromatase and estrogen receptor alpha (ERalpha) and overexpresses the multidrug transporter ABCB1 and the products of different genes implicated in cell proliferation and survival, like c-IAP-1, NAIP, survivin, and COX-2. Nevertheless, in cytotoxicity and cell death induction assays, we found that the antitumor activity of curcumin is substantial both in MCF-7 and in MCF-7R. We elaborated the diketone system of curcumin into different analogues; the benzyloxime and the isoxazole and pyrazole heterocycles showed remarkable increases in the antitumor potency both in the parental and in the MDR MCF-7 cells. Furthermore, curcumin or, more potently, the isoxazole analogue, produced early reductions in the amounts of relevant gene transcripts that were diverse (i.e., they were relative to Bcl-2 and Bcl-X(L) in MCF-7 and the inhibitory of apoptosis proteins and COX-2 in MCF-7R) in the two cell lines. Thus, the two compounds exhibited the remarkable property of being able to modify their molecular activities according to the distinct characteristics of the parental and MDR cells. We discuss also how curcumin may (1) exert antitumor effects in breast cancer through ER-dependent and ER-independent mechanisms; and (2) act as a drug transporter-mediated MDR reversal agent. Overall, the structure of curcumin may represent the basis for the development of new, effective anticancer agents in hormone-independent MDR breast cancer.