Treatment Of Basal-like Breast Cancer Research Articles

Abstract P6-04-08: Cytosolic phospholipase A2 (cPLA2) as a therapeutic target in basal-like breast cancer

Abstract Introduction: Basal-like breast cancer is frequently associated with triple negative phenotype, and there is a need for novel therapeutic strategies for this patient population. Inhibitors of cytosolic phospholipase A2 (cPLA2) have been suggested to block both MAPK and PI3K signalling and have a high potential for activity in basal-like breast cancer (Lin 1993, Wen 2013). In this study, we compared the expression of PLA2G4A between luminal B and basal-like breast cancer, both in patient-derived xenograft models (PDX) and human cancer tissue. In addition, we studied the effect of the novel cPLA2 inhibitor AVX235 on tumor growth in a basal-like PDX model. Materials and methods: Tumor tissue specimens were obtained from PDX models (n = 26) and a clinical breast cancer biobank (n = 32). Gene expression analysis was carried out on Agilent 8×60K microarrays. The expression of 54 genes directly involved in choline metabolism was examined. Differential expression of choline genes between basal-like and luminal B tumors was calculated by subtraction of log2 expression values. Mice carrying bilateral MAS98.12 basal-like xenografts (Bergamaschi 2009) were treated with the cPLA2 inhibitor AVX235 (30 mg/kg i.p. daily for 7 days then every second day for 14 days, n = 6) or drug-free vehicle (control group, n = 6). Results: The PDX models were subtyped into basal-like (n = 19) and luminal B (n = 7) subtypes based on the expression of 500 intrinsic genes [Sørlie 2003]. For the human cancer tissue, there were 18 basal-like and 14 luminal B tumors. There was a significant correlation between differential choline gene expression in basal-like vs luminal B tumors in PDX models and human cancer tissue (p<1.3*10−12). Both in PDX models (p<0.04) and human cancer tissue (p<0.0003), PLA2G4A was significantly higher expressed in basal-like than luminal B tumors. Treatment with AVX235 markedly reduced the growth rate of MAS98.12 xenografts compared to controls. After 19 treatmment days, the mean tumor volume (normalised to volume at start of treatment) in the treatment group was 36% of the tumor volume in the control group, the difference being statistically significant (p = 0.023). No signs of treatment-related adverse effects were observed. Conclusion: PLA2G4A is higher expressed in basal-like than in luminal B breast cancer. Treatment with the cPLA2 inhibitor AVX235 significantly inhibits tumor growth. These data suggest that cPLA2 inhibitors may be of particular value in treatment of basal-like breast cancer.

Combined Targeting of mTOR and AKT Is an Effective Strategy for Basal-like Breast Cancer in Patient-Derived Xenograft Models

Basal-like breast cancer is an aggressive disease for which targeted therapies are lacking. Recent studies showed that basal-like breast cancer is frequently associated with an increased activity of the phosphatidylinositol 3-kinase (PI3K) pathway, which is critical for cell growth, survival, and angiogenesis. To investigate the therapeutic potential of PI3K pathway inhibition in the treatment of basal-like breast cancer, we evaluated the antitumor effect of the mTOR inhibitor MK-8669 and AKT inhibitor MK-2206 in WU-BC4 and WU-BC5, two patient-derived xenograft models of basal-like breast cancer. Both models showed high levels of AKT phosphorylation and loss of PTEN expression. We observed a synergistic effect of MK-8669 and MK-2206 on tumor growth and cell proliferation in vivo. In addition, MK-8669 and MK-2206 inhibited angiogenesis as determined by CD31 immunohistochemistry. Biomarker studies indicated that treatment with MK-2206 inhibited AKT activation induced by MK-8669. To evaluate the effect of loss of PTEN on tumor cell sensitivity to PI3K pathway inhibition, we knocked down PTEN in WU-BC3, a basal-like breast cancer cell line with intact PTEN. Compared with control (GFP) knockdown, PTEN knockdown led to a more dramatic reduction in cell proliferation and tumor growth inhibition in response to MK-8669 and MK-2206 both in vitro and in vivo. Furthermore, a synergistic effect of these two agents on tumor volume was observed in WU-BC3 with PTEN knockdown. Our results provide a preclinical rationale for future clinical investigation of this combination in basal-like breast cancer with loss of PTEN.

Abstract 1134: RNAi screening of human kinome identified fibroblast growth factor receptor 4 (FGFR4) as potential molecular target in basal-like breast cancers (BLBC).

Abstract Global gene expression profiling has uncovered previously unrecognized subsets of human breast cancer, including the so-called "triple-negative" or "basal-like" tumours (BLBC) characterized by estrogen/progesterone receptor negativity, lack of Her-2/Neu amplification and high frequency of p53 mutation. These refractory tumours therefore are insensitive to hormonal therapy or trastuzumab-based therapy, and thus confer a markedly poor prognosis relative to other subtypes. To date, little progress has been made to identify specific molecular pathways associated with these refractory cancers that may be effectively targeted for therapeutic purposes. Using a human kinome RNAi library screen, we show that knock-down of endogenous human FGFR4 (but not FGFR1-3) induces significant tumor-specific cell death in basal-like breast cancer cells. Further analysis reveals that FGFR4 mediates the BLBC cells survival through activation of AKT as knock-down of endogenous FGFR4 in MDA-MB-468 and HCC1937 cells significantly reduced AKT phosphorylation. Consistently, ectopic expression of a constitutively active myristoylated AKT completely abrogates the apoptosis induced by FGFR4 knock-down. Interestingly, both MDA-MB-468 and HCC1937 also secrete fibroblast growth factor 19 (FGF19), a canonical ligand specific for FGFR4. RNAi-mediated silencing of FGF19 evidently suppresses the growth of these BLBC cells via AKT signalling as marked by diminished AKT phosphorylation following depletion of FGF19. Knockdown of FGF19 in addition triggers ERK1/2 activation to compensate for AKT signalling down-regulation in MDA-MB-468. Together, our results demonstrated the existence of a FGFR4-FGF19 autocrine loop which could potentially be developed as therapeutic target for future treatment of BLBC. Citation Format: Kai Hung Tiong, Boon Shing Tan, Heng Lungh Choo, Ammu Kutty Radhakrishnan, Rozita Rosli, Soon-Keng Cheong, Chee-Onn Leong. RNAi screening of human kinome identified fibroblast growth factor receptor 4 (FGFR4) as potential molecular target in basal-like breast cancers (BLBC). [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 1134. doi:10.1158/1538-7445.AM2013-1134

Abstract 869: PTEN/PI3K oncogenic pathway profiling informs an in vivo synergistic therapeutic model for basal-like breast cancer.

Abstract The basal-like breast cancer (BBC) subtype is generally characterized by positive staining for basal mammary epithelial cytokeratin markers, lack of hormone receptor and HER2 expression, and poor prognosis with currently no approved molecularly-targeted therapies. The oncogenic signaling pathways driving basal-like tumorigenesis, however, are not fully elucidated. In an analysis of phosphoinositide 3-kinase (PI3K) pathway-activating molecular aberrations in 116 unselected breast tumors, we find that three-quarters of cases had a potential oncogenic alteration to PIK3CA, PTEN, HER2, INPP4B, or EGFR, including 97% of estrogen receptor (ER)-negative tumors compared to 65% of ER-positive tumors (P<0.001). Interestingly, nearly all p53 mutant tumors had one or more of these alterations (93%, P=0.001). Loss of PTEN was significantly associated to EGFR overexpression, positivity for the BBC marker cytokeratin 5/14, and the BBC molecular subtype by mRNA gene expression profiling (all P<0.001), informing a potential therapeutic combination targeting the PTEN/PI3K and EGFR pathways in BBC. In BBC cell lines, combination treatment with the EGFR-inhibitor gefitinib plus PI3K-inhibitor LY294002 was synergistic and increased apoptosis. Similarly, in an in vivo BBC xenograft mouse model, gefitinib plus PI3K-inhibitor PWT-458 was more effective than either monotherapy. Our study supports the future clinical evaluation of EGFR and PI3K pathway inhibitors for the treatment of BBC. Citation Format: Lao H. Saal, Qing-Bai She, Sofia K. Gruvberger-Saal, Matthew Maurer, Mervi Jumppanen, Tao Su, Meaghan Dendy, Ying-Ka I. Lau, Lorenzo Memeo, Markus Ringner, Jorma Isola, Hanina Hibshoosh, Neal Rosen, Ramon Parsons. PTEN/PI3K oncogenic pathway profiling informs an in vivo synergistic therapeutic model for basal-like 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 869. doi:10.1158/1538-7445.AM2013-869

“Iron-saturated” bovine lactoferrin improves the chemotherapeutic effects of tamoxifen in the treatment of basal-like breast cancer in mice

BackgroundTamoxifen is used in hormone therapy for estrogen-receptor (ER)-positive breast cancer, but also has chemopreventative effects against ER-negative breast cancers. This study sought to investigate whether oral iron-saturated bovine lactoferrin (Fe-Lf), a natural product which enhances chemotherapy, could improve the chemotherapeutic effects of tamoxifen in the treatment of ER-negative breast cancers.MethodsIn a model of breast cancer prevention, female Balb/c mice treated with tamoxifen (5 mg/Kg) were fed an Fe-Lf supplemented diet (5 g/Kg diet) or the base diet. At week 2, 4T1 mammary carcinoma cells were injected into an inguinal mammary fat pad. In a model of breast cancer treatment, tamoxifen treatment was not started until two weeks following tumor cell injection. Tumor growth, metastasis, body weight, and levels of interleukin 18 (IL-18) and interferon γ (IFN-γ) were analyzed.ResultsTamoxifen weakly (IC50 ~ 8 μM) inhibited the proliferation of 4T1 cells at pharmacological concentrations in vitro. In the tumor prevention study, a Fe-Lf diet in combination with tamoxifen caused a 4 day delay in tumor formation, and significantly inhibited tumor growth and metastasis to the liver and lung by 48, 58, and 66% (all P < 0.001), respectively, compared to untreated controls. The combination therapy was significantly (all P < 0.05) more effective than the respective monotherapies. Oral Fe-Lf attenuated the loss of body weight caused by tamoxifen and cancer cachexia. It prevented tamoxifen-induced reductions in serum levels of IL-18 and IFN-γ, and intestinal cells expressing IL-18 and IFN-γ. It increased the levels of Lf in leukocytes residing in gut-associated lymphoid tissues. B, T and Natural killer (NK) cells containing high levels of Lf were identified in 4T1 tumors, suggesting they had migrated from the intestine. Similar effects of Fe-Lf and tamoxifen on tumor cell viability were seen in the treatment of established tumors.ConclusionsThe results indicate that Fe-Lf is a potent natural adjuvant capable of augmenting the chemotherapeutic activity of tamoxifen. It could have application in delaying relapse in tamoxifen-treated breast cancer patients who are at risk of developing ER-negative tumors.

The Forkhead Box Transcription Factor FOXC1 Promotes Breast Cancer Invasion by Inducing Matrix Metalloprotease 7 (MMP7) Expression

Therapeutic options for treatment of basal-like breast cancers are limited and identification of molecular targets for novel therapies to treat this aggressive cancer is urgently needed. Recently, FOXC1, a forkhead box transcription factor, was identified as a functionally important biomarker of breast cancer aggressiveness and the basal-like breast cancer subtype. However, the mechanism through which FOXC1 controls aggressiveness of basal-like breast cancer remains to be elucidated. Here, we identify matrix metalloprotease 7 (MMP7) as a key downstream effector of FOXC1-mediated invasiveness. Expression of FOXC1 and MMP7 is significantly correlated in breast cancer samples and cell lines at both the mRNA and protein levels. Transient expression of FOXC1 in nontransformed mammary epithelial cell lines resulted in significantly increased expression of MMP7 and an MMP7-dependent increase in invasiveness. In reciprocal experiments, silencing endogenous FOXC1 in basal-like breast cancer cell lines resulted in decreased expression of MMP7 without decreased expression of other matrix metalloproteinases. We also demonstrate that elevated co-expression of FOXC1 and MMP7 is an independent predictor of patient outcome in multivariate analyses of two breast cancer patient cohorts. Together, our findings identify MMP7 as a novel mechanism through which FOXC1 may regulate the basal-like breast cancer invasive phenotype and the propensity of these cancers to metastasize. Furthermore, our findings demonstrate for the first time a correlation between MMP7 expression and basal-like breast cancers, suggesting that MMP7 may be a useful therapeutic target for treatment of this disease.

Abstract 2861: Inhibition of mTOR and AKT as a potential strategy for the treatment of basal-like breast cancer

Abstract Basal-like breast cancer is an aggressive disease, for which effective targeted therapies are lacking. The frequent loss of phosphatase and tensin homolog (PTEN) and aberrant activation of phosphatidylinositol 3-kinase (PI3K)-AKT-mTOR pathway in basal-like breast cancer make it an attractive therapeutic strategy to target AKT and mTOR. To evaluate the effect of PTEN loss on tumor cell sensitivity to mTOR inhibition, we performed PTEN knockdown experiment in vitro using a basal-like breast cancer cell line WU-BC3, which was derived from a patient with triple negative breast cancer. A more dramatic reduction in cell proliferation was observed in WU-BC3 siPTEN compared to that in WU-BC3 siControl following 7 days of treatment with the mTOR inhibitor MK-8669 (p&amp;lt;0.001). We then tested MK-8669 and MK-2206 (an allosteric, selective pan-AKT inhibitor), either alone or in combination, in 2 Human Tumor in Mouse (HIM) models of basal-like breast cancer, WU-BC4 and WU-BC5, that are deficient in PTEN expression in vivo. In both models, MK-8669 alone significantly inhibited tumor growth (p&amp;lt;0.001). However, the combination of MK-8669 and MK-2206 was more effective than either MK-8669 (p&amp;lt;0.05) or MK-2206 (p&amp;lt;0.01) alone. Biomarker studies indicated that the combination therapy resulted in a more dramatic inhibition of tumor cell proliferation (as assessed by Ki67) and angiogenesis (as assessed by CD31 staining) than single agents alone in both models. In addition, treatment with MK-2206 abolished the MK-8669-induced AKT activation in vivo, suggesting the inhibition of the feedback loop between mTOR and AKT being a potential underlying mechanism for the enhanced anti-tumor effect observed with the combination therapy. Our results provide a preclinical rationale for future clinical investigation of this combination in basal-like breast cancer. 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 2861. doi:1538-7445.AM2012-2861

FOXC1 regulates the functions of human basal-like breast cancer cells by activating NF-κB signaling

Human basal-like breast cancer (BLBC) is an enigmatic and aggressive malignancy with a poor prognosis. There is an urgent need to identify therapeutic targets for BLBC, because current treatment modalities are limited and not effective. The forkhead box transcription factor FOXC1 has recently been identified as a critical functional biomarker for BLBC. However, how it orchestrates BLBC cells was not clear. Here we show that FOXC1 activates the transcription factor nuclear factor-κB (NF-κB) in BLBC cells by increasing p65/RelA protein stability. High NF-κB activity has been associated with estrogen receptor-negative breast cancer, particularly BLBC. The effect of FOXC1 on p65/RelA protein stability is mediated by increased expression of Pin1, a peptidyl-prolyl isomerase. FOXC1 requires NF-κB for its regulation of cell proliferation, migration and invasion. Notably, FOXC1 overexpression renders breast cancer cells more susceptible to pharmacological inhibition of NF-κB. These results suggest that BLBC cells may rely on FOXC1-driven NF-κB signaling. Interventions of this pathway may provide modalities for the treatment of BLBC.

Rapamycin synergizes cisplatin sensitivity in basal-like breast cancer cells through up-regulation of p73

Recent gene expression profiling studies have identified five breast cancer subtypes, of which the basal-like subtype is the most aggressive. Basal-like breast cancer poses serious clinical challenges as there are currently no targeted therapies available to treat it. Although there is increasing evidence that these tumors possess specific sensitivity to cisplatin, its success is often compromised due to its dose-limiting nephrotoxicity and the development of drug resistance. To overcome this limitation, our goal was to maximize the benefits associated with cisplatin therapy through drug combination strategies. Using a validated kinase inhibitor library, we showed that inhibition of the mTOR, TGFβRI, NFκB, PI3K/AKT, and MAPK pathways sensitized basal-like MDA-MB-468 cells to cisplatin treatment. Further analysis demonstrated that the combination of the mTOR inhibitor rapamycin and cisplatin generated significant drug synergism in basal-like MDA-MB-468, MDA-MB-231, and HCC1937 cells but not in luminal-like T47D or MCF-7 cells. We further showed that the synergistic effect of rapamycin plus cisplatin on basal-like breast cancer cells was mediated through the induction of p73. Depletion of endogenous p73 in basal-like cells abolished these synergistic effects. In conclusion, combination therapy with mTOR inhibitors and cisplatin may be a useful therapeutic strategy in the treatment of basal-like breast cancers.

Microarray-Based Gene Expression Profiling for Molecular Classification of Breast Cancer and Identification of New Targets for Therapy

Basal-like breast cancers represent approximately 15%–20% of all breast carcinomas, are aggressive, have variable responses to chemotherapy, and associated with poor clinical outcome. The molecular mechanisms governing the biological behavior of basal-like breast cancers are not well understood. Hence, it is difficult to predict which chemotherapeutics are most likely to be effective and to determine appropriate management strategies for individual patients. Transcriptomic analysis may allow further stratification of basal-like breast cancers enabling prediction of 1) cancer recurrence after surgery; 2) likelihood of metastatic spread; 3) probable tissue sites for metastatic spread; and 4) responses to specific therapies and treatment modalities. Furthermore, careful examination of microarray-based gene expression profiles may identify new molecular targets (or pathways) for the development of targeted therapeutics. Targeted therapies may prove to be more efficacious in basal-like breast cancer treatment than the cytotoxic chemotherapeutics currently employed.

Contemplating chemosensitivity of basal-like breast cancer based on BRCA1 dysfunction

Gene-expression profiling classified breast cancer to intrinsic subtypes, including luminal A and B, HER2 positive, normal-breast-like, and basal-like tumors. Of these, basal-like tumors that express basal cytokeratins and that are negative for estrogen receptor alpha, progesterone receptor, and HER2 show the most aggressive phenotype with a poor prognosis. Analyses of clinical samples and basic research indicate that basal-like breast cancer is caused by deficiencies in the breast cancer susceptibility protein, BRCA1. Indeed, conditionally deleting BRCA1 from the mammary gland causes mice to develop basal-like cancers at high rates. One of the major functions of BRCA1 is DNA double-strand break (DSB) repair, and its failure to perform causes increased sensitivity of cells to DNA damage-inducing agents, such as PARP inhibitors, DNA cross-linkers, or topoisomerase inhibitors. Therefore, BRCA1 dysfunction could be a principal target for therapeutic application of basal-like breast cancer. Recently, significant progress has been made in understanding the BRCA1 cascade in response to DSBs, where ubiquitin polymer formation plays critical roles. Ubiquitination was indeed found to be an apparent early response of breast cancer to neoadjuvant treatment with epirubicin and cyclophosphamide. Deducing the role of BRCA1 ubiquitin E3 ligase activity in this pathway is a critical challenge to further clarify its functional mechanism. In individualized treatment of breast cancer, evaluation of the DNA repair capacity by the BRCA1 pathway may be an important issue when determining proper treatment of basal-like breast cancer.

1,1-Bis(3'-indolyl)-1-(p-biphenyl)methane inhibits basal-like breast cancer growth in athymic nude mice

Introduction1,1-Bis (3'-indolyl)-1-(p-biphenyl) methane (CDIM9) has been identified as a new peroxisome proliferator-activated receptor (PPAR)-γ agonist that exhibits both receptor dependent and independent antitumor activities. CDIM9 has not previously been studied with respect to its effects against basal-like breast cancer. Our goal in the present study was to investigate the anti-basal-like breast tumor activity of CDIM9 in vitro and in vivo.MethodsThe effects of CDIM9 on cell protein and DNA syntheses were determined in basal-like breast cancer MDA-MB231 and BT549 cells in vitro. Maximum tolerated dose and dose-limited toxicity were determined in BalB/c mice, and antitumor growth activities were assessed in MDA-MB231 basal-like breast tumor xenografts in athymic nude mice.ResultsCDIM9 exhibited selective cell cytotoxicity and anti-proliferation effects on basal-like breast cancer lines. In MDA-MB231 cell, CDIM9 induced caveolin-1 and p27 expression, which was significantly downregulated by co-treatment with the PPAR-γ antagonist GW9662. Nonsteroidal anti-inflammatory drug-activated gene-1 and activating transcription factor-3 were upregulated by CDIM9 through a PPAR-γ independent pathway. CDIM9 (40 mg/kg daily, intraperitoneally, for 35 days) inhibited the growth of subcutaneous MDA-MB231 tumor xenografts by 87%, and produced a corresponding decrease in proliferation index. Nearly half of the treated mice (46%) had complete durable remissions, confirmed by histology. The growth of an established tumor was inhibited by CDIM9 treatment (64 mg/kg daily, intraperitoneally, for 10 days), with a mean tumor growth inhibition of 67% as compared with controls. CDIM9 induced increases in tumor caveolin-1 and p27 in vivo, which may contribute to its antitumor activity in basal-like breast cancer.ConclusionCDIM9 showed potent antiproliferative effects on basal-like breast cancer cell in tissue culture and dramatic growth inhibition in animal models at safe doses. These findings justify further development of this drug for treatment of basal-like breast cancer.

BRCA1 dysfunction in sporadic basal-like breast cancer

Basal-like breast cancers form a distinct subtype of breast cancer characterized by the expression of markers expressed in normal basal/myoepithelial cells. Breast cancers arising in carriers of germline BRCA1 mutations are predominately of basal-like type, suggesting that BRCA1 dysfunction may play a role in the pathogenesis of sporadic basal-like cancers. We analysed 37 sporadic breast cancers expressing the basal marker cytokeratin 5/6, and age- and grade-matched controls, for downregulation of BRCA1. Although BRCA1 promoter methylation was no more common in basal-like cancers (basal 14% vs controls 11%, P=0.72), BRCA1 messenger RNA expression was twofold lower in basal-like breast cancers compared to matched controls (P=0.008). ID4, a negative regulator of BRCA1, was expressed at 9.1-fold higher levels in basal-like breast cancer (P<0.0001), suggesting a potential mechanism of BRCA1 downregulation. BRCA1 downregulation correlated with the presence of multiple basal markers, revealing heterogeneity in the basal-like phenotype. Finally, we found that 63% of metaplastic breast cancers, a rare type of basal-like cancers, had BRCA1 methylation, in comparison to 12% of controls (P<0.0001). The high prevalence of BRCA1 dysfunction identified in this study could be exploited in the development of novel approaches to targeted treatment of basal-like breast cancer.