Metastatic Progression Of Breast Cancer Research Articles

Abstract 5161: Identification of epithelial stromal interaction 1 as a novel mediator of breast cancer cell invasion and metastasis downstream of Krüppel-like factor 8.

Abstract Epithelial stromal interaction 1 (EPSTI1) is a recently identified novel gene that is highly upregulated in breast cancer cells when co-cultured with the stromal fibroblasts and in invasive breast carcinomas as compared with normal breast biopsies. The regulation and function of EPSTI1 are largely unknown. Our studies showed that EPSTI1 expression is upregulated by Kruppel-like factor 8 (KLF8), a transcriptional factor that is highly over-expressed in breast cancer and critical for cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition (EMT) and metastatic progression of breast cancer, by directly activating EPSTI1 promoter. Also, we demonstrated that EPSTI1 was necessary and sufficient to promote breast cancer cell invasiveness by genetically modifying EPSTI1 expression and Matrigel invasion assays. We also found that EPSTI1 could interact with valosin containing protein (VCP) and subsequently activate NF-kB nuclear function. Finally, our mouse mammary fat pad injection and tail vein injection experiments indicated that ectopic overexpression of EPSTI1 in MCF7 cells could enhance the tumor invasion and metastasis in vivo. These findings suggest that EPSTI1 is a novel target of KLF8 and plays a critical role in the regulation of breast cancer invasion and metastasis. Citation Format: Tianshu Li, Heng Lu, Chao Shen, Satadru Lahiri, Melissa Watson, Debarati Mukherjee, Lin Yu, Jihe Zhao. Identification of epithelial stromal interaction 1 as a novel mediator of breast cancer cell invasion and metastasis downstream of Krüppel-like factor 8. [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 5161. doi:10.1158/1538-7445.AM2013-5161 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 863: Inhibition of breast cancer metastaticprogression by a novel uPAR targeted monoclonal antibody, ATN-658, correlateswith the uPAR expression.

Abstract The urokinase plasminogen activator receptor (uPAR) has been implicated in the progression of metastatic disease in a number of different solid tumor types including breast cancer. We have developed a therapeutic monoclonal antibody, ATN-658, that targets uPAR in a novel manner. ATN-658 does not inhibit the binding of uPA to uPAR but appears to have pleiotropic effects on other uPAR interactions including those with various integrins. In the present study, we utilized 231LM2-4H2N, a novel highly metastatic variant of the triple negative breast cancer cell line cell line, MDA-MB-231, to evaluate the effect of ATN-658 on metastasis. This variant has been transfected to also express HER2 and metastases in this model are resistant to targeted treatments such as trastuzumab and sunitinib. Orthotopic 231LM2-4H2N xenograft tumor spontaneously metastasizes to lung and liver and forms large macroscopic metastases that lead to death. We transfected 231LM2-4H2N cells with dTomato and Luc2 to allow for real time assessment of metastatic progression using IVIS imaging. Primary orthotopic tumors were staged to 500 mm3, at which time they were resected and the mice followed using bioluminescence to detect metastases, which typically appear in the lungs within 2-3 weeks post-resection. Mice were randomized once lung metastases were visible and treatment was initiated using vehicle control, lapatanib (30 mg/kg QD), ATN-658 (10 mg/kg Q3D) or the combination of lapatanib and ATN-658. Growth of metastases was estimated using average radiance measurements. ATN-658 inhibited metastatic progression by ∼90% and doubled median survival compared to the vehicle control in this study. Lapatanib had no effect on the size of the metastases or survival and the combination of ATN-658+lapatanib inhibited metastatic progression and survival to the same extent as ATN-658 monotherapy. The activation of ERK was significantly inhibited by ATN-658 in vivo although this alone was insufficient to inhibit tumor growth since lapatanib also inhibited ERK to the same extent suggesting the other effects were being mediated by ATN-658. The effect of ATN-658 monotherapy on metastatic tumor growth was much greater in the 231LM2-4H2N model than in the parental MDA-MB-231 model. In order to probe the reason for this difference in effect, we analyzed the expression of uPAR in the various MDA-MB-231 variants. uPAR expression was significantly upregulated in HER2-transfected MDA-MB-231 breast cancer cells suggesting that the level of uPAR expression may dictate the degree of response to ATN-658. If this hypothesis can be validated, this would provide a patient enrichment strategy for the humanized version of ATN-658, huATN-658, which is getting ready to go into the clinic. Citation Format: Irawati Kandela, Andrey Ugolkov, Giulio Francia, Shafaat Rabbani, Robert S. Kerbel, Andrew P. Mazar. Inhibition of breast cancer metastaticprogression by a novel uPAR targeted monoclonal antibody, ATN-658, correlateswith the uPAR expression. [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 863. doi:10.1158/1538-7445.AM2013-863

MiR-7 Suppresses Brain Metastasis of Breast Cancer Stem-Like Cells By Modulating KLF4

Despite significant improvement in survival rates of patients with breast cancer, prognosis of metastatic disease is still dismal. Cancer stem-like cells (CSC) are considered to play a role in metastatic progression of breast cancer; however, the exact pathologic role of CSCs is yet to be elucidated. In this report, we found that CSCs (CD24(-)/CD44(+)/ESA(+)) isolated from metastatic breast cell lines are significantly more metastatic than non-CSC populations in an organ-specific manner. The results of our microRNA (miRNA) profile analysis for these cells revealed that CSCs that are highly metastatic to bone and brain expressed significantly lower level of miR-7 and that this miRNA was capable of modulating one of the essential genes for induced pluripotent stem cell, KLF4. Interestingly, high expression of KLF4 was significantly and inversely correlated to brain but not bone metastasis-free survival of patients with breast cancer, and we indeed found that the expression of miR-7 significantly suppressed the ability of CSCs to metastasize to brain but not to bone in our animal model. We also examined the expression of miR-7 and KLF4 in brain-metastatic lesions and found that these genes were significantly down- or upregulated, respectively, in the tumor cells in brain. Furthermore, the results of our in vitro experiments indicate that miR-7 attenuates the abilities of invasion and self-renewal of CSCs by modulating KLF4 expression. These results suggest that miR-7 and KLF4 may serve as biomarkers or therapeutic targets for brain metastasis of breast cancer.

Abstract C24: Identification and characterization of serum biomarkers associated with metastatic progression of breast cancer

Abstract Breast cancer is the most prevalent form of cancer among women in the United States. Treatment is greatly complicated by metastasis of cells from the primary tumor site, which increases mortality; about 40 percent of breast cancer patients will relapse and develop metastatic disease. Eighty percent of breast cancer patients are treated with adjuvant chemotherapy to decrease their risk of getting metastasis, while as indicated previously only 40% of them will eventually relapse and develop metastatic disease. Despite this precautionary measure, adjuvant chemotherapy is shown to increase 15-years survival of such patients by only 3-10%. Further, about 40,000 women die of breast cancer every year due to undetected early metastasis. Clearly detection of metastatic disease has major clinical implications. Identifying specific blood serum biomarkers that accurately and reproducibly indicate the presence of metastatic disease could greatly improve treatment, as they could substitute for more invasive procedures that are in current use and be used to monitor remission at higher resolution. Analysis of protein content in serum has been widely used as a method for biomarker identification, but detection, identification, and analysis of short peptide biomarkers faces technical challenges that have prevented widespread application of this approach. We report here results from a proteomics study aimed at identifying and characterizing short peptide biomarkers in serum samples taken from patients with stage I and stage III breast cancer. Serum samples contain over 2500 distinct peaks representing distinct peptides. A preliminary analysis reveals that the abundance of 92 peptides are significantly reduced in samples from patients with stage III disease, while the abundance of 64 peptides show significant increase in stage III sera. A large number of additional peptides show smaller changes in abundance in sera from the two patient groups. Our future work will be to characterize the identity of the serum biomarkers identified and validate their appearance or disappearance in a large cohort of patients. Citation Format: Adhari Abdullah Al Za'abi, Steven Graves, Marc Hansen. Identification and characterization of serum biomarkers associated with metastatic progression of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C24.

Abstract C75: miR-7 suppresses brain metastasis of cancer stem-like cells by modulating Kruppel-like factor 4 in breast cancer

Abstract The majority of cancer death of breast cancer attributed to metastatic disease. Despite this clinical importance, the exact pathological process of tumor metastasis is as yet poorly understood, and deciphering the exact molecular mechanism of this process is of paramount importance in order to identify specific therapeutic targets for this devastating disease. Recent active research in microRNA identified a series of this type of molecules that are involved in tumor progression in various tumors as oncogenes and tumor suppressors. Cancer stem-like cells (CSCs) are considered to play a role in metastatic progression of breast cancer; however, the exact pathological role of CSCs is yet to be elucidated. In this report, we found that CSCs (CD24-/CD44+/ESA+) isolated from metastatic breast cell lines are significantly more metastatic than non-CSC populations in an organ specific manner. The results of our microRNA profile analysis for these cells revealed that CSCs that are highly metastatic to bone and brain expressed significantly lower level of miR-7 and that this microRNA was capable of modulating one of the essential genes for induced pluripotent stem cell, KLF4. Interestingly, high expression of KLF4 was significantly and inversely correlated to brain- but not bone-metastasis free survival of breast cancer patients, and we indeed found that the expression of miR-7 significantly suppressed the ability of CSCs to metastasize to brain but not to bone in our animal model. We also examined the expression of miR-7 and KLF4 in brain-metastatic lesions and found that these genes were significantly down- or up-regulated, respectively, in the tumor cells in brain. Furthermore, the results of our in vitro experiments indicate that miR-7 attenuates the abilities of invasion and self-renewal of CSCs by modulating KLF4 expression. These results suggest that miR-7 and KLF4 may serve as biomarkers or therapeutic targets for brain metastasis of breast cancer. Citation Format: Fei Xing, Hiroshi Okuda, Aya Kobayashi, Puspa R. Pandey, Puspa R. Pandey, Sambad Sharma, Yin-Yuan Mo, Yin Liu, Kerui Wu, Kounosuke Watabe. miR-7 suppresses brain metastasis of cancer stem-like cells by modulating Kruppel-like factor 4 in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C75.

Evaluating The Clinicopathologic Characteristics and Survival Outcome of Breast Cancer Patients with Isolated Brain Metastases after Adjuvant Treatment or at Initial Diagnosis

CNS metastases usually appears late in the progression of metastatic breast cancer. Classical approach is evaluating and treat ing them when symptoms become evident. We evaluated the survival and described clinicopathologic characteristics of patients in whom the brain metastases after adjuvant treatment or at initial diagnosis are the first and the only side. Authors retrospec­ tively evaluated about 3600 patients with breast cancer treated in two university hospitals. In those 31 patients with first and only metastases to brain and no other metastases were evaluated. ER, PR, cerbB2 status T, N stage, grade, adjuvant taxane, trastuzumab, hormonal treatment, trastuzumab and platine use after brain metastases didn’t effect the survival. Surgery and WBRT may be more effective in cerbB2 negative patients, WBRT in cerbB2 positive ones. (p=0.06). The survival outcome may be better in pre and perimenouposal women. The mOS of pre and perimenopausal, postmenopausal women were 17.7 months and 10.3 months respectively (p=0.06) and lapatinib may affect the mOS of patients with isolated brain metastases. Some prognostic factor may help us to foresee which group may benefit more from which treatment modality. The need for studies with larger groups of patients is obvious.

Investigational drug slows metastatic breast cancer progression

Investigational drug slows metastatic breast cancer progression

Allelic Variation and Differential Expression of the mSIN3A Histone Deacetylase Complex Gene Arid4b Promote Mammary Tumor Growth and Metastasis

Accumulating evidence suggests that breast cancer metastatic progression is modified by germline polymorphism, although specific modifier genes have remained largely undefined. In the current study, we employ the MMTV-PyMT transgenic mouse model and the AKXD panel of recombinant inbred mice to identify AT–rich interactive domain 4B (Arid4b; NM_194262) as a breast cancer progression modifier gene. Ectopic expression of Arid4b promoted primary tumor growth in vivo as well as increased migration and invasion in vitro, and the phenotype was associated with polymorphisms identified between the AKR/J and DBA/2J alleles as predicted by our genetic analyses. Stable shRNA–mediated knockdown of Arid4b caused a significant reduction in pulmonary metastases, validating a role for Arid4b as a metastasis modifier gene. ARID4B physically interacts with the breast cancer metastasis suppressor BRMS1, and we detected differential binding of the Arid4b alleles to histone deacetylase complex members mSIN3A and mSDS3, suggesting that the mechanism of Arid4b action likely involves interactions with chromatin modifying complexes. Downregulation of the conserved Tpx2 gene network, which is comprised of many factors regulating cell cycle and mitotic spindle biology, was observed concomitant with loss of metastatic efficiency in Arid4b knockdown cells. Consistent with our genetic analysis and in vivo experiments in our mouse model system, ARID4B expression was also an independent predictor of distant metastasis-free survival in breast cancer patients with ER+ tumors. These studies support a causative role of ARID4B in metastatic progression of breast cancer.

Monitoring the progression of metastatic breast cancer on nanoporous silica chips

Breast cancer accounted for 15 per cent of total cancer deaths in female patients in 2010. Although significant progress has been made in treating early-stage breast cancer patients, there is still no effective therapy targeting late-stage metastatic breast cancers except for the conventional chemotherapy interventions. Until effective therapy for later-stage cancers emerges, the identification of biomarkers for the early detection of tumour metastasis continues to hold the key to successful management of breast cancer therapy. Our study concentrated on the low molecular weight (LMW) region of the serum protein and the information it contains for identifying biomarkers that could reflect the ongoing physiological state of all tissues. Owing to technical difficulties in harvesting LMW species, studying these proteins/peptides has been challenging until now. In our study, we have recently developed nanoporous chip-based technologies to separate small proteins/peptides from the large proteins in serum. We used nanoporous silica chips, with a highly periodic nanostructure and uniform pore size distribution, to isolate LMW proteins and peptides from the serum of nude mice with MDA-MB-231 human breast cancer lung metastasis. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and biostatistical analysis, we were able to identify protein signatures unique to different stages of cancer development. The approach and results reported in this study possess a significant potential for the discovery of proteomic biomarkers that may significantly enhance personalized medicine targeted at metastatic breast cancer.

Fulvestrant (F) plus sorafenib (S) as salvage therapy for hormone receptor positive (HR+) metastatic breast cancer (MBC) failing prior aromatase inhibitor (AI) treatment.

e11042 Background: Upon MBC progression with AI treatment; F, an estrogen receptor (ER) antagonist, is often used. Furthermore, vascular endothelial growth factor (VEGF) remains a potential target in MBC. S is a multi-kinase inhibitor of VEGF, Raf, platelet-derived growth factor receptor beta, Flt-3, and c-KIT. Prior studies of S show activity with chemotherapy and as a single agent. Combination therapy with an anti-estrogen and a VEGF inhibitor may be more effective than either agent alone. This pilot study combines F + S for HR+ MBC. Methods: Upon disease progression with an AI, 8 pts were treated with F. Concurrent S was administered starting day 1 until disease progression or unacceptable toxicity. Assessment of tumor status with computed tomography and/or bone scan was performed at baseline and every 2 cycles until disease progression. Results: Median age = 58; 75 % <65. ER and PR + = 6 pts; ER + and PR - = 2 pts. 6 pts were treated with adjuvant chemotherapy and endocrine therapy, with subsequent AI for MBC. 2 pts were metastatic at presentation and treated with prior AI. Median number of days on treatment = 220; median number of cycles = 7. 75% had grade 2/3 HFS; 50% required S dose modification. However, HFS and rash/desquamation were the only grade 3 toxicities (3 pts) with no additional grade 3/4 toxicities or dose modifications for other toxicities. Other grade 2 toxicities: neutropenia, myalgias, arthralgias, fatigue, pruritis. Only 1 pt discontinued the study at investigator’s decision for grade 3 HFS. There were no deaths on study. The remaining 7 pts continued F + S until disease progression with stable disease over a range of 2 to 22 cycles. Median time to progression was 7.3 months. Conclusions: Upon progression with an AI, treatment with F + S may provide advantage for some HR+ MBC pts beyond treatment with F alone. Future studies involving S in MBC should focus on identifying the population that will benefit the most in exchange for toxicities associated with S. [Table: see text]

Abstract 3829: Inhibition of breast cancer metastasis by deguelin in an experimental model

Abstract Breast cancer is a second leading cause of cancer related deaths in women. Cancer related deaths in breast cancer patients are due to metastasis of disease. Thus new drugs which could successfully inhibit metastatic disease spread are highly desired. However till date there are only a few experimental models available to study metastatic progression of breast cancer. Murine 4T1 mammary breast cancer cells when transplanted s.c./or into mammary gland metastasizes to lungs, liver and bone similar to that observed in women with stage IV metastatic disease. Thus this model is highly appropriate for studies related to breast cancer metastasis. Previously we have shown that Deguelin, originally isolated from an African plant Mundulea sericea significantly inhibits the growth of triple negative breast cancer and that the Deguelin effect is mediated through inhibition of WNT signaling pathway. However their therapeutic effect in vivo is still unexplored. We evaluated the effects of Deguelin on growth (in vitro and in vivo) and lung metastasis of 4T1 cells. In vitro, Deguelin inhibited growth of 4T1 cells in time and concentration depended manner; optimum growth inhibition was obtained at 250nM Deguelin concentration after 72 hr. treatment. Toxicity study performed in 4-6 weeks old female BALB/c mice suggested that Deguelin administered as a suspension (saline/gum Arabic/Deguelin) daily for two weeks by intraperitoneal (i.p.) injection was well tolerated up to 16mg/kg body weight, and no toxicity (loss of appetite, body weight loss, mobility, morbidity, death) was observed in Deguelin treated animals. In vivo, as compared to vehicle treated animals, Deguelin (2 or 6mg/kg body weight) administered daily for 21 days i.p significantly (P<0.05) inhibited growth of 4T1 cells (0.5 million cells/mouse) transplanted s.c. in to 4-6 weeks old female BALB/c mice. Similarly Deguelin also reduced (27-33%, P< 0.02) the number of metastatic lesions in the lungs subsequent to i.v. injection of 7500 to 10,000 4T1 cells in syngeneic mice as compared to vehicle treated control group. Immunohistochemical analysis of control/Deguelin treated 4T1 cells, and lung metastatic lesions suggest that Deguelin inhibited Ki67 expression suggesting its antiproliferative action in this model. In addition, phospho AKT, cyclin D1, Cox-2 and Hif-1α protein expressions were down regulated. Our results suggest that Deguelin effect on the growth and metastasis may be mediated by targeting signaling molecules involved in pAKT, HIF-1, Cox-2 pathway. These results provide a promising lead for a potential therapeutic agent to treat metastatic breast cancer. This work was supported in part by the National Cancer Institute grant CA140321. 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 3829. doi:1538-7445.AM2012-3829

Abstract 5310: Regulation of metastatic breast cancer by E2F transcription factors

Abstract Metastasis is the leading cause of mortality in breast cancer and the purpose of this study was to examine the role of the E2F transcription factors in mediating the dissemination of the primary cancer. E2F transcription factors have previously been demonstrated to play a critical role in regulation of cell cycle progression. However, in prior work we have demonstrated that gene expression signatures for the E2Fs have variable levels of activation in a heterogeneous tumor model. Using predictive bioinformatic methods to assess the probability of gene pathway activation, we have predicted variable roles for the E2Fs in key mouse models of breast cancer, including Myc, PyMT and Neu. We then genetically tested these computational predictions by interbreeding E2F knockout mice to the various transgenic tumor models. These crosses have revealed that the E2Fs have unique roles in tumor initiation and progression depending upon the oncogene driving the initial tumor. There are differential effects on tumor latency with the loss of individual E2F alleles, including decreased latency with E2F2 loss in the PyMT background but increased latency and reduced incidence in the Myc background. Moreover, we have observed key differences in tumor progression, with loss of E2Fs affecting the heterogeneity of the resulting tumors. In addition to tumor initiation, we have observed striking differences in tumor progression with loss of E2Fs increasing metastasis in the Myc model but reducing metastasis in the PyMT model. In order to determine the mechanism of action for these effects, we have undertaken a gene expression analysis from both primary tumors and the metastases. Comparison of tumor models in the various backgrounds has allowed us to generate a list of E2F target genes that have effects on metastasis. Comparison of these genes to human breast cancer has revealed that the key genes also have critical roles in metastatic human breast cancer survival. These E2F targets include genes that have previously been identified as metastasis suppressors and are being validated in this process. Taken together, this work suggests a critical role for the E2F transcription factors, not only as mediators of cell cycle but in regulating the metastatic progression of metastatic breast cancer. These data also illustrate the power of combining various genomic and gene expression analysis techniques with both mouse models and clinical data associated with gene expression analysis in human breast cancer. Using this approach we are able to predict and test for the role of critical pathways, such as the E2Fs, in the progression of 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 5310. doi:1538-7445.AM2012-5310

Abstract 3247: Identification of a novel TAZ/TEAD/BMP4/Smad1,5 signaling axis

Abstract TAZ functions as a transcriptional co-activator critical for tissue development, mesenchymal stem cell differentiation, and embryonic stem cell renewal. Recently, TAZ has been identified as a major downstream component of the novel Hippo-LATS tumor suppressor pathway, where TAZ over-expression in immortalized mammary epithelial cells, MCF10A, leads to enhanced cell proliferation and migration, transformation, EMT, and resistance to the chemotherapeutic paclitaxel. However, the molecular mechanisms underlying these TAZ-induced phenotypes are largely unknown. In this study, we aimed to identify downstream targets responsible for TAZ-induced phenotypes. Through screening a 44K human genome microarray we have identified many potential targets, one of which is bone morphogenetic protein 4 (BMP4), a cytokine that regulates important processes such as cell proliferation, migration, and EMT. By qRT-PCR, western blotting, and an ELISA assay we verified that BMP4 is upregulated at the mRNA, protein, and secreted levels, respectively, in TAZ overexpressing MCF10A cells (MCF10A-TAZ) compared to control cells (MCF10A-WPI). To investigate whether BMP4 is a direct transcriptional target of TAZ, we used a dual luciferase assay to measure BMP4 promoter activity. When co-transfected with TAZ and TEAD4, a transcription factor mediating TAZ transactivation, BMP4 promoter activity was significantly increased. This TAZ/TEAD interaction is critical for BMP4 transcriptional activation as interaction mutants of TAZ and TEAD4 (TAZα72 and TEAD4-Y429H) both fail to activate BMP4 promotor activity. Significantly, we have identified two TEAD Response Elements (TRE) located in the BMP4 promoter where mutation of TRE1 alone completely abolished activation of BMP4 promoter activity by TAZ/TEAD4 and mutation of TRE2 had no effect. This suggests that TRE1 is responsible for TAZ/TEAD activation of BMP4. Through microarray analysis, we have identified many secreted cytokines leading us to suspect that they may promote EMT. Strikingly, MCF10A cells continuously treated with conditioned media from MCF10A-TAZ or exogenous BMP4 induced an obvious EMT phenotype, suggesting that BMP4 may be responsible for TAZ-induced EMT. Moreover, phosphorylation of Smad1,5 (pSmad1,5), two major mediators of BMP4 function, was also significantly increased in MCF10A-TAZ, but was completely abolished when MCF10A-TAZ cells were treated with Noggin, a BMP4 specific inhibitor, demonstrating that activation of Smad is specifically due to BMP4. Our results provide convincing evidence that BMP4 is a novel transcriptional target of TAZ, which may mediate the TAZ-induced EMT phenotype and thereby identify a novel TAZ/TEAD/BMP4/Smad1,5 signaling axis. Further functional characterization of this pathway will greatly facilitate our understanding of the roles of TAZ in the metastatic progression of 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 3247. doi:1538-7445.AM2012-3247

Changes in Keratin Expression during Metastatic Progression of Breast Cancer: Impact on the Detection of Circulating Tumor Cells

Circulating tumor cells (CTC) might function as early markers for breast cancer metastasis or monitoring therapy efficacy. Enrichment and identification of CTCs are based on epithelial markers that might be modulated during epithelial-mesenchymal transition. Little is known about the expression of keratins in CTCs and whether all CTCs can be detected with antibodies directed against a limited panel of keratins. Protein expression of keratin 2, 4-10, 13-16, 18, and 19 were assessed by a cocktail of antibodies (C11, AE1, AE3, and K7) and keratin antibodies C11 and A45-B/B3 alone in 11 breast cancer cell lines and 50 primary breast carcinomas and their lymph node metastases. Furthermore, CTCs were assessed in blood of 70 metastatic breast cancer patients. Claudin-low cell lines did not show expression of normal breast epithelial keratins but were positive for K14 and K16, detected by the cocktail only. Primary breast carcinomas showed changes in keratin expression during metastatic progression to the lymph nodes. In 35 of 70 patients CTCs were identified, of which 83%, 40%, and 57% were identified by the cocktail, C11 and A45-B/B3, respectively. Identification of CTCs by the cocktail was associated with shorter survival (P < 0.01). In silico analyses revealed association between KRT16 expression and shorter relapse-free survival in metastatic breast cancer. Breast cancer cells show a complex pattern of keratin expression with potential biologic relevance. Individual keratin antibodies recognizing only a limited set of keratins inherit the risk to miss biologically relevant CTCs in cancer patients, and antibody cocktails including these keratins are therefore recommended.

Implication of the Strand-Specific Imprinting and Segregation Model: Integrating in utero Hormone Exposure, Stem Cell and Lateral Asymmetry Hypotheses in Breast Cancer Aetiology.

Known genetic mutations and familial hereditary factors account for less than 20–25% of breast cancer cases in women, therefore, most instances have been classified as sporadic cases of unknown aetiologies. Single nucleotide polymorphisms (SNPs) were considered as breast cancer risk factors, but numerous studies have failed to support this assertion. Recent evidence correlates aberrant epigenetic mechanisms in the development and metastatic progression of breast cancer, yet there has been limited progress made to identify the primary aetiology underlying sporadic cases of breast cancer. This has led some researchers to consider alternative hypotheses including in utero exposure to deleterious chemical agents during early development, the immortal strand and the strand-specific imprinting and selective chromatid segregation hypotheses. Here, we integrate prominent alternate models to help guide future research on this very important topic concerning human health.

Anti-Transforming Growth Factor ß Antibody Treatment Rescues Bone Loss and Prevents Breast Cancer Metastasis to Bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.

CD1d-Expressing Breast Cancer Cells Modulate NKT Cell-Mediated Antitumor Immunity in a Murine Model of Breast Cancer Metastasis

BackgroundTumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.Methodology/Principal FindingsIn this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.Conclusions/SignificanceThe results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.

Optimizing outcomes in patients (pts) with HER2+ metastatic breast cancer (MBC) through continuous inhibition of HER2 activity: A single institution study.

e11067 Background: Anti-HER2 therapies are effective in HER2+ breast cancer; even if resistance occurs, continued HER2 inhibition is required for antitumor effect. There are no definitive data on the clinical benefit of continued trastuzumab (T) beyond progression in MBC and the optimal duration of T in pts with long-term control of disease. This study explores outcomes of MBC pts treated with T in multiple sequential lines. Methods: From 2001 to 2009 we evaluated OS and cardiac toxicity in 50 pts with HER2+ (ASCO/CAP criteria) MBC who received T-based therapy for ≥ 12 months. OS was measured from the beginning of T-based CT to the last follow up visit or death. Cardiac event was any decline in LVEF by >10% from baseline or drop to <50%, III/IV NYHA CHF, new onset angina myocardial infarction, significant arrhytmias or sudden cardiac death. Results: Median age was 59 (33-79), visceral disease in 60% and multiple site in 34%; 8 (16%) pts developed brain metastasis during T. All had overexpression of HER2 by IHC, FISH was centrally assessed in 78% and not amplified in 8%. T was administered for a median duration of 23 months (12-120). All pts received a median of 2 CT regimens (1-8); 9 out of 25 pts with endocrine responsive disease received endocrine therapy plus T after at least 1 CT regimen; 20 pts (40%) experienced CR and received T alone as maintenance for a median duration of 9 months (3-46); 23 (46%) pts received lapatinib, when the drug was licensed in Italy, after failure of at least two T-based CT lines. Median OS was 34 months (12-120). There were 3 cardiac events (6%) and consisted in asymptomatic decrease in LVEF to less than 50%; T-based CT was interrupted in 1 patient because of LVEF decrease to ≤ 40%. Conclusions: T in multiple sequential lines demonstrated highly favorable outcomes in MBC pts. Overall the incidence of cardiac dysfunction was low.

Abstract 2345: Identification of epithelial stromal interaction 1 as a novel target of KLF8

Abstract Epithelial stromal interaction 1 (EPSTI1) is a recently identified novel gene with two splicing variants that is highly upregulated in breast cancer cells when co-cultured with the stromal fibroblasts and in invasive breast carcinomas as compared with normal breast biopsies. The regulation and function of EPSTI1 are largely unknown. Interestingly, our microarray studies showed that EPSTI1 is one of the most upregulated genes in MCF-10A cells expressing ectopic Kruppel-like factor 8 (KLF8), a transcriptional factor is highly over-expressed in breast cancer and critical for cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition (EMT) and metastatic progression of breast cancer. Real time qRT-PCR and western blotting demonstrated that EPSTI1 mRNA and protein levels are upregulated in a panel of invasive breast cancer cell lines that are known for aberrant overexpression of KLF8 as compared to noninvasive cell lines. Promoter reporter analysis indicated that KLF8 may activate EPSTI1 gene promoter. These results identified EPSTI1 as a novel target of KLF8 and suggest that EPSTI1 may play an important role for KLF8 signaling in the regulation of breast cancer invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2345. doi:10.1158/1538-7445.AM2011-2345

Abstract 3795: Mitochondrial complex I modulation regulates autophagy and breast cancer progression

Abstract Tumor cells express altered metabolic activities often linked to mitochondrial dysfunction. Such mitochondrial defects can inhibit oxidative phosphorylation, change the cellular redox state (NAD+/NADH), increase production of reactive oxygen species (ROS), and cause DNA damage that further supports tumorigenesis and a metastatic phenotype. Our study identified mitochondrial complex I (NADH dehydrogenase) as a modulator of tumorigenesis and metastatic progression in breast cancer. We used NDI1, the NADH dehydrogenase from yeast, to augment complex I activity in metastatic human breast cancer cells. The NDI1 protein is known to integrate into the inner mitochondrial membrane of mammalian cells, function as NADH dehydrogenase, pass electrons into the respiratory chain, and thereby fully augment intrinsic complex I activity. Thus, NDI1 identifies critical involvement of complex I NADH dehydrogenase activity in cellular functions that are altered upon its expression. We investigated a panel of aggressive human breast cancer cell lines, including MDA-MB 231, and found that augmentation of NADH dehydrogenase activity through NDI1 resulted in an enhanced NAD+/NADH ratio and inhibition of ROS production. Most importantly, NDI1 expression inhibited metastasis and tumor growth in the mammary fad pad of immune deficient mice, seen by non-invasive imaging and histology. The same effects were achieved by treatment with NAD precursors, demonstrating that normalization of the NAD+/NADH ratio is critical for inhibition of breast cancer progression. We found that the underlying mechanism is based on activation of PTEN, inhibition of the AKT/mTOR survival pathway, and stimulation of autophagy. Autophagy is responsible for NDI1-mediated inhibition of breast cancer progression, as knock-down of ATG5 reversed the anti-metastatic effect of NDI1. Our results demonstrate that tumorigenesis and metastasis in breast cancer are critically influenced by mitochondrial complex I activity. We show that augmentation of tumor cell complex I function can interfere with tumor formation and breast cancer spreading through modulating the NAD+/NADH ratio. Thus, normalization of complex I function appears as a promising new target for prevention and treatment of breast progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3795. doi:10.1158/1538-7445.AM2011-3795