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Supplementary Data from Sustained c-Jun-NH2-Kinase Activity Promotes Epithelial-Mesenchymal Transition, Invasion, and Survival of Breast Cancer Cells by Regulating Extracellular Signal-Regulated Kinase Activation

The c-Jun NH2-terminus kinase (JNK) mediates stress-induced apoptosis and the cytotoxic effect of anticancer therapies. Paradoxically, recent clinical studies indicate that elevated JNK activity in human breast cancer is associated with poor prognosis. Here, we show that overexpression of a constitutively active JNK in human breast cancer cells did not cause apoptosis, but actually induced cell migration and invasion, a morphologic change associated with epithelial-mesenchymal transition (EMT), expression of mesenchymal-specific markers vimentin and fibronectin, and activity of activator protein transcription factors. Supporting this observation, mouse mammary tumor cells that have undergone EMT showed upregulated JNK activity, and the EMT was reversed by JNK inhibition. Sustained JNK activity enhanced insulin receptor substrate-2–mediated ERK activation, which in turn increased c-Fos expression and activator protein activity. In addition, hyperactive JNK attenuated the apoptosis of breast cancer cells treated by the chemotherapy drug paclitaxel, which is in contrast to the requirement for inducible JNK activity in response to cytotoxic chemotherapy. Blockade of extracellular signal-regulated kinase activity diminished hyperactive JNK-induced cell invasion and survival. Our data suggest that the role of JNK changes when its activity is elevated persistently above the basal levels associated with cell apoptosis, and that JNK activation may serve as a marker of breast cancer progression and resistance to cytotoxic drugs. Mol Cancer Res; 8(2); 266–77

Purpose: To elucidate molecular pathways contributing to metastatic cancer progression and poor clinical outcome in serous ovarian cancer.Experimental Design: Poor survival signatures from three different serous ovarian cancer datasets were compared and a common set of genes was identified. The predictive value of this gene signature was validated in independent datasets. The expression of the signature genes was evaluated in primary, metastatic, and/or recurrent cancers using quantitative PCR and in situ hybridization. Alterations in gene expression by TGF-β1 and functional consequences of loss of COL11A1 were evaluated using pharmacologic and knockdown approaches, respectively.Results: We identified and validated a 10-gene signature (AEBP1, COL11A1, COL5A1, COL6A2, LOX, POSTN, SNAI2, THBS2, TIMP3, and VCAN) that is associated with poor overall survival (OS) in patients with high-grade serous ovarian cancer. The signature genes encode extracellular matrix proteins involved in collagen remodeling. Expression of the signature genes is regulated by TGF-β1 signaling and is enriched in metastases in comparison with primary ovarian tumors. We demonstrate that levels of COL11A1, one of the signature genes, continuously increase during ovarian cancer disease progression, with the highest expression in recurrent metastases. Knockdown of COL11A1 decreases in vitro cell migration, invasion, and tumor progression in mice.Conclusion: Our findings suggest that collagen-remodeling genes regulated by TGF-β1 signaling promote metastasis and contribute to poor OS in patients with serous ovarian cancer. Our 10-gene signature has both predictive value and biologic relevance and thus may be useful as a therapeutic target. Clin Cancer Res; 20(3); 711–23. ©2013 AACR.

PDF file 831K, Supplementary Figures. Supplementary Fig. 1. A schematic diagram of the methods, datasets and patient samples used in this study. Supplementary Fig. 2. Correlogram of the 10 poor outcome. signature genes. Supplementary Fig. 3. Validation of the 10-gene signature - Kaplan-Meyer plot. Supplementary Fig. 4. Real-time PCR of poor outcome signature genes in normal, primary, and metastatic ovarian cancer samples. Supplementary Fig. 5. Increase in COL11A1 expression in recurrent tumors in comparison to primary ovarian tumors. Supplementary Fig. 6. Confinement of COL11A1 expression to intra- and peri-tumoral stroma - immunohistochemical detection. Supplementary Fig. 7. Knockdown of COL11A1 in the A2780 ovarian cancer cell line - qPCR and Western blot

XLS file 139K, Supplementary Tables. Supplementary Table 1A-C. Poor prognosis genes identified from 3 different datasets (A) TCGA, (B) GSE26712, (C) Karlan. Supplementary Table 2. Functional annotation of poor prognosis genes in three datasets. Supplementary Table 3. PCR primer sequences. Supplementary Table 4. Genes and corresponding probes used for Oncomine analysis of expression in primary tumors and metastases. Supplementary Table 5.Matched primary, metastatic and recurrent tumor samples

Supplementary Figures 1-8 from FOXC1 Is a Potential Prognostic Biomarker with Functional Significance in Basal-like Breast Cancer

Gene expression signatures for a basal-like breast cancer (BLBC) subtype have been associated with poor clinical outcomes, but a molecular basis for this disease remains unclear. Here, we report overexpression of the transcription factor FOXC1 as a consistent feature of BLBC compared with other molecular subtypes of breast cancer. Elevated FOXC1 expression predicted poor overall survival in BLBC (P = 0.0001), independently of other clinicopathologic prognostic factors including lymph node status, along with a higher incidence of brain metastasis (P = 0.02) and a shorter brain metastasis–free survival in lymph node–negative patients (P < 0.0001). Ectopic overexpression of FOXC1 in breast cancer cells increased cell proliferation, migration, and invasion, whereas shRNA-mediated FOXC1 knockdown yielded opposite effects. Our findings identify FOXC1 as a theranostic biomarker that is specific for BLBC, offering not only a potential prognostic candidate but also a potential molecular therapeutic target in this breast cancer subtype. Cancer Res; 70(10); 3870–6. ©2010 AACR.

Supplementary Methods, Tables 1-6, Figure Legends 1-8 from FOXC1 Is a Potential Prognostic Biomarker with Functional Significance in Basal-like Breast Cancer

Purpose: Much effort has been focused on understanding breast cancer tumorigenesis. However, the regulation of human breast cancer development by gene mutations, hormone perturbation, external chemicals, and environmental cues is poorly understood at the molecular and cellular level. Although mouse models have been invaluable in advancing the knowledge of breast cancer tumorigenesis and progression, human breast ex vivo models comprising the breast tissue microenvironment are needed to help elucidate the underlying mechanisms of breast cancer risk factors. Here, we report a new human breast tissue mimetic organoid model. Methods: Fresh human breast tissue specimens from reduction mammaplasty reduction and prophylactic surgeries were cut and digested overnight. The breast microtissue containing stroma components were collected via centrifugation. They were then cultured in 3D using a matrix mixture containing Matrigel, collagen I, and specially formulated organoid medium. Using culture inserts, a “sandwich” method was utilized, in which we added 1 layer of matrix mixture, followed by 2 layers of organoids and matrix mixture, and a final layer of matrix mixture. Imaging analyses, including immunohistochemistry, immunofluorescence, and highly-multiplexed CODEX imaging, were used to study cellular marker expression in the organoids. Results: Our 3D culture system is distinct from previous models that reconstitute 3D mammary microstructures using isolated individual cell populations and is capable of growing patient-derived breast organoids for 3 weeks and longer. Via CODEX imaging we spatially localized more than 25 antigens for different cell types, allowing us to produce detailed characterizations of the organoid microarchitecture. Breast tissue microstructures with ducts and lobules along with stromal cell types normally found in the human breast are present. For the first time, we found that different stromal cell types grow alongside the mammary epithelium in the organoid system. The cultured breast organoids are hormone responsive and can be subcultured. We also found that BRCA1 mutant organoids are more sensitive to estrogen and progesterone treatment compared with BRCA1 wildtype, as indicated by organoid size changes. Furthermore, BRCA1 mutant organoids display unique gene expression profiles related to oxidative stress and DNA damage response. Conclusion: We have developed a 3D human breast tissue mimetic culture model that phenocopies the breast tissue microenvironment. This model will serve as the foundation for refining a breast organoid system that can faithfully recapitulate the physiology of human breast tissue. It will pave the way for a novel screening system that will test the pathogenic effects of breast cancer risk factors, providing a useful tool for elucidating the biological mechanisms of breast cancer tumorigenesis. Citation Format: Tian-Yu Lee, Nadya Nikulina, Srivarshini C. Mohan, Edward Ray, Armando Giuliano, Oliver Braubach, Xiaojiang Cui. Generation of a human breast tissue mimetic organoid model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2965.

Patients with breast cancer metastases to the brain on average survive less than one year. These tumors tend to be resistant to current therapies, and the majority of targeted therapeutics are unable to breach the blood brain barrier (BBB) to reach these tumors, thus improved alternatives are urgently needed. Elevated cell surface levels of the human epidermal growth factor receptor subunit 3 (HER3) is associated with metastatic breast tumors, including those that spread to the brain. Elevated HER3 is also associated with resistance to a number of targeted therapies currently used in the clinic, including inhibitors of EGFR (lapatinib), HER2 (lapatinib, trastuzumab, T-DM1), HER2-3 (pertuzumab), and combination therapy. Whereas a number of targeted therapies are currently used to combat peripheral breast tumors, the delivery of these molecules to brain metastases is limited by the blood brain barrier (BBB). This is exemplified by HER2+ breast tumors that metastasize to the brain: these tumors, while targetable outside of the central nervous system (CNS) by HER2 antibodies such as trastuzumab, are unreachable by these same antibodies because the HER2 subunit, though present on the brain endothelium, does not mediate antibody transcytosis across the blood vessel wall. HER3, on the other hand, undergoes rapid transcytosis across the brain endothelium upon ligand binding, which normally occurs to mediate the delivery of neuregulin growth factors for neural growth and maintenance. We have developed a self-assembling nanobiological particle, HerMn, which uses HER3 as a portal for targeted entry of toxic molecules into tumor cells. HerMn is a 10-20 nm diameter serum-stable particle comprised of a HER3-targeted cell penetration protein non-covalently assembled with a sulfonated manganese(III) corrole (S2Mn or Mn-corrole). Tumor-targeted toxicity by HerMn occurs by mitochondria membrane disruption and superoxide-mediated damage to the cytoskeleton. HerMn can also elicit tumor-selective detection by magnetic resonance imaging (MRI) due to the paramagnetic property of the corrole. HerMn distributes to the brain after systemic injection in mice, in addition to showing preferential homing and toxicity to subcutaneous tumors expressing the HER2-3 dimer. Interestingly, the Mn corrole is known to exhibit neuroprotective effects due to its antioxidant activity on normal tissue. Consistent with this, we have found that HerMn supports human cardiac cell survival ex vivo. Our studies interrogating the therapeutic potential of HerMn suggest that this nanobiologic bears the capacity for targeting toxicity to brain-metastatic breast tumors while sparing off-target tissue due to both its targeting capacity and ability to provide beneficial protective effects to normal tissue such as the brain and heart. Citation Format: Medina-Kauwe L, Sims J, Taguiam M, Hanson C, Alonso-Valenteen F, Cui X, Wagner S, Sorasaenee K, Moats R, Marban E, Chung A, Gray H, Gross Z, Giuliano A. A corrole nanobiologic crosses the blood-brain-barrier and recognizes triple negative breast cancer: Implications for targeting brain metastases. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-17-05.

Background: Radiation is the current choice treatment for non-operable metastatic breast-brain cancer. When cancer lesions are located in sensitive areas like the brain or have excessive amounts of metastatic sites, radiation usually proves to be a more viable option than excision. Ionizing (X-ray and gamma) radiation is non-selective and affects all the tissue it penetrates. In order to concentrate the dose on tumors, high energy radiation from multiple directions is typically used, reaching the highest dose where the radiation crosses. This type of multiple angle treatment minimizes the dose to normal tissue by increasing overall normal tissue irradiation. The objective is to achieve sufficient radiation in the tumor tissue to cause the DNA strands to break and to disrupt the reproduction and maintenance of cancer cells while keeping the damage to normal tissue in a reasonable range for tissue preservation. Metal nanoparticles have shown promising results for reinforcing the radiation dose effect. High atomic number (Z) elements absorb a greater amount of radiation because the higher density raises the probability of interaction. The metal nanoparticles interact with the energy of the ionizing radiation by either scattering or absorbing, or accumulating the energy, thus increasing the number of DNA strand breaks in the nucleus of cells. Methods: Four breast cancer cell lines (BT-474, MDA-231, BT-549 and MCF-7) were incubated with 1-2 nm platinum nanoparticles (0-1000 μg/mL) produced with a cysteine coating. 24 hours later cells were exposed to 2 Gy radiation with a C-arm (Toshiba Infinix VF-i/SP) using 125 KVP to deliver a spectrum of KeV low energy X-rays. After 24 hours the cells were washed and analyzed using a bioluminescence assay to assess cell proliferation based on ATP production. Results: Of the four cell lines tested the BT-474 and BT-549 demonstrated limited reduction in cell proliferation at up to the highest treatment concentration 1000 μg/mL with no radiation exposure. As a result of the limited toxicity of the platinum nanoparticles the effect from increased radiation can be more readily observe when 2 Gy radiation is added resulting a in platinum nanoparticle dose dependent decrease in proliferation in the BT-474 cell line. Nanoparticle Toxicity Concentration of Platinum Nanoparticles (μ/mL) 02505007501000MDA-2311.000±0.0050.995±0.0120.974±0.0130.979±0.0140.777±0.014BT-5491.000±0.0131.003±0.0091.003±0.0170.969±0.0170.894±0.009MCF-71.000±0.0140.960±0.0150.927±0.0220.851±0.0220.769±0.032BT-4741.000±0.0240.961±0.0290.957±0.0330.965±0.0630.985±0.065Table 1: Indexed values for cell proliferation for the BT-474 cell Radiation Toxicity Concentration of Platinum Nanoparticles (μ/mL) 02505007501000*0 Gy1.000±0.0240.961±0.0290.957±0.0330.965±0.0630.985±0.0652 Gy1.027±0.0380.966±0.0230.908±0.0340.870±0.0310.799±0.037Table 2: Indexed values for cell proliferation for the BT-474 cell line 0 and 2 Gy radiation doses, 6 averages. * Student T-TEST P Conclusions: At moderate doses of low energy radiation, a reduction in cell proliferation can be detected. This data supports follow-up experiments to add a targeting protein to facilitate uptake by cancer cells based on cell receptor expression. Experiments are current being done to utilize the HER2+ cell receptor upregulation to increase internalization of the particles to achieve a greater effect. Citation Format: Wagner S, Yue Y, Cui X, Zhang G, Bingchen H, Li D, Medina-Kauwe L. Radiation enhancement with cysteine coated platinum nanoparticles. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-12-13.

Human mammary gland development and differentiation are tightly regulated by hormones, growth factors, and microenvironmental cues. Rodent models have been used to help gain knowledge about mammary gland biology, but there are significant structural and hormonal response differences between the human and rodent mammary glands. Moreover, cultured immortalized human mammary cell lines have been widely used for in vitro experiments to study epithelial cell biology, but it has been questioned whether they faithfully recapitulate normal breast cells. Conditional reprogramming has recently emerged as an efficient method to induce rapid and inexhaustible in vitro proliferation of primary epithelial cells from normal and malignant tissues in two-dimensional (2D) culture conditions. However, studies using this method have not shown whether conditionally reprogrammed mammary epithelial cells can form defined structures in three-dimensional (3D) culture conditions. Therefore, our goal is to develop an appropriate in vitro model using conditional reprogramming to study human mammary cell and tissue function under 2D and 3D culture conditions. We cultured primary human mammary cells from normal prophylactic tissues or breast tumors using this method. Cell type heterogeneity, cellular marker expression, and structural arrangement were examined using immunofluorescence staining. We found that normal breast cells grown under this culture condition exhibited morphologic features of luminal cells (CK18, desmoglein 3, and CK19) and myoepithelial cells (vimentin, p63, and CK14), indicating maintenance of in vivo heterogeneity. CD49f and EpCAM double staining is commonly used to separate luminal, basal, and progenitor populations. Immunofluorescence and FACS analysis further revealed subpopulations with varying CD49f and EpCAM expression profiles in the normal primary cultures, as well as detectable expression of ERα in earlier passages. Treatment with estradiol also stimulated cellular proliferation as detected by positive EdU staining. When grown in Matrigel/Collagen I gel, normal primary cells self-organize into two distinct 3D structures that are composed of densely packed cells or a spherical structure containing a lumen, which express either luminal or myoepithelial cell markers, respectively. CK8-positive luminal cells that form the lumen can differentiate into milk-producing cells in the presence of a prolactogenic growth condition. Tumor cells extracted from breast cancer patients showed expression for either basal (CK18 and FOXC1) or luminal (CK14 and ER-positive) markers in 2D cultures. Our ongoing work entails delineating the long-term culture effect on primary mammary cell fate and function and the tumorigenic property of primary breast tumor cells. The current findings uncover an in vitro model that may be a valuable tool to study mammary cell function and can potentially be used to elucidate mechanisms involved in mammary tumorigenesis. Citation Format: Stacey Chung, Liting Jin, Ying Qu, Liliana J. Gomez, Bingchen Han, Bowen Gao, Xuefeng Liu, Farin Amersi, Catherine Dang, Armando E. Giuliano, Xiaojiang Cui. Assessment of conditional reprogramming to generate 2D and 3D primary human mammary cell culture models [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A59.

Gene expression signatures for a basal-like breast cancer (BLBC) subtype have been associated with poor clinical outcomes, but a molecular basis for this disease remains unclear. Here, we report overexpression of the transcription factor FOXC1 as a consistent feature of BLBC compared with other molecular subtypes of breast cancer. Elevated FOXC1 expression predicted poor overall survival in BLBC (P = 0.0001), independently of other clinicopathologic prognostic factors including lymph node status, along with a higher incidence of brain metastasis (P = 0.02) and a shorter brain metastasis–free survival in lymph node–negative patients (P < 0.0001). Ectopic overexpression of FOXC1 in breast cancer cells increased cell proliferation, migration, and invasion, whereas shRNA-mediated FOXC1 knockdown yielded opposite effects. Our findings identify FOXC1 as a theranostic biomarker that is specific for BLBC, offering not only a potential prognostic candidate but also a potential molecular therapeutic target in this breast cancer subtype. Cancer Res; 70(10); 3870–6. ©2010 AACR.

The c-Jun NH2-terminus kinase (JNK) mediates stress-induced apoptosis and the cytotoxic effect of anticancer therapies. Paradoxically, recent clinical studies indicate that elevated JNK activity in human breast cancer is associated with poor prognosis. Here, we show that overexpression of a constitutively active JNK in human breast cancer cells did not cause apoptosis, but actually induced cell migration and invasion, a morphologic change associated with epithelial-mesenchymal transition (EMT), expression of mesenchymal-specific markers vimentin and fibronectin, and activity of activator protein transcription factors. Supporting this observation, mouse mammary tumor cells that have undergone EMT showed upregulated JNK activity, and the EMT was reversed by JNK inhibition. Sustained JNK activity enhanced insulin receptor substrate-2–mediated ERK activation, which in turn increased c-Fos expression and activator protein activity. In addition, hyperactive JNK attenuated the apoptosis of breast cancer cells treated by the chemotherapy drug paclitaxel, which is in contrast to the requirement for inducible JNK activity in response to cytotoxic chemotherapy. Blockade of extracellular signal-regulated kinase activity diminished hyperactive JNK-induced cell invasion and survival. Our data suggest that the role of JNK changes when its activity is elevated persistently above the basal levels associated with cell apoptosis, and that JNK activation may serve as a marker of breast cancer progression and resistance to cytotoxic drugs. Mol Cancer Res; 8(2); 266–77

Progesterone action contributes to the signaling of many growth factor pathways relevant to breast cancer tumor biology, including the insulin-like growth factor (IGF) system. Previous work has shown that insulin receptor substrate-2 (IRS-2) but not IRS-1 levels were regulated by progestin in progesterone receptor-B (PR-B) isoform expressing MCF-7 cells (C4-12 PR-B). Furthermore, type 1 IGF receptor (IGF1R) signaling via IRS-2 correlated with the increased cell migration observed in a number of breast cancer cell lines. Consequently, in this study, we examined whether the elevation of IRS-2 protein induced by progestin was sufficient to promote IGF-I–stimulated cell motility. Treatment of C4-12 PR-B cells with progestin shifted the balance of phosphorylation from IRS-1 to IRS-2 in response to IGF-I. This shift in IRS-2 activation was associated with enhanced migration in C4-12 PR-B cells pretreated with progestin, but had no effect on cell proliferation or survival. Treatment of C4-12 PR-B cells with RU486, an antiprogestin, inhibited IGF-induced cell migration. Attenuation of IRS-2 expression using small interfering RNA resulted in decreased IGF-stimulated motility. In addition, IRS-2 knockdown resulted in an abrogation of PKB/Akt phosphorylation but not mitogen-activated protein kinase. Consequently, LY294002, a phosphoinositide-3-kinase inhibitor, abolished IGF-induced cell motility in progestin-treated C4-12 PR-B cells. These data show a role for the PR in functionally promoting growth factor signaling, showing that levels of IRS proteins can determine IGF-mediated biology, PR-B signaling regulates IRS-2 expression, and that IRS-2 can mediate IGF-induced cell migration via phosphoinositide-3-kinase in breast cancer cells. (Mol Cancer Res 2008;6(9):1491–8)

The epidermal growth factor (EGF) and insulin-like growth factor (IGF) signaling pathways are critically involved in cancer development and progression. However, how these two signals cross-talk with each other to regulate cancer cell growth is not clearly understood. In this study, we found that EGF remarkably induced expression of major IGF signaling components, insulin receptor substrate (IRS)-1 and IRS-2, an effect that could be blocked by EGF receptor (EGFR) tyrosine kinase inhibitors. Although both extracellular signal-regulated kinase and c-Jun NH2-terminal kinase (JNK) signaling pathways were involved in the EGF up-regulation of IRS-1, the IRS-2 induction by EGF was specifically mediated by JNK signaling. Consistent with this, EGF increased IRS-2 promoter activity, which was associated with recruitment of activator protein-1 (AP-1) transcription factors and was inhibited by blocking AP-1 activity. Moreover, EGF treatment enhanced IGF-I and integrin engagement-elicited tyrosine phosphorylation of IRS and their downstream signaling, such as binding to phosphatidylinositol 3′-kinase regulatory subunit p85. Finally, repressing the induction of IRS-2 levels abolished the EGF enhancement of cell motility, suggesting that increased IRS-2 is essential for the EGF regulation of breast cancer cell migration. Taken together, our results reveal a novel mechanism of cross-talk between the EGF and IGF signaling pathways, which could have implications in therapeutic applications of targeting EGFR in tumors. Because AP-1 activity is involved in breast cancer progression, our work may also suggest IRS-2 as a useful marker for aggressive breast cancer. (Cancer Res 2006; 66(10): 5304-13)

Blockade of the estrogen receptor (ER) with antiestrogens and aromatase inhibitors is effective in the treatment of breast cancer. Why ER plays such a dominant role in breast cancer and represents such an excellent target remains to be defined. The ability of ER to respond to multiple inputs and to control expression of multiple downstream genes may be one of the reasons why ER is such a powerful target for breast cancer treatment. The recent modest performance of a number of targeted therapies in breast cancer has raised the question whether we will ever develop therapies that have such success as antiestrogens. Targeted therapies tend to inhibit a single pathway that is probably altered in only a subset of patients. Even within this subset, only a limited number of patients respond. The evidence that virtually all pathways can cross-talk and that they exhibit several layers of redundancy reveals a complexity of signaling networks that may defy the generation of targeted therapies with efficacy similar to antiestrogens. However, there are clearly regulatory nodes that can integrate multiple upstream inputs and elicit diverse downstream outputs. We provide evidence and rationales for integrins, insulin receptor substrates (IRSs), and cyclin D1 as potential therapeutic targets. These proteins, similar to ER, can integrate and coordinate multiple signals in breast cancer cells and thus mediate diverse aspects of breast cancer progression. New treatment targets will emerge in light of more global models of signal transduction that fully integrate all aspects of cell biology such as the role of the extracellular matrix and will hopefully result in the development of targeted therapies that show efficacy similar to antiestrogens.

Introduction: Triple-negative breast cancer (TNBC) is a highly diverse group of cancers, and may benefit from molecular-targeted therapies. This study aims to stratify prognosis of TNBC patients using pre-treatment 18F-FDG PET/CT, alone and with correlation to immunohistochemistry biomarkers. Method: 200 consecutive TNBC breast cancer patients treated between 2008 and 2012 who received lumpectomy or mastectomy as primary treatment were retrieved. Among the full cohort, 79 patients had pre-treatment 18F FDG PET/CT scans. Immunostaining status (percentage and intensity) of basal biomarkers (EGFR, CK5/6), Ki-67, P53, and other clinicopathological variables (age, tumor size, pathological T/N stage, nuclear grade, and lymph node metastasis) were obtained. Three PET image features were evaluated: maximum uptake values (SUVmax), mean uptake (SUVmean) and target volume (SUVvol) defined by SUV>2.5. The relationships among tumor metabolic activities and clinicopathological factors were evaluated. All variables were analyzed versus disease-free survival (DFS) using univariate and multivariate Cox analysis, Kaplan-Meier curves and log-rank tests. The optimal cutoff points of variables were estimated using time-dependent survival receiver operating characteristic (ROC) analysis. Results: All PET features significantly correlated with proliferation marker Ki-67 (all p3.5, AUC=0.654, p=0.006). Basal biomarkers EGFR and CK5/6 and image features SUVmax, SUVmean, SUVvol were significant associated with DFS in univariate Cox analysis, whereas SUVmax (p=0.001) and EGFR (p=0.001) were also significant in multivariate Cox analysis. To integrate prognosis of biological and imaging markers, patients were first stratified by EGFR into low (≤15%) and high (>15%) risk groups. Further, SUVmax was used as a variable to stratify the two EGFR groups. In the high EGFR group, patients with high FDG uptake (SUVmax>3.5) had worse survival outcome (median DFS=7.6 months) than those patients with low FDG uptake (SUVmax≤3.5, median DFS=11.6 months). In the low EGFR group, high SUVmax also indicated worse survival outcome (17.2 months) than low SUVmax (22.8 months). The risk stratification with integrative EGFR and PET was statistically significant with log-rank p< Multivariate Cox analysis for disease-free survivalVariablesHR (95% CI)p-valuePathology, T stage, ≤ 3 vs >32.337(0.428-7.384)0.148EGFR, ≤15% vs > 15%9.109(1.997-41.55)0.004CK5/6, ≤ 50% vs > 50%1.471(0.598-3.614)0.401SUVmax, ≤3.5 vs > 3.53.883(1.13-13.32)0.031 TNBC patient risk groups stratified by EGFR and SUVmax (with the median values of variables)Risk groups (EGFR>15, SUVmax>3.5)patient#DFS monthsEGFR %SUVmaxSUVmeanSUVvolKi-67%1 (-, -)1222.852.00.60.2342 (-, +)1517.258.94.37.2673 (+, -)1311.6502.72.60.9354 (+, +)377.66011.35.210.960 Conclusions: Pre-treatment 18F-FDG PET/CT imaging has significant prognostic value for predicting survival outcome of TNBC patients. Integrated with basal-biomarker EGFR, PET imaging can further stratify patient risks in the pre-treatment stage, and help select appropriate treatment strategies for individual patients. Citation Format: Yue Y, Cui X, Bose S, Audeh W, Zhang X, Fraass B. Stratifying triple-negative breast cancer prognosis using 18F-FDG-PET/CT imaging. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-01-03.

[Purpose] Luminal B breast cancer patients are significantly at the risk of relapse with in 5 years. Acquired resistance to the receptor tyrosine kinase HER2 targeting antibody trastuzumab is a major clinical problem in the treatment of recurrent HER2-positive breast cancer. The molecular mechanisms leading to trastuzumab resistance (TZR) remain unclear in luminal B breast cancer. Here, we investigated the mechanism of TZR in HER2-positive luminal B breast cancer cells with acquired resistance. [Methods and Results] We generated trastuzumab resistant breast cancer cell line (BT-474-R) by treating BT-474 cells with trastuzumab for over long-term. Initial validation showed that HER2 overexpression in the BT-474-R cells remained unaltered suggesting that probably HER2 independent signaling pathway is involved in the development of TZR. We demonstrated that NF-κB is constitutively activated in the BT-474-R cells by quantitative realtime RT-PCR, western blotting and immunofluorescent microscopy. Expression of pro-inflammatory cytokines were also demonstrated. Pharmacologic inhibition of NF-κB improved sensitivity to trastuzumab in BT-474-R cells. We identified both IKK-α and IKK-β as the major determinants of the activation of the NF-κB signaling in the resistant cells. [Conclusion] Our study suggests by activating NF-κB pathway, luminal B cells may facilitate acquiring basal-like, Her2+ phenotype a notion consistent with “progression through grade” hypothesis. Finally, our study suggests that blocking NF-κB will provide a new treatment option to limit relapse in HER2 positive breast cancer patients treated with trastuzumab. Citation Format: Hirotaka Kanzaki, Nishit Makhopadhya, Xiaojiang Cui, Krishnan V. Ramanujan, Tatsuaki Takeda, Yoshihisa Kitamura, Toshiaki Sendo, Ramachandran Murali. Constitutive NF-kB-activation loop enhances resistance to trastuzumab in HER2 positive luminal B breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 726. doi:10.1158/1538-7445.AM2015-726

HER2-positive breast cancers represent almost a quarter of invasive breast cancers and are indicative of poor patient survival. Although many patients with HER2-positive breast cancer initially respond to anti-HER2 treatments, such as trastuzumab, a significant portion of them develop resistance to these therapies. Consequently, there is a great need to develop new drugs that are effective against these HER2+ tumors that are non-responsive or have become resistant to these therapies. Recently, it has been shown that another member of the HER family, HER3, is commonly upregulated in these drug-resistant cancers. This observation led us to develop a novel drug delivery protein, called HerPBK10, that specifically targets another member of the HER receptor family, HER3. HerPBK10, once it has bound to the HER3 receptor, triggers rapid endocytosis and endosomal penetration, enabling it to deliver a toxic payload to the cell, resulting in cell death. We hypothesized that cytotoxic drugs delivered by HerPBK10 would induce significant targeted cell death in trastuzumab-resistant HER2+ breast cancers due to the high levels of surface HER3 and would therefore provide an effective treatment for patients who have developed resistance to traditional therapies. First, we verified that cell surface levels of HER3 are elevated in trastuzumab-resistant cell lines compared to trastuzumab-sensitive cells. We then demonstrated, through competitive inhibition with free HER3 ligand, that HerPBK10 binds specifically to HER3 on multiple HER2+ cell lines. We assembled our targeted molecule, HerPBK10 with the chemotherapeutic doxorubicin. The resulting nanoparticle, called HerDox, was used to treat HER2+ breast cancer cell lines that were either inherently resistant to trastuzumab or had acquired resistance to trastuzumab. We demonstrated that HerDox caused significant cell death in both types of resistant cells. We also compared the effect of the HerDox nanoparticle to trastuzumab, pertuzumab, and the two drugs together and showed that it caused superior cell death in all three instances. In addition, we combined our nanoparticle with trastuzumab, and showed that together, they have an additive effect on cell death. These results indicate that our HER3 targeting nanoparticle, HerDox, efficiently targets and kills cancer cells that have become resistant to trastuzumab, and has the potential to be used either as a single drug or as part of a combinatorial therapy in eliminating drug-resistant HER2+ breast cancers. We are in the process of verifying these findings in vivo in order to demonstrate the potential of HerDox as a treatment for patients who have become non-responsive to traditional anti-HER2 therapies. Citation Format: Jessica Sims, Michael Taguaim, Chris Hanson, Xiaojiang Cui, Lali K. Medina-Kauwe. Targeting trastuzumab-resistant HER2+ breast cancer with a HER3-targeting nanoparticle. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4487. doi:10.1158/1538-7445.AM2014-4487

Basal-like breast cancer (BLBC), an aggressive subtype of breast cancer, are usually associated with high histologic grade, high recurrence rate, short recurrence-free survival, younger patient age, poor outcome, and a propensity to metastasize to the brain and lung. So far, developing targeted therapies against BLBC still remains a great challenge. Uncovering the underlying molecular events and identify therapeutic target becomes highly urgent. Forkhead Box C1 (FOXC1), a biomarker for BLBC, is associated with poor prognosis in patients with BLBC. Here, we found that FOXC1 increases cancer stem cells (CSCs) properties in BLBC cells in vitro and in vivo. FOXC1 activates SMO-independent non-canonical Hedgehog (Hh) signaling pathway, which mediates the effects of FOXC1 on CSCs properties in BLBC cells. Further study showed that the N-terminal of FOXC1 bind directly to the Gli2 transcription factor, which has the highest transcriptional activity in Hh signaling pathway. Here we also found that the elevated expression of FOXC1 induces anti-Hh pathway drug resistance. All of these data demonstrate a novel mechanism underlying the regulation of CSC properties and the poor prognosis of BLBC patients, and may provide new insight into anti-CSC therapy resistance. Thus, our study provides a strong rationale for developing FOXC1-targeted therapy for treating BLBC patients. Citation Format: Bingchen Han, Yanli Jin, Shikha Bose, Xiao Zhang, Ying Qu, Beth Y. Karlan, Armando E. Giuliano, Xiaojiang Cui. FOXC1 regulates cancer stem cells properties via inducing SMO-independent Gli activation and confers anti-Hedgehog drug resistance in basal-like breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1906. doi:10.1158/1538-7445.AM2014-1906

It is now known that tumors are complex ‘organs’ consisting of multiple malignant and nonmalignant cells, blood vessels, and many other cell types, which are frequently lumped together as tumor stroma because their individual characteristics and roles in tumor progression and metastasis are not well defined. Despite the universal presence of cancer-associated stroma in solid tumors and wide clinical use as a prognostic marker, the origin of stromal cells in tumors is still the subject of extensive debate. The dynamic bi-directional interaction between the cancer cells and stroma is also not well understood although it is becoming increasingly apparent that the stroma can modify the aggressiveness of tumor cells and that tumor cells re-program the stroma to generate a nurturing microenvironment that is crucial for tumor survival, progression, and metastasis. Because of the prominent role of stroma in most aspects of tumor progression, it is believed that rational anticancer therapy design should not only target the cancer cells but also the stroma. Therefore, a better understanding of the cancer-stroma interaction and the identification of molecular events whose disruption may undermine tumor progression are required steps toward successful anticancer therapy. Most public expression profile databases contain information from tumors with variable amounts of stroma. Thus, a snapshot of cross-signaling between cancer and stroma is captured in such transcription profiles although it is difficult to deconvolve the source and direction of the signals. The intensities of the signals are also difficult to determine because analysis of total mRNA from tissues dampens signals from rare cell types, such as cancer stem cells and activated fibroblasts. Expression profile databases that contain laser capture-microdissected samples or single-cell samples provide more information about specific cell types but typically suffer from a small number of patient samples. Through integration of multiple expression profile databases, we identified a set of highly co-regulated genes that are associated with poor patient survival. These genes are induced as a result of the epithelial-stromal interaction and define a microenvironment that serves as a favorable niche for ovarian cancer stem cells, thereby leading to chemotherapy resistance and metastasis and, ultimately, poor overall survival. Citation Format: Dong-Joo Cheon, Jessica Beach, Yunguang Tong, Xiaojiang Cui, Mourad Tighiouart, Zhenqiu Liu, Ann E. Walts, Beth Y. Karlan, Sandra Orsulic. Using publicly available databases for the functional characterization of cancer genes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA11.

Basal-like breast cancer (BLBC) is an aggressive malignancy that is associated with high histological grade, aggressive clinical behavior, a high rate of metastasis to the lung and brain, and poor prognosis. Currently, chemotherapy is the only systemic therapy for BLBC, which underexpresses estrogen receptor (ER), progesterone receptor (PR), and HER2. To date, little is known about the molecular basis of BLBC. We and others recently reported that the forkhead box C1 (FOXC1) transcription factor is a critical maker for BLBC and predicts poor clinical outcome in human breast cancer. Overexpression of FOXC1 increases BLBC cell growth, migration, invasion, epithelial-mesenchymal transition, and chemoresistance. However, how its expression is induced exclusively in BLBC is not understood. Previous studies have shown that EGFR is overexpressed in BLBC and is associated with poor prognosis in breast cancer patients. We thus hypothesize that activated EGFR signaling may regulate FOXC1 expression. In this study, we show that EGF treatment upregulates FOXC1 expression in BLBC cells at the transcription level through MEKK1/ERK and PI3K/AKT pathways. AKT3, which was recently found to be enriched in ER/PR/HER2 triple-negative breast cancer, is a potent activator of FOXC1 transcription. Overexpression of EGFRvIII, a truncated constitutively active form of EGFR that does not bind to the ligand, also induced FOXC1 expression. Pharmacologic inhibition of EGFR suppressed FOXC1 expression in BLBC cells. Immunohistochemistry and microarray analysis demonstrated that FOXC1 expression was positively, significantly correlated with EGFR expression in human BLBC tumors at protein and RNA levels. Moreover, we identified the nuclear factor κB (NF-κB) transcription factor as a pivotal regulator of EGF-induced FOXC1 expression, downstream of AKT and ERK. NF-κB directly activates FOXC1 transcription through binding to the FOXC1 promoter. Deletion or mutation of the NF-κB binding sites in the FOXC1 promoter abolished the EGF induction of FOXC1 expression. Knockdown of FOXC1 levels in BLBC cells by RNA interference markedly attenuated EGF-elicited cell proliferation, migration and invasion, suggesting that FOXC1 mediates the effects of EGF on BLBC cell functions. Taken together, our findings uncovered a novel mechanism of FOXC1 regulation and shed light on the role of EGFR-NF-κB-FOXC1 signaling in BLBC pathogenesis. Intervention of this signaling pathway would provide potential modalities for BLBC treatment. FOXC1 levels may serve as a readout of EGFR activity and a marker for selecting breast cancer patients who may benefit from anti-EGFR therapy. Citation Format: Yanli Jin, Bingchen Han, Wolf Ruprecht Wiedemeyer, Sandra Orsulic, Beth Karlan, Armando Giuliano, Xiaojiang Cui. FOXC1 is a critical mediator of EGFR function in 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 5443. doi:10.1158/1538-7445.AM2013-5443

Despite the clinical and histopathological similarities in initial disease presentation, patients with advanced ovarian cancer rapidly develop chemoresistance and succumb to the disease. In order to identify genes that are associated with poor patient survival, we analyzed microarray datasets of 795 advanced ovarian cancer patients and correlated gene expression levels with clinical data. We identified a 60-gene signature that was strongly associated with poor survival. Many genes in the signature were stromal genes, including POSTN, FN1, COL11A1, COL6A1, COL3A1, LOX, TIMP3, and VCAN. The same stromal signature was enriched in ovarian cancer metastases and in a cisplatin-resistant A2780 human ovarian cancer cell line. Expression of many stromal genes was induced by TGFβ1 and the effect was reversed by A83-01, a potent TGFβ1 inhibitor. Interestingly, we found that ovarian cancer stem cells (CSC) also express TGFβR2 and several TGFβ1-target genes, including COL6A1 and COL3A1, which were previously implicated in cisplatin resistance. Culturing of A2780 cells on collagen substrate resulted in increased cisplatin resistance and expansion of the CSC population. Our results suggest that TGFβ1 signaling induces stromal gene expression and forms a favorable microenvironment for ovarian CSC, thereby leading to cisplatin resistance and poor patient survival. Citation Format: Dong-Joo Cheon, Yunguang Tong, Myung-Shin Sim, Xiaojiang Cui, Dror Berel, Mourad Tighiouart, Wolf Ruprecht Wiedemeyer, Beth Karlan, Sandra Orsulic. Stromal gene signature regulated by TGFβ signaling predicts poor clinical outcome in ovarian 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 1511. doi:10.1158/1538-7445.AM2013-1511

The human epidermal growth factor receptor subunit (HER2), or ErbB2, is receptor tyrosine kinase that is amplified in approximately 20-25% of invasive breast cancers. Anti-HER2 therapies such as trastuzumab (Herceptin®) have become important in the management of aggressive and metastatic breast cancer. Although many patients with HER2-positive breast cancer initially respond to anti-HER2 treatments, such as trastuzumab, a significant portion of them develop resistance to these therapies. Consequently, there is a great need to develop therapies that will treat these tumors once they become resistant. We have developed a unique drug delivery protein (HerPBK10) that specifically targets the cell surface receptor HER3, which has been shown to be elevated in trastuzumab-resistant HER2+ breast cancers. HerPBK10, once it has bound to the HER3 receptor subunit, triggers rapid endocytosis and endosomal penetration, enabling it to deliver a toxic payload to the cell, resulting in cell death. We hypothesized that cytotoxic drugs delivered by HerPBK10 would induce significant targeted cell death in trastuzumab-resistant Her2+ breast cancers and will provide an effective treatment for patients who have developed these resistant tumors. We have demonstrated that HerPBK10 binds specifically to HER3 in vitro and binds to the cell surface of three different breast cancer cell lines. Importantly, this binding can be competitively inhibited by free HER3 ligand, indicating that HerPBK10 binds specifically to HER3. We also examined whether cells that have become resistant to trastuzumab have altered levels of HER receptors on their surface when compared to parental, trastuzumab-responsive, cancer cells. We demonstrated in multiple trastuzumab-resistant cell lines that HER3 receptor levels are significantly increased. We then assembled HerPBK10 with a cytotoxic gallium corrole, which we have previously demonstrated to be toxic once taken up into a cell. The resulting nanoparticle, called HerGa, was used to treat three different aggressive breast cancer cell lines (one that is susceptible to trastuzumab treatment and two that have become resistant to trastuzumab) and demonstrated that HerGa caused cell death in all three cell lines, but at a greater level and at a lower dosage in the resistant cell lines. We also compared the effect of the HerGa nanoparticle to trastuzumab and showed that it caused greater overall cell death. Together, these results indicate that our HER3-targeting nanoparticle, HerGa, efficiently targets and kills cancer cells that have become resistant to trastuzumab, and warrants further in vivo testing to investigate its potential as a treatment for patients who have become non-responsive to traditional anti-HER2 therapies. Citation Format: Jessica D. Sims, Michael Taguaim, Chris Hanson, Xiaojiang Cui, Lali K. Medina-Kauwe. Treating trastuzumab-resistant HER2+ breast cancers with a HER3-targeted nanoparticle. [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 A101.

The Forkhead-box transcription factor FOXC1 has recently been identified as a critical marker for human basal-like breast cancer, which lacks or under-expresses estrogen receptor-α (ERα). Here we show a consistent inverse correlation between FOXC1 expression and ERα expression in multiple cDNA microarray data sets of human breast cancer. Overexpression of FOXC1 in ERα-positive breast cancer cells induces cell growth, migration, and invasion, but downregulates ERα mRNA and protein levels and consequently reduces cellular responses to estradiol and tamoxifen. This could explain why tamoxifen-refractory breast cancer cells exhibit higher FOXC1 levels as compared with tamoxifen-sensitive parental cells. We also found that FOXC1 induces NF-κB activation, which is known to be associated with ER-negative breast cancer, by increasing Pin1-mediated p65 protein stability and thus p65 protein levels. NF-κB mediates, at least in part, the regulation of ERα by FOXC1, because inhibition of NF-κB by an IκBα super-repressor and small-molecule inhibitors attenuated the suppression of ERα expression by FOXC1. Furthermore, the importance of the NF-κB pathway in FOXC1-stimulated cell growth was demonstrated by increased cell sensitivity to pharmacologic inhibition of NF-κB. Taken together, these results reveal a novel ERα-regulating mechanism that may explain the loss or low expression of ERα in basal-like breast cancer and also may provide new insight into mechanisms for antiestrogen resistance in breast cancer. 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 3138. doi:10.1158/1538-7445.AM2011-3138

Cancer cells usually generate and maintain higher levels of reactive oxygen species (ROS) compared to normal cells, in part because of their higher metabolic rate and defects in the electron transport chain. Because most tumor cells possess stronger antioxidative defense mechanisms to counterbalance excessive ROS, redox proteins such as thioredoxins (TRXs) and glutaredoxins (GRXs) are potential targets of anticancer therapy. We recently found that a novel redox protein called thioredoxin-like 2 (TXNL2) is required for embryonic stem cell functions and is consistently overexpressed in many different cancers. It has a TRX-homology domain and two GRX-homology domains, suggesting that it may have redox function. Using immunohistochemical and cDNA microarray analyses, we found that levels of TXNL2 were significantly higher (P = 0.00036) in human breast cancers than in normal breast tissues; levels also were higher in human breast cancer cell lines than in normal mammary epithelial cell lines. Knockdown of TXNL2 by shRNA in breast cancer cell lines induced a 5-fold increase of ROS levels and inhibited cell proliferation, survival, invasion, and the expression of genes critically involved in these cellular processes. Breast cancer stem-like cell populations and stem-like properties such as mammosphere formation were reduced by TXNL2 silencing and induced by TXNL2 overexpression. Surprisingly, TXNL2 depletion in normal mammary epithelial cells did not significantly affect ROS levels and cell survival. Xenograft models of orthotopic injection indicated that TXNL2 knockdown inhibited mammary tumor growth and metastasis to the lung. Cell signaling studies showed that TXNL2 knockdown inhibited GSH synthase (GSS) expression, and thereby decreased GSH levels and increased ROS levels; N-acetylcysteine (NAC), a GSH precursor, partially reversed these effects. In addition, TXNL2 knockdown suppressed NF-κB activity by inactivating IKK and p65 via glutathionylation, whereas TXNL2 overexpression markedly increased NF-κB activity. Blockade of NF-κB using its small-molecule inhibitors and IκB overexpression mimicked the effect of TXNL2 depletion. Our results suggest that TXNL2 is a potentially promising target for ROS-based anticancer therapies and is a key regulator of the NF-κB pathway. Because high cellular antioxidant capacity is well-known to impair cellular responses to chemotherapy and radiation therapy, our study may also open up a new avenue of maximize the efficacy of conventional anticancer therapies by targeting TXNL2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1794.

Although basal-like breast cancer (BLBC) is a distinct molecular subtype, it has no defining gene or protein marker that can be used as the basis for therapy. Estrogen receptor (ER) and HER2 receptor guide treatment of luminal and HER2 breast cancer subtypes, respectively, but chemotherapy is still the only systemic treatment available for BLBC. In view of its poor clinical outcomes, high proliferation rates and preferential metastasis to the brain, there is an urgent need for effective targeted therapy of BLBC. In a previous study using cDNA microarray analyses and immunohistochemistry of archival breast cancer tissue specimens, we showed consistent and exclusive overexpression of FOXC1 in BLBC versus other molecular subtypes. FOXC1 overexpression was significantly associated with poor overall survival and brain metastasis. In the present study, we show that overexpression of FOXC1 in breast cancer cells with low endogenous FOXC1 levels increased cellular proliferation, migration, and invasion, and induced epithelial-mesenchymal transition. Knockdown of FOXC1 by shRNA in breast cancer cells with high endogenous FOXC1 expression had the opposite effect. Cell signaling studies showed that FOXC1 activated the NF-κB pathway by upregulating the peptidyl-prolyl isomerase Pin1 and downregulating the ubiquitin ligase SOCS-1. Pin1 binds to p65, inhibits the p65 interaction with IκBα, and thus enhances p65 nuclear localization and protein stability. SOCS-1 directly interacts with p65 and promotes its degradation. FOXC1 knockdown reduced p65 protein levels and NF-κB activity. The importance of the NF-κB pathway for FOXC1 function was confirmed by the increased sensitivity to NF-κB inhibitors in cells that overexpressed FOXC1. Because BLBC under-expresses ER, FOXC1 was inversely associated with ER, as expected. Interestingly, FOXC1 repressed ER transcription and activity via NF-κB signaling, and FOXC1 overexpression switched MCF-7 breast cancer cell growth from estrogen-dependent to estrogen-independent. This may explain why ER is not normally detected in BLBC. These results suggest that FOXC1 may be a specific diagnostic and prognostic biomarker for BLBC and may play an important role in regulating aggressive traits associated with BLBC. It might also serve as a potential molecular therapeutic target for BLBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3742.

Abstract #5075 The c-Jun N-terminal kinase (JNK) is a critical mediator of stress-induced apoptosis and is required for the cytotoxic effect of anticancer therapies. Interestingly, recent clinical studies showed that high JNK activity is associated with poor prognosis in human breast cancer. The molecular basis of this potential dual function of JNK in breast cancer is poorly understood. Here we report that overexpression of a constitutively active JNK in breast cancer cells increased cell migration and invasion. In agreement with this, fibroblast-specific markers like the intermediate filament vimentin and the extracellular matrix protein fibronectin were up-regulated. AP-1 transcription factor, which induces expression of these two proteins, was markedly increased by JNK overexpression. In addition, hyperactive JNK reduced Akt1 activation but enhanced Erk activation in breast cancer cells. In relevance, we also found that JNK levels are significantly higher in invasive breast cancers including those with distant metastases (n = 235, p < 0.0001, Wilcoxon rank sum test) than in normal breast tissues (n = 69). Most surprisingly, an increase of JNK attenuated the apoptosis of breast cancer cells treated with chemotherapy drugs doxorubicin and taxol. This suggests that the role of JNK changes when its activity/expression increases above the basal levels associated with apoptosis. Our data, together with a recent finding that cells undergoing cytotoxic agent-induced apoptosis can induce compensatory hyper-proliferation of proximal cancer cells, suggest a novel mechanism of breast cancer therapeutic resistance. Therapy-elicited apoptosis of tumor cells with basal JNK activity may release mitogens that induce hyperactive JNK in neighboring cells to promote growth and invasion. Thus JNK activation may serve as a marker of breast cancer progression. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5075.