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At late stage, nearly all breast cancers are heterogeneous and refractory to treatment, like metaplastic breast cancer is at an early stage. These rare carcinomas are highly aggressive and de-differentiated. They are enriched for mesenchymal and stem cell features and essentially fail current therapies. As metaplastic tumors provide a time-compressed picture of breast cancer progression early on, understanding these tumors will yield insight into mechanisms that drive breast cancer into advanced stages and treatment resistance. To investigate a genetic basis for heterogeneity in metaplastic breast cancer, we established a progression model comprising three cell lines. The cell lines were derived from a primary tumor, a local recurrence and a pleural effusion of a 40-year old patient. The primary tumor was a stage III invasive metaplastic, triple negative, inflammatory breast cancer, resected after neoadjuvant chemotherapy (capecitabine and taxotere, then adriamycin and one cycle of bevacizumab). The local recurrence, biopsied seven months post mastectomy, developed after the patient received adjuvant carboplatin and gemcitabine for 3 cycles and then radiation to the chest wall. At this time, the patient had lung metastases and was treated with taxol and bevacizumab yielding a mixed response. Local invasive growth continued and a malignant pleural effusion developed four months later. Analyzing the genetic and molecular characteristics of this progression model in vitro, its tumorigenicity and metastasis in vivo, and interrogating lead findings in a growing collection of metaplastic tumors helps us to dissect the genetic heterogeneity in breast cancer, and potentially to identify the cell types that drive disease progression and treatment resistance. Our gene expression analyses and genomic evaluations identified epithelial to mesenchymal transition (EMT) as a key characteristic in the progression and treatment resistance of this cancer. Major changes in cytoskeletal genes, chemokines and their receptors, amplification of drug transporter proteins, metalloproteinases and matrix proteins seen with increasing motility and invasiveness along with recruitment of host inflammatory responses in the in vivo model, loss of chromosomal regions harboring known and putative tumor suppressors, and deletions of genes encoding proteins for metabolic inactivation of sex hormones in the breast tissue, along with specific loss of clusters of desmosomal genes are guiding our understanding of metaplastic breast cancer progression. The results provide insight into the development, the extremely invasive nature, and treatment resistance of these tumors. Our collaborative network of clinicians, pathologists, translational genomic researchers and bioinformatics specialists will enable us to identify and prioritize genetic events as disease drivers, prognostic biomarkers of disease progression, and determinants of treatment resistance. Our goal is to identify molecular and functional targets for effective therapy and evaluate them in the clinic. Lessons learned from metaplastic breast cancer will improve our understanding of breast cancer progression in general, and could translate into effective treatments for advanced breast cancer where current standard of care is failing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD03-07.

A large number of somatic mutations accumulate during the process of tumorigenesis. A subset of these mutations contribute to tumor progression (known as “driver” mutations) whereas the majority of these mutations are effectively neutral (known as “passenger” mutations). The ability to differentiate between drivers and passengers will be critical to the success of upcoming large-scale cancer DNA resequencing projects. Here we show a method capable of discriminating between drivers and passengers in the most frequently cancer-associated protein family, protein kinases. We apply this method to multiple cancer data sets, validating its accuracy by showing that it is capable of identifying known drivers, has excellent agreement with previous statistical estimates of the frequency of drivers, and provides strong evidence that predicted drivers are under positive selection by various sequence and structural analyses. Furthermore, we identify particular positions in protein kinases that seem to play a role in oncogenesis. Finally, we provide a ranked list of candidate driver mutations. [Cancer Res 2008;68(6):1675–82]

The shift from a normal to high glycolytic state (Warburg effect) is a central hallmark of almost every cancer and a strong driving force for cancer cell survival and disease progression. Importantly, studies - including our own - have shown that normalization of metabolic state in cancer cells can halt or reverse malignancy. To date, the intrinsic and extrinsic factors that drive metabolic shifts in cancer cells are not entirely clear. A better understanding of how the metabolic state of cancer cells is regulated will give us important tools to inhibit disease progression. We have established a novel model of breast cancer progression consisting of three cancer cell lines established from the primary tumor (TES1) and two sites of metastasis (TES2b and TESPE) of the same patient. We compared gene expression profiles between primary and recurrent tumor cells to identify factors that regulate disease progression and dissemination. Key differences between cells derived from primary and recurrent disease were an activation of genes involved in glycolysis and genes involved in activation and maintenance of the epithelial to mesenchymal transition (EMT) program, a powerful driver of cancer metastasis. Our analyses on a functional relationship between EMT and activation of glycolysis uncovered a direct link between the malignancy-driving EMT mechanism in breast cancer progression and metabolic reprogramming of the tumor cells. Specifically, we identified Twist1, a pro-metastatic master regulator of EMT as a direct modulator of a key glycolytic enzyme known to modulate the rate of glycolysis, 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3 (PFKFB3). We found that Twist1 binds directly to the promoter region of PFKFB3 and induces expression of the enzyme. Additionally, inhibition of Twist1 expression in TES2b (TES2b-shTwist1) cells decreased glycolytic flux, while Twist1 overexpression in HMLE immortalized human breast epithelial cells (HMLE-Twist1) increased glycolytic activity and simultaneously decreased cellular respiration. Metabolomics analyses of HMLE vs. HMLE-Twist1 cells using Gas Chromatography Mass Spectroscopy (GCMS) indicated that Twist1 expression is sufficient to induce drastic changes in the overall metabolic state of the cells, including glutamine metabolism and availability of TCA cycle intermediates and amino acids. Together, our findings are significant as they underscore the importance and potency of EMT activation in cancer and uncover a novel mechanism by which EMT activation through Twist1 drives metabolic alterations that fuel cancer progression and metastasis. Results from this study will ultimately aid in the discovery and development of new paradigms to fight cancer mortality rates around the world. Citation Format: Esmeralda Casas, Antonio F. Santidrian, Mihaela Lorger, Ali Torkamani, Boris Ratnikov, Sheena M. Saayman, Julie B. Steele, Smith W. Jeffrey, Brunhilde Felding. Twist1 modifies the metabolic state of breast cancer cells through transcriptional activation of a key regulator of glycolysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 57.

CXCL14 (BRAK) is a cytokine implicated in inflammatory responses, but its contribution to cancer is largely unknown. Recent studies in clinical breast cancer indicate an association between CXCL14 expression and shortened time to metastatic progression, as well as the presence of CXCL14 in a subset of breast epithelial cells with a stem-like phenotype. Using global gene expression profiling, we identified CXCL14 as the most prominently upregulated cytokine in a novel progression model of triple-negative inflammatory and metaplastic breast cancer that we generated. The model comprises tumor cells established from a locally aggressive primary tumor obtained from a patient at the time of initial surgery (TES-1), and tumor cells from the same site at the time of local failure in the chest wall after chemo and radiation therapy (TES-2b). In contrast to TES-1, TES-2b cells are tumorigenic in immuno deficient mice and can be followed and quantified by non-invasive bioluminescence imaging after injecting the F-luc tagged cancer cells into the 4th mammary fat pad. Illumina microarray analysis revealed a 66-fold increase in CXCL14 expression in TES-2b cells over TES-1 cells, ranking it as one of the top 25 genes most differentially expressed among over 25,000 identified gene transcripts. We validated CXCL14 overexpression in TES-2b cells by real time PCR (TaqMan) (64 fold), and found a significant increase in CXCL14 protein secretion in cultured TES-2b cells (3.8 fold) by immuno-capture ELISA. To address a functional role in CXCL14 in the progression of these aggressive breast cancer cells, we stably reduced CXCL14 expression in TES-2b cells by lentiviral transduction with small interfering RNA and confirmed an 80% reduction in gene expression. CXCL14 protein production was concomitantly reduced 2.7 fold. Importantly, xenograft studies revealed a critical role of CXCL14 in the in vivo growth behavior of these breast cancer cells, as the tumor growth rate in the mammary fat pad of CXCL14 knock-down cells was significantly reduced compared to scrambled control vector treated cells. After injecting 5×105 F-luc tagged tumor cells, 4/6 mice had tumors measuring 10 mm in diameter after 11 weeks (requiring sacrifice of the animals) compared with 1/6 in the CXCL14 knock-down group. These initial results indicate a critical role of tumor cell derived CXCL14 in the in vivo survival and proliferation of these breast cancer cells. Our findings provide a basis for further analyses of CXCL14 in breast cancer progression in vivo and for detailed studies of the mechanisms through which CXCL14 may promote disease progression of highly aggressive triple-negative 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 5251. doi:10.1158/1538-7445.AM2011-5251