Abstract SY20-02: Insights into mechanisms of breast cancer metastasis
Authors: Hynes Nancy E. 
Published: May 2015

Breast carcinoma is the most frequent cancer in women, affecting one million yearly. In the past decades there have been great improvements in surgery, screening and therapy. These have led to dramatic changes in the 5-year overall survival rates, which have gone from 40% in the 1950's up to 86% in the past decade (Maxmen 2012). However, many patients who present with early stage disease die from metastases, despite complete surgical removal of primary breast tumors. Indeed, if a tumor has spread to distant organs at the time of presentation, the 5-year overall survival is only 23% compared to 90% or higher for patients with localized tumors. Today, primary tumors are efficiently removed by surgical intervention, but there are essentially no successful therapeutic options for metastatic disease.

Metastasis is a complex process whereby tumor cells detach from the primary site, acquire migratory properties, enter and survive in the circulation, and colonize and grow at distant sites (Gupta and Massague 2006). Metastatic relapse can occur years after surgery, suggesting that tumor cells have the ability to survive in a dormant state in metastatic niches (Karrison, Ferguson et al. 1999). Typical sites of relapse of breast cancer are the bone, lungs, liver and brain (Weigelt, Peterse et al. 2005; Nguyen, Bos et al. 2009). Thus, the most relevant clinical and experimental questions in the breast cancer field revolve around metastasis. Important research areas are: the identification of specific molecules and pathways contributing to metastatic spread, tumor cell survival and growth in distant sites, as well as uncovering metastatic therapies. My presentation will cover our recent work on: 1-targeting receptor tyrosine kinases (RTKs) in metastatic breast cancer models; 2- Memo, a novel enzyme that affects intracellular ROS production, and is essential for in vivo metastasis; 3- analyses aimed at uncovering molecular mechanisms involved in crosstalk between tumor cells and the bone marrow (BM) environment.

1-Targeting RTKs with antibodies or small molecular inhibitors is a clinically validated approach for cancer therapy. In breast cancer, the ErbB2/HER2 specific antibody trastuzumab is routinely given in combination with chemotherapy to ErbB2/HER2-positive breast cancer patients and has had a significant impact on patient mortality (Gianni, Dafni et al., 2011). Only a sub-set of patients, however, are eligible for this treatment, making it essential to uncover additional RTKs that could be useful in for therapy; the Ret kinase might be an interesting new target. Ret was discovered as an outlier kinase in breast cancer, with unexpectedly high expression levels detected in many breast tumors (Esseghir, Todd et al. 2007; Boulay, Breuleux et al. 2008; Kothari, Wei et al. 2013). Unlike thyroid or lung tumors that carry oncogenic Ret, as fusion proteins or with activating mutations, Ret appears to be wild type in breast cancer. We have shown that elevated Ret levels are found in different sub-types of human breast cancers and that high Ret correlates with decreased metastasis-free survival. Using a metastatic breast cancer model, we showed that in vivo blockade of Ret inhibits tumor outgrowth and metastatic potential (Gattelli, Nalvarte et al. 2013). Our results suggest that Ret has an important role in tumor growth and metastasis.

Multiple RTKs are often active in cancer cells and combining RTK inhibitors has been shown to have strong anti-tumor activity (e.g., Stommel, Kimmelman et al. 2007). Considering that breast tumors often co-express ErbB and FGFRs (Cancer Genome Atlas Network, 2012), we have tested the impact of blocking both in metastatic breast cancer models. We have recently shown that only when both RTKs are blocked do we observe: blockade of the ERK and PI3K pathways, prolonged tumor-stasis and a significant decrease in metastasis (Dey, Bianchi et al. 2010; Issa, Gill et al. 2013).

2-Our group originally identified Memo as being essential for breast cancer cell motility in response to several RTKs (Marone, Hess et al. 2004; Meira, Masson et al. 2009). The 2.1 Å crystal structure of Memo revealed its structural homology with bacterial enzymes (Qiu, Lienhard et al. 2008); we have recently discovered that Memo is a metal-binding enzyme that requires Cu(II) for its oxidase-like activity. Memo knock-down (KD) in breast cancer models affects localized intracellular ROS production, cell migration and invasion, as well as in vivo spontaneous lung metastasis from xenografts. To investigate if Memo plays a role in human breast disease, immunohistochemistry (IHC) was carried out on breast cancer tissue microarrays (TMAs). Memo expression is low in non-neoplastic breast tissue and exhibits significantly higher expression in &gt; 40% of tumors. Memo is diffusely localized to the cytoplasm and the nucleus and accumulates at the membrane upon growth factor stimulation (Schlatter, Meira et al. 2012). To examine if the sub-cellular localization of Memo correlates with clinical or histopathological parameters, we categorized the breast tumor samples according to expression and localization of Memo. We found that elevated Memo was prognostic of poor outcome; elevated levels of cytoplasmic Memo, but not nuclear Memo, significantly predicted early metastasis and death at 5 years in a multivariable Cox model. These data suggest that Memo has distinct roles in different cellular compartments and that its cytoplasmic function regulates pathways that promote tumor dissemination.

3- Bone marrow is particularly permissive for disseminated tumor cells since structural and environmental features needed to allow trafficking of hematopoietic stem cells and their progeny can also be employed by the tumor cells. The fenestration of bone sinusoidal capillaries facilitates the entrance of tumor cells. Moreover, bone stroma is a potent source of CXCL12 that attracts tumor cells expressing its receptor CXCR4, making BM a perfect soil for circulating tumor cells (Morrison and Spradling 2008). Metastatic tumors show different molecular features compared to primary tumors, which could reflect tumor-cell selection for particular conditions in the distant site and/or the influence of the surrounding stroma on a disseminated tumor cells (Kang, Siegel et al. 2003; Minn, Gupta et al. 2005). The BM niche is a complex, multicellular structure composed of osteoblasts, osteoclasts, stromal fibroblasts, endothelial cells, and mesenchymal progenitors, all of which contribute to general bone homeostasis (Mendez-Ferrer, Michurina et al. 2010; Ehninger and Trumpp 2011). Considering the strong interplay between these elements, it has been suggested that blocking the crosstalk between tumor and the bone environment would be a potential strategy to inhibit tumor expansion. To uncover molecular mechanisms involved in the crosstalk between tumor cells and the BM environment, we compared the transcriptome of different cell populations from the BM environment from tumor-bearing and tumor-free mice. For this, we used purified stromal cell subsets of: endothelial and osteoblastic cells, as well as mesenchymal and osteogenic progenitors. These analyses revealed that BM is strongly affected by breast cancer cell dissemination. Indeed, several components of key molecular pathways involved in tumor development, including TGFBR, PDGFRB, EGFR, HGFR, IGFR and JAK/Stat, are modulated in the BM of mice with tumor cells.

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Citation Format: Nancy E. Hynes. Insights into mechanisms of breast cancer metastasis. [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 SY20-02. doi:10.1158/1538-7445.AM2014-SY20-02