Proteomics of Tissue Tropism in Breast Cancer mostly

Proteomics of Tissue Tropism in Breast Cancer (mostly) Lauren Barney Peyton Lab March 31, 2014

Peyton Lab ‘Omics’ Approaches • High throughput approaches: higher dimensionality data sets, requires bioinformatics approaches • Often reveals more questions than answers due to heterogeneity across samples and complexity of datasets • Success stories: targeted therapeutics such as Herceptin and gefitinib Vucic et al. , 2012; Sidransky 2002

Peyton Lab Large-Scale Example: Matched Primary and Bone Met from Patient Many proteins identified as up- or down-regulated: • Up-regulated in bone met: – Collagen IV, Cathepsin G, laminin subunits gamma 1 and alpha 4 • Down-regulated in bone met: Dumont et al. , 2012 – Vitronectin, beta 1 integrin, collagen XIV, alpha 2 integrin, alpha 5 integrin

Peyton Lab Proteomics of tropism generally studied on a smaller scale • One to tens of proteins, not whole proteome – Immunohistochemistry staining, western blotting, functional assays (antibodies, si. RNA, overexpression, etc) • Most studies focus on one site of metastasis, so it is still hard to know what is specific to bone, brain, or lung metastasis Many studies use tropic selection in 231 s or 4 T 1 mouse mammary carcinoma cells Kang et al. , 2003

Peyton Lab Tenascin C • Breast cancer cell production of Tenascin C supports lung metastasis, correlates with aggressiveness of tumors TNC in human lung metastasis (brown) Immunohistochemical analysis of TNC expression in lung metastatic foci of various sizes formed by MDA 231 -LM 2 cells in mice. TNC accumulation at the invasive front in larger metastatic foci. Arrows, TNC expression. Scale bar, 50 μm Oskarsson et al. , 2011

Peyton Lab Bone marrow derived cells create a pre-metastatic niche in the lung • Tumor-specific growth factors upregulate fibroblast production of fibronectin to create a permissive niche for lung colonization – Prior to arrival of BMDCs, but at future site of metastatic niche location WT lung Kaplan et al. , 2005 Pre-metastatic lung, day 3 Before arrival of BMDCs Maximal FN expression on day 14

Peyton Lab ECM Proteins in Bone Metastasis • Osteopontin (OPN) and bone sialoprotein (BSP) expression are both present in breast cancer bone metastasis – OPN: promotes invasive behavior of breast cancer cells, recruits macrophages, initiates downstream signaling – BSP: facilitates adhesion, survival, metastasis – Potential “osteomimicry” Ibrahim et al. , 2001 Wai et al. , 2004 Kruger et al. , 2014 Diel et al. , 1999

Peyton Lab Integrins • avb 3 is required for bone metastasis in many types of cancer • Binds vitronectin, fibronectin, osteopontin, bone sialoprotein, others in present bone & bone marrow Liapis et al. , 1996 Mc. Cabe et al. , 2007 Prostate cancer example

Peyton Lab Integrins • a 3 b 1 mediates initial pulmonary arrest of HT 1080 cells (fibrosarcoma) – Binds laminin and thrombospondin • Silencing a 3 integrin expression prevented lung metastasis in 4 T 1 cells – Tail vein injection Wang et al. , 2004 Zhou et al. , 2014

Peyton Lab HER 2 and Brain Metastasis • HER 2 is a prognostic factor for brain metastasis clinically HER 2 – HER 2+ tumors have a predisposition for brain metastasis – Brain may be a “sanctuary” for Herceptin-treated tumor cells Palmieri et al. , 2007 Lin and Winer, 2007

Peyton Lab Metadherin • Cell surface protein involved in angiogenesis – Known oncogene – Mediates lung metastasis in 4 T 1 cells; initially identified via phage display to lung vasculature Brown and Ruoslahti, 2004

Peyton Lab Osteoactivin • Cell surface glycoprotein • Upregulated in bone metastasis • Overexpression promotes bone metastasis in weakly bone metastatic cells Rose et al. , 2007

TGFb is released during bone remodeling, stimulates “vicious cycle” Peyton Lab • TGFb stimulates bone metastatic cell growth • Blocking TGFb signaling prevents PTHr. P secretion by breast cancer cells and prevents bone metastasis Parental 231 -Br 231 -Bo Yoneda et al. , 2001 Yin et al. , 1999

Interleukins • IL-8 in a 231 variant correlates with bone metastasis in mice • Bone tropic cells produce more IL-11 • 231 brain metastatic variants release more IL-8 and VEGF-A Bendre et al. , 2002 Kim et al. , 2004 Kang et al. , 2003 Solid: MDA-231 Peyton Lab Dashed: MDA-MET Metastatic lesion Normal brain

VEGF-C Peyton Lab Lung Lymph node • VEGF-C overexpression increases lymph node and lung metastasis in breast cancer via increased lymphangiogenesis Skobe et al. , 2001

Peyton Lab CXCR 4 • Inhibiting CXCR 4 impairs ability to metastasize to lymph nodes and lung • Important in bone metastasis – Bone homing (RNA quantification) Muller et al. , 2001 Kang et al. , 2003 Liang et al. , 2005

Peyton Lab Proteases • MMP 2, MMP 3, MMP 9 higher in brain metastasis than primary tumor in rat model • Cross-talk with astrocytes increases MMP 2 expression and invasion in vitro • MMP 3 is up-regulated in bone tropic variant of 4 T 1 cells Mendes et al. , 2005 Mendes et al. , 2007 Rose et al. , 2007

Peyton Lab Proteases • ADAMTS 1 and MMP 1 together are required for bone metastasis • Paracrine signaling to modulate bone microenvironment and promote metastasis Lu et al. , 2009 Clinical IDC Tumor

Peyton Lab Serpins & Brain Metastasis • Metastatic cells rarely survive in the brain – Plasmin (serine protease) from the reactive brain stroma is a defense against metastatic invasion – Brain tropic cells express plasminogen activator (PA) inhibitory serpins to facilitate colonization m. RNA quantification, confirmed similar protein expression Valiente et al. , 2014

Peyton Lab Src Activity Necessary for Bone Colonization Mesenchymal signals in primary tumor select for bone metastatic seeds with high Src activity Zhang et al. , 2009 Zhang et al. , 2014

Peyton Lab Where we are trying to fit in • Collaboration with Mario Niepel (Harvard Medical School) – Connecting proteomics with cell phenotypes (large scale) – Proteomics of bone, brain, lung tropic cells – Connection between lapatinib-induced osteoactivin upregulation with phenotype Niepel et al. , 2013

Peyton Lab Conclusions • Tissue-specific metastasis is mediated, in part, by microenvironment-related proteins – Mechanisms are largely unknown, likely complicated. – Most studies do not compare sites, so it is impossible to know if many of these things are specific! • Large-scale proteomic analysis of metastases (in human patients) would give best insight. – This could at least be done more easily in mice!

Peyton Lab Questions?

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Peyton Lab Bone Metastasis

Peyton Lab Methods • Mass Spectrometry – Large number of proteins – Sample prep, data analysis important; state-of-the -art MS required • Gel-based – Low throughput • • 2 DE DIGE Tagging techniques Protein microarrays Immunohistochemistry Staining Brennan et al. , 2010