Kate M. Moore

Mouse extraembryonic arterial vessels harbor precursors capable of maturing into definitive HSCs

During mouse development, definitive hematopoietic stem cells (dHSCs) emerge by late E10.5 to E11 in several hematopoietic sites. Of them, the aorta-gonad-mesonephros (AGM) region drew particular attention owing to its capacity to autonomously initiate and expand dHSCs in culture, indicating its key role in HSC development. The dorsal aorta contains characteristic hematopoietic clusters and is the initial site of dHSC emergence, where they mature through vascular endothelial (VE)-cadherin(+)CD45(-)CD41(low) (type 1 pre-HSCs) and VE-cadherin(+)CD45(+) (type 2 pre-HSCs) intermediates. Although dHSCs were also found in other embryonic niches (placenta, yolk sac, and extraembryonic vessels), attempts to detect their HSC initiating potential have been unsuccessful to date. Extraembryonic arterial vessels contain hematopoietic clusters, suggesting that they develop HSCs, but functional evidence for this has been lacking. Here we show that umbilical cord and vitelline arteries (VAs), but not veins, contain pre-HSCs capable of maturing into dHSCs in the presence of exogenous interleukin 3, although in fewer numbers than the AGM region, and that pre-HSC activity in VAs increases with proximity to the embryo proper. Our functional data strongly suggest that extraembryonic arteries can actively contribute to adult hematopoiesis.

Therapeutic Targeting of Integrin αvβ6 in Breast Cancer

Background Integrin αvβ6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of αvβ6 has yet to be elucidated in breast cancer. Methods Protein expression of integrin subunit beta6 (β6) was measured in breast cancers by immunohistochemistry (n > 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of β6 in breast cell lines, the role of αvβ6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ≥ 3), respectively. A student’s t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni’s Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided. Results High expression of either the mRNA or protein for the integrin subunit β6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of β6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P < .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts. Conclusions Targeting αvβ6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients.

Suppression of TGFβ and Angiogenesis by Type VII Collagen in Cutaneous SCC

BACKGROUND Individuals with severe generalized recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering disorder caused by mutations in the COL7A1 gene, develop unexplained aggressive squamous cell carcinomas (SCC). Here we report that loss of type VII collagen (Col7) in SCC results in increased TGFβ signaling and angiogenesis in vitro and in vivo. METHODS Stable knockdown (KD) of Col7 was established using shRNA, and cells were used in a mouse xenograft model. Angiogenesis was assessed by immunohistochemistry, endothelial tube-forming assays, and proteome arrays. Mouse and zebrafish models were used to examine the effect of recombinant Col7 on angiogenesis. Findings were confirmed in anonymized, archival human tissue: RDEB SCC tumors, non-EB SCC tumors, RDEB skin, normal skin; and two human RDEB SCC cell lines. The TGFβ pathway was examined using immunoblotting, immunohistochemistry, biochemical inhibition, and siRNA. All statistical tests were two-sided. RESULTS Increased numbers of cross-cut blood vessels were observed in Col7 KD compared with control xenografts (n = 4 to 7 per group) and in RDEB tumors (n = 21) compared with sporadic SCC (n = 24, P < .001). Recombinant human Col7 reversed the increased SCC angiogenesis in Col7 KD xenografts in vivo (n = 7 per group, P = .04). Blocking the interaction between α2β1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGFβ signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGFβ markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGFβ markers, n = 6; VEGF, n = 21). Inhibition of TGFβ receptor signaling using siRNA resulted in decreased endothelial cell tube formation (n = 9 per group, mean tubes per well siC = 63.6, SD = 17.1; mean tubes per well siTβRII = 29.7, SD = 6.1, P = .02). CONCLUSIONS Type VII collagen suppresses TGFβ signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy.

PHLDA1 Mediates Drug Resistance in Receptor Tyrosine Kinase-Driven Cancer

Summary Development of resistance causes failure of drugs targeting receptor tyrosine kinase (RTK) networks and represents a critical challenge for precision medicine. Here, we show that PHLDA1 downregulation is critical to acquisition and maintenance of drug resistance in RTK-driven cancer. Using fibroblast growth factor receptor (FGFR) inhibition in endometrial cancer cells, we identify an Akt-driven compensatory mechanism underpinned by downregulation of PHLDA1. We demonstrate broad clinical relevance of our findings, showing that PHLDA1 downregulation also occurs in response to RTK-targeted therapy in breast and renal cancer patients, as well as following trastuzumab treatment in HER2+ breast cancer cells. Crucially, knockdown of PHLDA1 alone was sufficient to confer de novo resistance to RTK inhibitors and induction of PHLDA1 expression re-sensitized drug-resistant cancer cells to targeted therapies, identifying PHLDA1 as a biomarker for drug response and highlighting the potential of PHLDA1 reactivation as a means of circumventing drug resistance.

Integrin αVβ6-EGFR crosstalk regulates bidirectional force transmission and controls breast cancer invasion

The mechanical properties of the extracellular matrix within tumours control multiple cellular functions that drive cancer invasion and metastasis. However, the mechanisms controlling microenvironmental force sensation and transmission, and how these regulate transcriptional reprogramming and invasion, are unclear. Our aim was to understand how mechanical inputs are transmitted bidirectionally and translated into biochemical and transcriptional outputs to drive breast cancer progression. We reveal that adhesion receptor and growth factor receptor crosstalk regulates a bidirectional feedback mechanism co-ordinating force-dependent transcriptional regulation and invasion. Integrin αVβ6 drives invasion in a range of carcinomas and is a potential therapeutic target. αVβ6 exhibits unique biophysical properties that promote force-generation and increase matrix rigidity. We employed an inter-disciplinary approach incorporating proteomics, biophysical techniques and multi-modal live-cell imaging to dissect the role of αVβ6-EGFR crosstalk on transmission of mechanical signals bidirectionally between the extracellular matrix and nucleus. We show that αVβ6 expression correlates with poor prognosis in triple-negative breast cancer (TNBC) and drives invasion of TNBC cells. Moreover, our data show that a complex regulatory mechanism exists involving crosstalk between αVβ6 integrin and EGFR that impacts matrix stiffness, force transmission to the nucleus, transcriptional reprogramming and microenvironment rigidity. αVβ6 engagement triggers EGFR & MAPK signalling and αVβ6-EGFR crosstalk regulates mutual receptor trafficking mechanisms. Consequently, EGF stimulation suppresses αVβ6-mediated force-application on the matrix and nuclear shuttling of force-dependent transcriptional co-activators YAP/TAZ. Finally, we show that crosstalk between αVβ6 & EGFR regulates TNBC invasion. We propose a model whereby αVβ6-EGFR crosstalk regulates matrix stiffening, but also the transmission of extracellular forces into the cell in order to co-ordinate transcriptional reprogramming and invasion. To exploit adhesion receptors and receptor tyrosine kinases therapeutically, it will be essential to understand the integration of their signalling functions and how crosstalk mechanisms influence invasion and the response of tumours to molecular therapeutics.

Abstract B046: Therapeutic targeting of integrin αvβ6 in high-risk breast cancer

Background: Integrin αvβ6 promotes migration, invasion and survival of cancer cells, however, the relevance and role of αvβ6 has yet to be elucidated in breast cancer. Methods: Protein expression of integrin subunit beta6 (β6) was measured in over 2000 breast cancers by immunohistochemistry and ITGB6 mRNA expression measured in the METABRIC dataset. Overall survival was assessed using Kaplan-Meier curves and bioinformatics statistical analyses were performed in R statistical environment v2.14.1. Using antibody (264RAD; supplied by AZ-Medimmune) blockade and siRNA knockdown of β6 in breast cell lines, the role of αvβ6 in HER2 biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT assays and Transwell invasion assays and xenografts, respectively. All statistical tests were two-sided. Results: High expression of either the mRNA or protein for the integrin subunit β6 correlated with very poor survival (HR=1.99, P=2.9x10-6) and increased metastases to distant sites (P=0.02). Co-expression of β6 and HER2 gave a worse prognosis (HR=3.43, P=4x10-12). HER2-driven invasion was mediated by αvβ6 in an Akt2-dependent manner. Monotherapy with 264RAD or trastuzumab, slowed growth of MCF-7/HER2-18 and BT-474 xenografts to a similar degree (P Conclusions: Targeting αvβ6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients, giving hope to the 70% of women treated who have, or develop, resistance. Moreover, routine determination of the level of expression of αvβ6 on breast cancers would stratify women into higher-risk categories, requiring enhanced therapeutic intervention. Citation Format: Kate M. Moore, Gareth J. Thomas, Stephen W. Duffy, Jane Warwick, Rhian Gabe, Patrick Chou, Ian O. Ellis, Andrew R. Green, Syed Haider, Kellie Brouilette, Antonio Saha, Sabari Vallath, Rebecca Bowen, Claudia Chelala, Diana Eccles, William J. Tapper, Alastair M. Thompson, Phillip Quinlan, Lee Jordan, Cheryl Gillett, Adam Brentnall, Sheila Violette, Paul Weinreb, Jane Kendrew, Simon T. Barry, Ian R. Hart, Louise Jones, John F. Marshall. Therapeutic targeting of integrin αvβ6 in high-risk breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B046.

Abstract P4-15-01: Integrin avb6 is a therapeutic target for high-risk breast cancer and enhances trastuzumab efficacy

The integrin avβ6 promotes migration, invasion and survival of cancer cells, but the biological relevance has yet to be ascertained in breast cancer. Our immunhistochemical analysis of over 2000 breast cancers has revealed that high expression of the protein for the integrin subunit beta6 (β6) is associated with very poor survival (HR = 1.99, P = 2.9×10-6) and increased metastases to distant sites (P = 0·02). This correlation was confirmed at the mRNA level via bioinformatic analysis of the 2000 women in the METABRIC cohort. Furthermore, co-expression of HER2 gave a significantly worse prognosis (HR = 3.43, P = 4×10-12), which we investigated further. We report from in vitro studies that HER2-driven invasion is mediated by αvβ6 in an Akt2-dependent manner. Using the well-tolerated αvβ6-blocking antibody 264RAD in vivo we show that antibody-blockade of this integrin suppressed growth of BT-474 and MCF-7/HER2-18 human breast cancer xenografts similarly to trastuzumab alone (P 264RAD or trastuzumab prolonged survival to a similar degree (14.3% and 33.33% treated mice alive after 100d, respectively, no significant difference) but again, when both drugs were combined 85.7% of mice were alive after 100d, a highly significant response compared with PBS (P Since 70% of women treated with trastuzumab either have, or develop resistance, we suggest combined targeting of αvβ6 and HER2 could provide an important novel therapy for thousands of women with breast cancer. In fact, over 39,000 American women annually (NIH statistics) will develop HER2+ breast cancers for which no specific therapies exist. Our data shows that in excess of 40% of these women with trastuzumab-resistant disease are also likely to express high levels of αvβ6. Our data also suggest that routine determination of the level of expression of αvβ6 on breast cancers would be a valuable clinical tool as it identifies novel high-risk groups of women that require enhanced therapeutic intervention. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-01.

P2-01-05: Integrin avb6 Mediates HER2−Driven Invasion in Breast Cancer.

We have shown strong expression of integrin αvβ6 reduces the 5 year survival of HER2−positive breast cancers from 66% (HR 1.84) for moderate/low αvβ6 expressors to 54% (HR 2.18) in cases with strong expression (2063 cases, unpublished). This is in direct comparison to HER2/αvβ6-double negative cases, where strong αvβ6 expression reduces survival from 86% (HR 1.00) to 77% (HR 1.20). The biological mechanism underlying these observations was investigated in two isogenic breast cancer models: MCF-7/neo-1 and MCF-7/HER2−18 (a gift from Prof. M-C. Hung, USA) and MCF10A and MCF10A.CA1a. Flow cytometry showed MCF-7/HER2−18 expressed high levels of both HER2 and αvβ6 whereas MCF-7/neo-1 expressed low levels of both receptors. MCF10A and MCF10A.CA1a both expressed high levels of αvβ6 whereas only MCF10A.CA1a expressed elevated levels of HER2. In charcoal-stripped (cs)-serum, comparing MCF-7/neo-1 and MCF-7/HER2−18, HRGβ1(1μM), which stimulates HER2/HER3 heterodimers, increased proliferation by 50.2%±9% (P=0.048) and 66.2%±5.5% (P=0.003), in MCF-7/neo-1 and MCF-7/HER2−18 cells respectively. In contrast, Herceptin reduced proliferation by 32.3%±13.4% (P=0.003) and 15.2%±3.4% (P=0.028), respectively. MCF10A and MCF10A.CA1a proliferation remained unchanged with HRGβ 1 treatment and antibody-blockade of αvβ6 did not affect proliferation of any cell line. (NB, in complete serum there was no effect on proliferation of any of the above treatments). Invasion through Matrigel of MCF-7/HER2−18 was inhibited by antibody blockade (10μg/ml) of αvβ6 (mAb 10D5; 38.6%±20.8%, P=0.005) or HER2 (Herceptin, 10μg/ml; 40.1%±28.6%, P=0.01). The same trend was observed in MCF10A.CA1a invasion (83%±30.2% (P=0.025) with 10D5 and 80.4%±8.7% (P=0.022) with Herceptin). Combination of both antibodies had no additional effect. siRNA knockdown of αvβ6 or HER2 in MCF-7/HER2−18 and MCF10A.CA1a cells also reduced invasion to a similar extent as the blocking antibodies. This suggests that HER2 driven breast carcinoma invasion is mediated by αvβ6. To investigate this further HER2/3 was stimulated with HRGβ1, which consistently increased invasion by 111.5%±35.4% (P=0.011) in MCF-7/HER2−18 cells and by 57%±34% (P=0.042) in MCF10A.CA1a cells; an increase that was abrogated by co-treatment with 10D5 or Herceptin. To determine the mechanism through which HER2 and αvβ6 co-operate we examined several signalling pathways. Analysis of total or activated Akt, ERKI/II, c-Jun or Src in the MCF-7 model showed no changes. However, elevated total and phospho-Stat3 in MCF-7/HER2−18 were observed and siRNA knockdown, or small-molecule inhibition, of Stat3 suppressed invasion of MCF-7/HER2−18 cells (54.5%±27.3% (P=0.008) and 55.3%±33.3% (P=0.01) respectively), possibly suggesting that activation of Stat3 may link αvβ6 and HER2 co-operative signalling in this model. Interestingly, Akt was constitutively phosphorylated in MCF10A.CA1a cells and, moreover, 10D5 reduced these levels suggesting αvβ6 may influence HER2 signalling via Akt in these cells. These data confirm HER2−driven invasion is αvβ6-mediated and provide a mechanistic explanation for our clinical observations. We suggest HER2 and αvβ6 should be considered as dual targets for future therapy of some breast cancers. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-05.