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 multimodal 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. Regulation of TP53 Activity through Phosphorylation Regulation of TP53 ActivityRNA Polymerase II Transcription Gene expression (Transcription) Transcriptional Regulation by TP53 Generic Transcription Pathway Costimulation by the CD28 family CD28 dependent PI3K/Akt signaling CD28 co-stimulation VEGFR2 mediated vascular permeability Intracellular signaling by second messengers PIP3 activates AKT signaling Constitutive Signaling by AKT1 E17K in Cancer AKT-mediated inactivation of FOXO1A Regulation of TP53 Activity through Association with Co-factors AKT phosphorylates targets in the nucleus RUNX2 regulates genes involved in cell migration AKT phosphorylates targets in the cytosol Downregulation of ERBB2:...

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<0.001), the antibody used for treating HER2-positive cancers (both 10mg/kg, bi-weekly). Moreover, when 264RAD was co-administered it significantly enhanced the ability of trastuzumab to suppress BT-474 tumor growth with a reduction in mean tumor volume of 94.8%+/-1.18% compared to 70.8%+/-5.98% observed with trastuzumab alone (P<0.0001) after 2 weeks treatment. This trend was reproduced even in the MCF-7/HER2-18 trastuzumab-resistant breast cancer tumors where a 76.24%+/-10.15% reduction was observed with combination therapy (P<0.0001) compared with only 44.62%+/-10.43% (P = 0.0006) and 46.6%+/-14.71% (P = 0.0004) reductions in final volume with 264RAD and trastuzumab respectively. The combination therapy was so effective it almost eradicated 100mm3 BT-474 tumors and completely eliminated small (10-20mm3) MCF-7/HER2-18 tumors. 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<0.0001) or monotherapies (264RAD: P<0.0001, trastuzumab: P<0.0001). Post-therapy biochemistry revealed residual tumors expressed significantly reduced αvβ6, HER2, HER3 and downstream signaling molecules including Akt2 and Smad2, essentially a much lower ‘grade’ tumour. 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.

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.