SUMMARY Endothelial cells (ECs) form cell-cell adhesive junctional structures maintaining vascular integrity. This barrier is dynamically regulated by vascular endothelial growth factor (VEGF) receptor signaling. We created an inducible knockin mouse model to study the contribution of the integrin-associated focal adhesion tyrosine kinase (FAK) signaling on vascular function. Here we show that genetic or pharmacological FAK inhibition in ECs prevents VEGF-stimulated permeability downstream of VEGF receptor or Src tyrosine kinase activation in vivo. VEGF promotes tension-independent FAK activation, rapid FAK localization to cell-cell junctions, binding of the FAK FERM domain to the vascular endothelial cadherin (VE-cadherin) cytoplasmic tail, and direct FAK phosphorylation of β-catenin at tyrosine-142 (Y142) facilitating VE-cadherin-β-catenin dissociation and EC junctional breakdown. Kinase inhibited FAK is in a closed conformation that prevents VE-cadherin association and limits VEGF-stimulated β-catenin Y142 phosphorylation. Our studies establish a role for FAK as an essential signaling switch within ECs regulating adherens junction dynamics.

The transition from ductal carcinoma in situ (DCIS) to invasive breast cancer (IBC) is a crucial step in breast cancer progression. The specific alterations that govern this transition have not been elucidated. HER2/neu is frequently overexpressed in DCIS but is less common in IBC, thereby suggesting additional requirements for transformation. To identify genes capable of cooperating with HER2/neu to fully transform mammary epithelial cells, we used an insertional mutagenesis screen on cells isolated from wild-type neu expressing mice and identified the E3 ligase HACE1 as HER2 cooperative tumor suppressor gene. Loss of HACE1 expression is commonly seen in clinical breast cancer data sets. HACE1 downregulation in normal human mammary epithelial cells (HMECs) results in the accumulation of the activated GTP-bound Rac1 partially transforming these cells. Overexpression of HER2 activates Rac1, which further accumulates upon HACE1 loss resulting in Rac1 hyperactivation. Although the knockdown of HACE1 or overexpression of HER2 alone in HMECs is not sufficient for tumorigenesis, HER2 overexpression combined with HACE1 downregulation fully transforms HMECs resulting in robust tumor formation. The pharmaceutical interference of Rac function abrogates the effects of HACE1 loss both in vitro and in vivo, resulting in marked reduction in tumor burden. Our work supports a critical role for HACE1 in breast cancer progression and identifies patients that may benefit from Rac-targeted therapies.

Resistance to chemotherapy represents a major limitation in the treatment of colorectal cancer. Novel strategies to circumvent resistance are critical to prolonging patient survival. Rac1b, a constitutively activated isoform of the small GTPase Rac1, is upregulated with disease progression and promotes cell proliferation and inhibits apoptosis by activation of NF-kB signaling. Here, we show that Rac1b overexpression correlates with cancer stage and confirmed Rac1b expression is associated with increased growth through enhancing NF-kB activity. Rac1b knockdown reduced cellular proliferation and reduced NF-kB activity. Surprisingly, Rac1b expression and NF-kB activity were upregulated in cells treated with chemotherapeutics, suggesting that Rac1b facilitates chemo-resistance through activation of NF-kB signaling. Knockdown of Rac1b or Rac inhibition increases the sensitivity of the cells to oxaliplatin. When used in combination, inhibition of Rac prevents the increase in NF-kB activity associated with chemotherapy treatment and increases the sensitivity of the cells to oxaliplatin. Although Rac inhibition or oxaliplatin treatment alone reduces the growth of colorectal cancer in vivo, combination therapy results in improved outcomes compared with single agents alone. We provide the first evidence that Rac1b expression confers resistance to chemotherapy in colorectal cancer. Additionally, we show that the use of a Rac inhibitor prevents chemoresistance by blocking activation of chemotherapy induced NF-kB signaling, providing a novel strategy to overcome resistance to chemotherapy in colorectal cancer.

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Cisplatin is a major chemotherapeutic that continues to have a significant impact in the treatment of more than 50% of all cancers. Since its Food and Drug Administration approval in 1978 for the treatment of advanced ovarian and bladder cancer, this chemotherapeutic has made significant strides and its application has been extended to a large variety of other cancers. However, the vast majority of patients who receive cisplatin therapy often suffer from nephrotoxicity, neurotoxicity, nausea, and ototoxicity. Numerous methods currently exist for overcoming nephrotoxicity-and nausea-related side effects, but there is no clear prevention to fight ototoxicity and neurotoxicity. In this work, we examined Platin-A, a prodrug of cisplatin and aspirin, using preclinical mouse-and guinea pig-based models and demonstrated its efficacy with reduced ototoxicity. In addition, in vitro studies documented that when Platin-A is used in combination with a clinically relevant dose of radiation, its efficacy can further be improved by attacking cellular bioenergetic profiles, producing multiple modes of DNA damage, and delaying repair of damaged DNA. These studies demonstrated novel properties of the prodrug, Platin-A, highlighting its superior efficacy with reduced toxicity.