VEGF is unique among angiogenic growth factors because it disrupts endothelial barrier function. Therefore, we considered whether this property of VEGF might contribute to tumor cell extravasation and metastasis. To test this, mice lacking the Src family kinases Src or Yes, which maintain endothelial barrier function in the presence of VEGF, were injected intravenously with VEGF-expressing tumor cells. We found a dramatic reduction in tumor cell extravasation in lungs or livers of mice lacking Src or Yes. At the molecular level, VEGF compromises the endothelial barrier by disrupting a VE-cadherin–β-catenin complex in lung endothelium from wild-type, but not Yes-deficient, mice. Disrupting the endothelial barrier directly with anti–VE-cadherin both amplifies metastasis in normal mice and overcomes the genetic resistance in Yes-deficient mice. Pharmacological blockade of VEGF, VEGFR-2, or Src stabilizes endothelial barrier function and suppresses tumor cell extravasation in vivo. Therefore, disrupting Src signaling preserves host endothelial barrier function providing a novel host-targeted approach to control metastatic disease.

Integrin ␣ ␤3 is found on a subset of tumor blood vessels where it is associated with angiogenesis and malignant tumor growth. We designed an ␣ ␤3-targeted nanoparticle (NP) encapsulating the cytotoxic drug doxorubicin (Dox) for targeted drug delivery to the ␣ ␤3-expressing tumor vasculature. We observed real-time targeting of this NP to tumor vessels and noted selective apoptosis in regions of the ␣ ␤3-expressing tumor vasculature. In clinically relevant pancreatic and renal cell orthotopic models of spontaneous metastasis, targeted delivery of Dox produced an antimetastatic effect. In fact, ␣ ␤3-mediated delivery of this drug to the tumor vasculature resulted in a 15-fold increase in antimetastatic activity without producing drug-associated weight loss as observed with systemic administration of the free drug. These findings reveal that NP-based delivery of cytotoxic drugs to the ␣ ␤3-positive tumor vasculature represents an approach for treating metastatic disease.antiangiogenic ͉ intravital microscopy ͉ pancreatic cancer ͉ renal cell carcinoma ͉ liposome A ngiogenesis contributes to tumor malignancy and is linked to a wide variety of inflammatory and ischemic diseases. Integrin ␣v␤3, an internalization receptor for a number of viruses (1, 2), was shown to be preferentially expressed on the angiogenic endothelium in malignant or diseased tissues (3, 4). These characteristics of integrin ␣v␤3 make it an attractive targeting molecule for molecular imaging and delivery of therapeutics for cancer. Previous studies have shown that ␣v␤3-targeted nanoparticles (NPs) coupled to contrast agents can readily image the tumor vasculature revealing ''hot spots'' of angiogenesis within the tumor (5, 6). Therapeutic studies using the ␣v integrin-targeting peptide, RGD-4C, demonstrated that this peptide effectively targeted doxorubicin (Dox) to the tumor neovasculature and enhanced efficacy in human breast cancer xenografts in mice (7). In another study, an ␣v␤3-targeted NP delivering a suicide gene to angiogenic blood vessels was capable of producing an anticancer response (8). Although integrin ␣v␤3 is a marker of angiogenic endothelium, histological analysis of breast cancer biopsy tissue revealed that ␣v␤3 was a primary marker of blood vessels within the most malignant tumors (4). In fact, a strong correlation was established between the percent of ␣v␤3-positive vessels within the tumor and disease progression (9).Here, we report the design and characterization of an ␣v␤3-targeted NP capable of delivering various pharmacological agents to the ␣v␤3-expressing tumor vasculature. Evidence is provided that an ␣v␤3-targeted NP carrying the cytotoxic drug Dox is capable of controlling the metastatic behavior of pancreatic and renal cell cancer in mice. Importantly, targeted delivery of Dox to the tumor vasculature provided a 15-fold increase in the efficacy of the drug while producing few, if any, side effects. Results Design of NPs Targeted to Angiogenic Endothelium.A schematic representation of the targeted NP (RGD-NP) (Fig. ...

Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin αvβ3 is associated with progression of a variety of human tumors. Here, we reveal a novel adhesion-independent role for integrin αvβ3 in pancreatic cancer and other carcinomas. Specifically, αvβ3 expressed in carcinoma cells enhanced anchorage-independent tumor growth in vitro and increased lymph node metastases in vivo. This required recruitment of c-src to the β3 integrin cytoplasmic tail, leading to c-src activation, crk-associated substrate (CAS) phosphorylation and tumor cell survival that, surprisingly, was independent of cell adhesion or focal adhesion kinase (FAK) activation. Reduced expression of endogenous αvβ3 or c-src not only suppressed anchorage-independent growth, but also decreased metastasis in vivo, yet did not affect migration/invasion. These data define an unexpected role for an integrin as a mediator of anchorage-independence suggesting that an αvβ3/c-src signaling module may account for the aggressive behavior of αvβ3-expressing tumors in man.

• CC-122 is a novel agent for DLBCL with antitumor and immunomodulatory activity.• CC-122 binds CRBN and degrades Aiolos and Ikaros resulting in a mimicry of IFN signaling and apoptosis in DLBCL.Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of 2 common substrates, transcription factors Aiolos and Ikaros.Here we report that CC-122, a new chemical entity termed pleiotropic pathway modifier, binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo, and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or short hairpin RNA-mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon (IFN)-stimulated genes independent of IFN-a, -b, and -g production and/or secretion and results in apoptosis in both activated B-cell (ABC) and germinal center B-cell DLBCL cell lines. Our results provide mechanistic insight into the cell-of-origin independent antilymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide. (Blood. 2015;126(6):779-789)

Focal adhesion kinase (FAK) plays a critical role during vascular development because knockout of FAK in endothelial cells (ECs) is embryonic lethal. Surprisingly, tamoxifen-inducible conditional knockout of FAK in adult blood vessels (inducible EC–specific FAK knockout [i-EC-FAK-KO]) produces no vascular phenotype, and these animals are capable of developing a robust growth factor–induced angiogenic response. Although angiogenesis in wild-type mice is suppressed by pharmacological inhibition of FAK, i-EC-FAK-KO mice are refractory to this treatment, which suggests that adult i-EC-FAK-KO mice develop a compensatory mechanism to bypass the requirement for FAK. Indeed, expression of the FAK-related proline-rich tyrosine kinase 2 (Pyk2) is elevated and phosphorylated in i-EC-FAK-KO blood vessels. In cultured ECs, FAK knockdown leads to increased Pyk2 expression and, surprisingly, FAK kinase inhibition leads to increased Pyk2 phosphorylation. Pyk2 can functionally compensate for the loss of FAK because knockdown or pharmacological inhibition of Pyk2 disrupts angiogenesis in i-EC-FAK-KO mice. These studies reveal the adaptive capacity of ECs to switch to Pyk2-dependent signaling after deletion or kinase inhibition of FAK.

Tyrosine kinase receptors and integrins play essential roles in tumor cell invasion and metastasis. Previously, we showed that epidermal growth factor (EGF) stimulation of pancreatic carcinoma cells led to invasion and metastasis that was blocked by antagonists of integrin A v B 5 . Here, we show that EGF stimulates metastasis of carcinoma cells via a Srcdependent phosphorylation of p130 CAS leading to activation of Rap1, a small GTPase involved in integrin activation. Specifically, EGF receptor (EGFR)-induced Src activity leads to phosphorylation of a region within the CAS substrate domain, which is essential for Rap1 and A v B 5 activation. This pathway induces A v B 5 -mediated invasion and metastasis in vivo yet does not influence primary tumor growth or activation of other integrins on these cells. These findings show cross-talk between a tyrosine kinase receptor and an integrin involved in carcinoma cell invasion and metastasis and may explain in part how inhibitors of EGFR affect malignant disease. [Cancer Res 2009;69(4):1383-91]

Recent studies have shown that lymphangiogenesis or the growth of lymphatic vessels at the periphery of tumors promotes tumor metastasis to lymph nodes. We show here that the fibronectin-binding integrin α4β1 and its ligand fibronectin are novel functional markers of proliferative lymphatic endothelium. Tumors and lymphangiogenic growth factors, such as vascular endothelial growth factor-C (VEGF-C) and VEGF-A, induce lymphatic vessel expression of integrin α4β1. Integrin α4β1 then promotes growth factor and tumor-induced lymphangiogenesis, as genetic loss of integrin α4β1 expression in Tie2Cre+ α4 loxp/loxp mice or genetic loss of α4 signaling in α4Y991A knock-in mice blocks growth factor and tumor-induced lymphangiogenesis, as well as tumor metastasis to lymph nodes. In addition, antagonists of integrin α4β1 suppress lymphangiogenesis and tumor metastasis. Our studies show that integrin α4β1 and the signals it transduces regulate the adhesion, migration, invasion, and survival of proliferating lymphatic endothelial cells. As suppression of α4β1 expression, signal transduction, or function in tumor lymphatic endothelium not only inhibits tumor lymphangiogenesis but also prevents metastatic disease, these results show that integrin α4β1-mediated tumor lymphangiogenesis promotes metastasis and is a useful target for the suppression of metastatic disease.

Pancreas cancer is one of the most lethal malignancies and is characterized by activating mutations of Kras, present in 95% of patients. More than 60% of pancreatic cancers also display increased c-Src activity, which is associated with poor prognosis. Although loss of tumor suppressor function (for example, p16, p53, Smad4) combined with oncogenic Kras signaling has been shown to accelerate pancreatic duct carcinogenesis, it is unclear whether elevated Src activity contributes to Kras-dependent tumorigenesis or is simply a biomarker of disease progression. Here, we demonstrate that in the context of oncogenic Kras, activation of c-Src through deletion of C-terminal Src kinase (CSK) results in the development of invasive pancreatic ductal adenocarcinoma (PDA) by 5–8 weeks. In contrast, deletion of CSK alone fails to induce neoplasia, while oncogenic Kras expression yields PDA at low frequency after a latency of 12 months. Analysis of cell lines derived from Ras/Src-induced PDA’s indicates that oncogenic Ras/Src cooperativity may lead to genomic instability, yet Ras/Src-driven tumor cells remain dependent on Src signaling and as such, Src inhibition suppresses growth of Ras/Src-driven tumors. These findings demonstrate that oncogenic Ras/Src cooperate to accelerate PDA onset and support further studies of Src-directed therapies in pancreatic cancer.