Activation induced cytidine deaminase (AID) is required for somatic hypermutation and immunoglobulin class switching in activated B cells. Because AID possesses no known target site specificity, there have been efforts to identify non-immunoglobulin AID targets. We show that AID acts promiscuously, generating widespread DNA double strand breaks (DSB), genomic instability and cytotoxicity in B cells with diminished homologous recombination (HR) capability. We demonstrate that the HR factor XRCC2 suppresses AID-induced off-target DSBs, promoting B cell survival. Finally, we suggest that aberrations affecting human chromosome 7q36, including XRCC2, correlate with genomic instability in B cell cancers. Our findings demonstrate that AID has promiscuous genomic DSB-inducing activity, identify HR as a safeguard against off-target AID action, and have implications for genomic instability in B cell cancers.

Extracellular matrix (ECM) remodeling regulates angiogenesis. However, the precise mechanisms by which structural changes in ECM proteins contribute to angiogenesis are not fully understood. Integrins are molecules with the ability to detect compositional and structural changes within the ECM and integrate this information into a network of signaling circuits that coordinate context-dependent cell behavior. The role of integrin ␣v␤3 in angiogenesis is complex, as evidence exists for both positive and negative functions. The precise downstream signaling events initiated by ␣v␤3 may depend on the molecular characteristics of its ligands. Here, we identified an RGD-containing cryptic collagen epitope that is generated in vivo. Surprisingly, rather than inhibiting ␣v␤3 signaling, this collagen epitope promoted ␣v␤3 activation and stimulated angiogenesis and inflammation. An antibody directed to this RGDKGE epitope but not other RGD collagen epitopes inhibited angiogenesis and inflammation in vivo. The selective ability of this RGD epitope to promote angiogenesis and inflammation depends in part on its flanking KGE motif. Interestingly, a subset of macrophages may represent a physiologically relevant source of this collagen epitope. Here, we define an endothelial cell mechano-signaling pathway in which a cryptic collagen epitope activates ␣v␤3 leading to an Src and p38 MAPK-dependent cascade that leads to nuclear accumulation of Yes-associated protein (YAP) and stimulation of endothelial cell growth. Collectively, our findings not only provide evidence for a novel mechano-signaling pathway, but also define a possible therapeutic strategy to control ␣v␤3 signaling by targeting a proangiogenic and inflammatory ligand of ␣v␤3 rather than the receptor itself.

Endoglin is a type III TGFβ auxiliary receptor that is upregulated in endothelial cells during angiogenesis and, when mutated in humans, results in the vascular disease hereditary hemorrhagic telangiectasia (HHT). Though endoglin has been implicated in cell adhesion, the underlying molecular mechanisms are still poorly understood. Here we show endoglin expression in endothelial cells regulates subcellular localization of zyxin in focal adhesions in response to BMP9. RNA knockdown of endoglin resulted in mislocalization of zyxin and altered formation of focal adhesions. The mechanotransduction role of focal adhesions and their ability to transmit regulatory signals through binding of the extracellular matrix are altered by endoglin deficiency. BMP/TGFβ transcription factors, SMADs, and zyxin have recently been implicated in a newly emerging signaling cascade, the Hippo pathway. The Hippo transcription coactivator, YAP1 (yes-associated protein 1), has been suggested to play a crucial role in mechanotransduction and cell-cell contact. Identification of BMP9-dependent nuclear localization of YAP1 in response to endoglin expression suggests a mechanism of crosstalk between the two pathways. Suppression of endoglin and YAP1 alters BMP9-dependent expression of YAP1 target genes CCN1 (cysteine-rich 61, CYR61) and CCN2 (connective tissue growth factor, CTGF) as well as the chemokine CCL2 (monocyte chemotactic protein 1, MCP-1). These results suggest a coordinate effect of endoglin deficiency on cell matrix remodeling and local inflammatory responses. Identification of a direct link between the Hippo pathway and endoglin may reveal novel mechanisms in the etiology of HHT.

Evidence suggests that stromal cells play critical roles in tumor growth. Uncovering new mechanisms that control stromal cell behavior and their accumulation within tumors may lead to development of more effective treatments. We provide evidence that the HU177 cryptic collagen epitope is selectively generated within human ovarian carcinomas and this collagen epitope plays a role in SKOV-3 ovarian tumor growth in vivo. The ability of the HU177 epitope to regulate SKOV-3 tumor growth depends in part on its ability to modulate stromal cell behavior because targeting this epitope inhibited angiogenesis and, surprisingly, the accumulation of a-smooth muscle actineexpressing stromal cells. Integrin a10b1 can serve as a receptor for the HU177 epitope in a-smooth muscle actineexpressing stromal cells and subsequently regulates Erkdependent migration. These findings are consistent with a mechanism by which the generation of the HU177 collagen epitope provides a previously unrecognized a 10 b 1 ligand that selectively governs angiogenesis and the accumulation of stromal cells, which in turn secrete protumorigenic factors that contribute to ovarian tumor growth. Our findings provide a new mechanistic understanding into the roles by which the HU177 epitope regulates ovarian tumor growth and provide new insight into the clinical results from a phase 1 human clinical study of the monoclonal antibody D93/TRC093 in patients with advanced malignant tumors. (Am J Pathol 2016 http://dx

Models of tumor angiogenesis have played a critical role in understanding the mechanisms involved in the recruitment of vasculature to the tumor mass, and have also provided a platform for testing antiangiogenic potential of new therapeutics that combat the development of malignant growth. In this regard, the chorioallantoic membrane (CAM) of the developing chick embryo has proven to be an elegant model for investigation of angiogenic processes. Here, we describe methods for effectively utilizing the preestablished vascular network of the chick CAM to investigate and quantify tumor-associated angiogenesis in a breast tumor model.

Proteolytic remodeling of extracellular matrix (ECM) results in structural changes that facilitate the generation of cryptic regulatory sites that promotes angiogenesis, tumor growth and metastasis. While alterations in the biophysical characteristic of the ECM can help create a tumor permissive microenvironment, little is known concerning whether structural changes in the ECM contributes to the ability of tumors to escape immune control. Molecular insight into the signaling pathways operating in stromal cells has contributed to the development of new cancer therapies. While clear progress has been made, as indicated by the recent approvals of new therapies such as immune checkpoint inhibitors, the overall survival of patients with metastatic disease remains alarmingly low. Accumulating evidence suggests that stromal components of the tumor microenvironment may contribute to the development of multiple resistance mechanisms including adaptive immune resistance. Thus, there is an urgent need for a more detailed understanding of how immune and inflammatory mechanisms govern tumor progression in order to enhance long-term durable responses with current therapies in a larger percentage of patients. Tumor-associated macrophages (TAMs) have been suggested to play roles in tumor growth and metastasis by multiple mechanisms including structural remodeling of the ECM. TAMs may also contribute to the development of resistance to current anti-cancer therapies. Our studies indicate that distinct subsets of macrophage may facilitate the generation of the RGDKGE containing XL313 cryptic collagen epitope that promotes angiogenesis and inflammation in vivo. Here we provide the first evidence that cellular interactions with the XL313 collagen epitope may regulate immune checkpoint molecules by a αVβ3 integrin-associated mechanism. Cellular interactions with the XL313 epitope and denatured forms of collagen, which are present within the tumor microenvironment, enhanced the levels of the immune checkpoint molecules PD-L1 and LAG-3. Selective targeting of the XL313 collagen epitope with a monoclonal antibody inhibited tumor growth and metastasis and tumors from these mice exhibited reduced levels of immune checkpoint molecules. Importantly, anti-XL313 epitope antibody significantly enhanced the anti-tumor activity of anti-PD-L1 therapy in vivo. These data suggest that the XL313 epitope may play a functional role in promoting immune suppression in tumors and that selective targeting of this cryptic collagen epitope may reduce immune suppression and significantly enhance the efficacy of immune checkpoint inhibitors. Taken together, our studies are consistent with the possibility that the endogenously generated XL313 epitope may regulate tumor growth in part by facilitating the escape of tumors from immune control. Citation Format: Jennifer M. Caron, Liangru Contois, Jacquelyn Ames, Peter C. Brooks. The XL313 cryptic collagen epitope regulates immune checkpoint molecules by a αVβ3-integrin-associated mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5131.

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Accumulating evidence indicates that the malignant behavior of tumors depends not only on tumor cells themselves, but also on the stromal cells that comprise the malignant tumor mass. Experimental findings suggest that stromal cells such as cancer-associated fibroblast (CAF) may play important roles in promoting tumor growth and metastasis as well as regulating the efficacy of certain chemotherapeutic drugs. However, developing novel clinical strategies that selectively and simultaneously impacts tumor and stromal cells remains challenging. Alterations in the integrity and molecular composition of the extracellular matrix (ECM) are hallmarks of tumor progression. Our previous studies have shown that structural remodeling of the ECM can result in localized triggering of what we have termed “biomechanical ECM switches” or changes in the three-dimensional structure of pre-existing ECM molecules. A humanized antibody (TRC093/D93) specifically directed to the HU177 cryptic collagen epitope that is selectively exposed following triggering of a biomechanical ECM switch has been developed, and a human phase-I clinical trial was recently completed with encouraging results. Here we provide evidence that the HU177 biomechanical ECM switch is triggered within human ovarian tumors resulting in the exposure of the HU177 cryptic collagen epitope. The relative exposure of the HU177 cryptic site was significantly (P<0.05) enhanced in biopsies of malignant ovarian tumors as compared to benign ovarian lesions. Selective targeting of the HU177 cryptic collagen epitope by Mab D93 significantly (P<0.05) inhibited SKOV-3 tumor growth by approximately 70% as compared to controls. Tumors from these mice exhibited reduced angiogenesis, elevated levels of apoptosis and a significant reduction in infiltration of alpha smooth muscle cell actin (αSMC-actin) positive stromal fibroblasts. Importantly, while Mab D93 inhibited SKOV-3 tumor cell adhesion to denatured collagen type-I and enhanced the expression of the cyclin dependent kinase inhibitor P27KIP1, it also selectively inhibited (80%) fibroblast migration on denatured collagen type-I that was induced by either FGF-2 or SKOV-3 condition medium. Collectively these studies provide evidence that specific targeting of the HU177 cryptic collagen epitope may represent a highly selective strategy to inhibit ovarian tumor growth in part by limiting stromal cell invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1480. doi:1538-7445.AM2012-1480