Liangru Contois

Integrins as “functional hubs” in the regulation of pathological angiogenesis

It is well accepted that complex biological processes such as angiogenesis are not controlled by a single family of molecules or individually isolated signaling pathways. In this regard, new insight into the interconnected mechanisms that regulate angiogenesis might be gained by examining this process from a more global network perspective. The coordination of signaling cues from both outside and inside many different cell types is required for the successful completion of angiogenesis. Evidence is accumulating that the multifunctional integrin family of cell adhesion receptors represent an important group of molecules that play active roles in sensing, integrating, and distributing a diverse set of signals that regulate many cellular events required for angiogenesis. Given the ability of integrins to bind numerous extracellular ligands and transmit signals in a bi-directional fashion, we will discuss the multiple ways by which integrins may serve as a functional hub during pathological angiogenesis. In addition, we will highlight potential imaging and therapeutic strategies based on the expanding new insight into integrin function.

Endoglin Regulates Cancer–Stromal Cell Interactions in Prostate Tumors

Endoglin is an accessory receptor for TGF-β that has been implicated in prostate cancer cell detachment, migration, and invasiveness. However, the pathophysiologic significance of endoglin with respect to prostate tumorigenesis has yet to be fully established. In this study, we addressed this question by investigation of endoglin-dependent prostate cancer progression in a TRAMP (transgenic adenocarcinoma mouse prostate) mouse model where endoglin was genetically deleted. In this model, endoglin was haploinsufficient such that its allelic deletion slightly increased the frequency of tumorigenesis, yet produced smaller, less vascularized, and less metastatic tumors than TRAMP control tumors. Most strikingly, TRAMP:eng(+/-)-derived tumors lacked the pronounced infiltration of carcinoma-associated fibroblasts (CAF) that characterize TRAMP prostate tumors. Studies in human primary prostate-derived stromal cells (PrSC) confirmed that suppressing endoglin expression decreased cell proliferation, the ability to recruit endothelial cells, and the ability to migrate in response to tumor cell-conditioned medium. We found increased levels of secreted insulin-like growth factor-binding proteins (IGFBP) in the conditioned medium from endoglin-deficient PrSCs and that endoglin-dependent regulation of IGFBP-4 secretion was crucial for stromal cell-conditioned media to stimulate prostate tumor cell growth. Together, our results firmly establish the pathophysiologic involvement of endoglin in prostate cancer progression; furthermore, they show how endoglin acts to support the viability of tumor-infiltrating CAFs in the tumor microenvironment to promote neovascularization and growth.

Insulin-like growth factor binding protein-4 differentially inhibits growth factor-induced angiogenesis.

Background: We examined the impact of insulin-like growth factor binding protein-4 (IGFBP-4) on growth factor-induced angiogenesis in vivo. Results: IGFBP-4 inhibited IGF-1 and FGF-2, but not VEGF-induced angiogenesis, and this inhibition depended on p38 MAPK activity. Conclusion: The anti-angiogenic activity of IGFBP-4 depends in part on p38 MAPK. Significance: New insight is provided into how blood vessels respond to endogenous inhibitors during growth factor-stimulated angiogenesis. An in-depth understanding of the molecular and cellular complexity of angiogenesis continues to advance as new stimulators and inhibitors of blood vessel formation are uncovered. Gaining a more complete understanding of the response of blood vessels to both stimulatory and inhibitory molecules will likely contribute to more effective strategies to control pathological angiogenesis. Here, we provide evidence that endothelial cell interactions with structurally altered collagen type IV may suppress the expression of insulin-like growth factor binding protein-4 (IGFBP-4), a well documented inhibitor of the IGF-1/IGF-1R signaling axis. We report for the first time that IGFBP-4 differentially inhibits angiogenesis induced by distinct growth factor signaling pathways as IGFBP-4 inhibited FGF-2- and IGF-1-stimulated angiogenesis but failed to inhibit VEGF-induced angiogenesis. The resistance of VEGF-stimulated angiogenesis to IGFBP-4 inhibition appears to depend on sustained activation of p38 MAPK as blocking its activity restored the anti-angiogenic effects of IGFBP-4 on VEGF-induced blood vessel growth in vivo. These novel findings provide new insight into how blood vessels respond to endogenous inhibitors during angiogenesis stimulated by distinct growth factor signaling pathways.

Inhibition of tumor-associated αvβ3 integrin regulates the angiogenic switch by enhancing expression of IGFBP-4 leading to reduced melanoma growth and angiogenesis in vivo

A more complete understanding of the mechanisms that regulate the angiogenic switch, which contributes to the conversion of small dormant tumors to actively growing malignancies, is important for the development of more effective anti-angiogenic strategies for cancer therapy. While significant progress has been made in understanding the complex mechanisms by which integrin αvβ3 expressed in endothelial cells governs angiogenesis, less is known concerning the ability of αvβ3 expressed within the tumor cell compartment to modulate the angiogenic output of a tumor. Here we provide evidence that αvβ3 expressed in melanoma cells may contribute to the suppression of IGFBP-4, an important negative regulator of IGF-1 signaling. Given the multiple context-dependent roles for αvβ3 in angiogenesis and tumor progression, our novel findings provide additional molecular insight into how αvβ3 may govern the angiogenic switch by a mechanism associated with a p38 MAPK and matrix metalloproteinases-dependent regulation of the endogenous angiogenesis inhibitor IGFBP-4.

Identification of an Endogenously Generated Cryptic Collagen Epitope (XL313) That May Selectively Regulate Angiogenesis by an Integrin Yes-associated Protein (YAP) Mechano-transduction Pathway.

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 αvβ3 in angiogenesis is complex, as evidence exists for both positive and negative functions. The precise downstream signaling events initiated by αvβ3 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 αvβ3 signaling, this collagen epitope promoted αvβ3 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 αvβ3 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 αvβ3 signaling by targeting a pro-angiogenic and inflammatory ligand of αvβ3 rather than the receptor itself.

The HU177 Collagen Epitope Controls Melanoma Cell Migration and Experimental Metastasis by a CDK5/YAP-Dependent Mechanism

Stromal components not only help form the structure of neoplasms such as melanomas, but they also functionally contribute to their malignant phenotype. Thus, uncovering signaling pathways that integrate the behavior of both tumor and stromal cells may provide unique opportunities for the development of more effective strategies to control tumor progression. In this regard, extracellular matrix-mediated signaling plays a role in coordinating the behavior of both tumor and stromal cells. Here, evidence is provided that targeting a cryptic region of the extracellular matrix protein collagen (HU177 epitope) inhibits melanoma tumor growth and metastasis and reduces angiogenesis and the accumulation of α-SMA-expressing stromal cell in these tumors. The current study suggests that the ability of the HU177 epitope to control melanoma cell migration and metastasis depends on the transcriptional coactivator Yes-associated protein (YAP). Melanoma cell interactions with the HU177 epitope promoted nuclear accumulation of YAP by a cyclin-dependent kinase-5-associated mechanism. These findings provide new insights into the mechanism by which the anti-HU177 antibody inhibits metastasis, and uncovers an unknown signaling pathway by which the HU177 epitope selectively reprograms melanoma cells by regulating nuclear localization of YAP. This study helps to define a potential new therapeutic strategy to control melanoma tumor growth and metastasis that might be used alone or in combination with other therapeutics.

Abstract 5131: The XL313 cryptic collagen epitope regulates immune checkpoint molecules by a αVβ3-integrin-associated mechanism

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.

Abstract 4984: Generation of an RGD containing soluble pro-inflammatory form of the XL313 cryptic collagen epitope during tumor growth and inflammation.

Evidence indicates that malignant tumor progression depends not only on tumor cells themselves, but also on stromal cells of the malignant lesion including tumor-associated macrophages (TAMs). TAMs have been suggested to contribute to changes in the extracellular matrix (ECM) through remodeling of architectural proteins like type-I collagen. Our previous studies have shown that remodeling of the ECM results in localized triggering of what we have termed “biomechanical ECM switches” resulting in exposure of cryptic ECM epitopes. In this regard, collagen has a number of Arg-Gly-Asp (RGD) containing cryptic sites and soluble RGD containing reagents are currently being developed for the treatment of malignant tumors. However, it is not known if the distinct cryptic RGD elements in type-I collagen are functionally redundant or if specific flanking sequences convey differential control of cellular behavior. In this regard, we previously generated a series of monoclonal antibodies (Mabs) that target distinct RGD containing cryptic sites of type-I collagen. One of these Mabs designated XL313 exhibits selective recognition of the conserved RGDKGE cryptic element and significantly (P Citation Format: Jacquelyn J. Ames, Jennifer Caron, Liangru Contois, Peter Brooks. Generation of an RGD containing soluble pro-inflammatory form of the XL313 cryptic collagen epitope during tumor growth and inflammation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4984. doi:10.1158/1538-7445.AM2013-4984

Abstract 1480: Regulation of ovarian tumor growth and stromal cell infiltration by the HU177 biomechanical ECM switch

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 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

Abstract 3485: Insulin-like growth factor binding protein-4 (IGFBP-4) differentially inhibits growth factor induced angiogenesis in vivo

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Angiogenesis contributes to the development and progression of malignant tumors. An expanding body of evidence also suggests that the response of both vascular and tumor cells to growth factor stimulation depends in part on cellular communication with the extracellular matrix (ECM), and studies have documented important roles for the integrin family of ECM receptors in modulating growth factor signaling. Therefore, uncovering molecular mechanisms by which integrin-mediated cellular communication with the ECM modulate the specificity of growth factor signaling during pathological angiogenesis may provide unique insight into malignant tumor progression. Here we provide evidence that disruption of αvβ3-dependent interaction with the ECM enhance expression of Insulin-like growth factor binding protein-4 (IGFBP-4), a well-known regulator of IGF-1 signaling. Interestingly, IGFBP-4 significantly (P<0.05) inhibited IGF-1 and bFGF-induced angiogenesis by greater than 50%. In contrast, IGFBP-4 alone failed to inhibit VEGF-induced angiogenesis in chick CAM. Levels of phosphorylated p38 MAP kinase were elevated in chick CAM tissues treated with VEGF as compared to those in tissues stimulated with either IGF-1 or bFGF. Surprisingly, while the p38 MAP kinase inhibitor SB 202190 alone had little effect on VEGF-induced angiogenesis, a combination of IGFBP-4 and the p38 MAP kinase inhibitor significantly (P<0.05) inhibited VEGF-induced angiogenesis. These novel findings are consistent with a role for elevated levels of activated p38 MAP kinase in modulating the anti-angiogenic activity of IGFBP-4 in vivo. Taken together, our experimental findings provide additional molecular insight into how integrin-mediated interactions with the local ECM microenvironment may modulate the responses of cells to distinct growth factor signaling pathways during angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3485. doi:10.1158/1538-7445.AM2011-3485