Virginia Morello

Integrins and Signal Transduction

Integrin signaling has a critical function in organizing cells in tissues during both embryonic development and tissue repair. Following their binding to the extracellular ligands, the intracellular signaling pathways triggered by integrins are directed to two major functions: organization of the actin cytoskeleton and regulation of cell behaviour including survival, differentiation and growth. Basic research conducted in the past twelve years has lead to remarkable breakthroughs in this field. Integrins are catalytically inactive and translate positional cues into biochemical signals by direct and/or functional association with intracellular adaptors, cytosolic tyrosine kinases or growth factor and cytokine receptors. The purpose of this chapter is to highlight recent experimental and conceptual advances in integrin signaling with particular emphasis on the ability of integrins to regulate Fak/Src family kinases (SFKs) activation and the cross-talk with soluble growth factors receptors and cytokines.

β1 integrin controls EGFR signaling and tumorigenic properties of lung cancer cells

Lung cancer is the leading cause of cancer death worldwide. The epidermal growth factor receptor (EGFR) represents the main target for non-small cell lung cancer (NSCLC) therapy, as its overexpression or constitutive activation contributes to malignancy and correlates with poor prognosis. Our previous work demonstrated that in epithelial cells β1 integrin is required for propagating EGFR signaling from the plasma membrane to the nucleus. In this study, we silenced β1 integrin in human NSCLC A549 cells. The β1 integrin-silenced cells show a defective activation of the EGFR signaling cascade, leading to decreased in vitro proliferation, enhanced sensitivity to cisplatin and Gefitinib, impaired migration and invasive behavior. Inhibitory effects on tumor growth and on the EGFR pathway were also observed in in vivo experiments. Moreover, β1 integrin silencing increases the amount of EGFR on the cell surface, suggesting that β1 integrin is required for efficient constitutive EGFR turnover at the cell membrane. Although the rate of EGF internalization and recycling is not affected in silenced cells, EGFR signaling is recovered only by expression of the Rab-coupling protein RCP, indicating that β1 integrin sustains the endocytic machinery required for EGFR signaling. Overall, these results show that β1 integrin is an essential regulator of EGFR signaling and tumorigenic properties of lung cancer cells, and that its silencing might represent an adjuvant approach to anti-EGFR therapy.

Convergence of integrins and EGF receptor signaling via PI3K/Akt/FoxO pathway in early gene Egr-1 expression†

The early gene early growth response (Egr‐1), a broadly expressed member of the zing‐finger family of transcription factors, is induced in many cell types by a variety of growth and differentiation stimuli, including epidermal growth factor (EGF). Here we demonstrate that Egr‐1 expression is mainly regulated by integrin‐mediated adhesion. Integrin‐dependent adhesion plays a dual role in Egr‐1 regulation, either being sufficient “per se” to induce Egr‐1, or required for EGF‐dependent expression of Egr‐1, which occurs only in adherent cells and not in cells in suspension. To dissect the molecular basis of integrin‐dependent Egr‐1 regulation, we show by FLIM‐based FRET that in living cells beta1‐integrin associates with the EGF receptor (EGFR) and that EGF further increases the extent complex formation. Interestingly, Egr‐1 induction depends on integrin‐dependent PI3K/Akt activation, as indicated by the decrease in Egr‐1 levels in presence of the pharmacological inhibitor LY294002, the kinase‐defective Akt mutant and Akt1/2 shRNAs. Indeed, upon adhesion activated Akt translocates into the nucleus and phosphorylates FoxO1, a Forkhead transcription factors. Consistently, FoxO1silencing results in Egr‐1‐increased levels, indicating that FoxO1 behaves as a negative regulator of Egr‐1 expression. These data demonstrate that integrin/EGFR cross‐talk is required for expression of Egr‐1 through a novel regulatory cascade involving the activation of the PI3K/Akt/Forkhead pathway. J. Cell. Physiol. 218: 294–303, 2009.

Four individually druggable MET hotspots mediate HGF-driven tumor progression.

Activation of MET by HGF plays a key role in tumor progression. Using a recently developed llama platform that generates human-like immunoglobulins, we selected 68 different antibodies that compete with HGF for binding to MET. HGF-competing antibodies recognized 4 distinct hotspots localized in different MET domains. We identified 1 hotspot that coincides with the known HGF β chain binding site on blades 2-3 of the SEMA domain β-propeller. We determined that a second and a third hotspot lie within blade 5 of the SEMA domain and IPT domains 2-3, both of which are thought to bind to HGF α chain. Characterization of the fourth hotspot revealed a region across the PSI-IPT 1 domains not previously associated with HGF binding. Individual or combined targeting of these hotspots effectively interrupted HGF/MET signaling in multiple cell-based biochemical and biological assays. Selected antibodies directed against SEMA blades 2-3 and the PSI-IPT 1 region inhibited brain invasion and prolonged survival in a glioblastoma multiforme model, prevented metastatic disease following neoadjuvant therapy in a triple-negative mammary carcinoma model, and suppressed cancer cell dissemination to the liver in a KRAS-mutant metastatic colorectal cancer model. These results identify multiple regions of MET responsible for HGF-mediated tumor progression, unraveling the complexity of HGF-MET interaction, and provide selective molecular tools for targeting MET activity in cancer.

The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression

Whether hERG1 potassium channels promote proliferation or metastasis in breast cancer cells depends on channel conformation. Channeling proliferation or metastasis The hERG1 potassium channel is best known for its role in repolarizing excitable cells such as cardiomyocytes, but the abundance of this cardiac channel is aberrantly high in cancer cells. Becchetti et al. investigated the interaction of hERG1 with the β1 integrin subunit, a member of a family of adhesion molecules. Forms of hERG1 with mutations that fixed the channel in the open conformation more weakly interacted with β1 integrin in cells and enhanced proliferation when expressed in breast cancer cells injected into mice. However, K+ flow through open hERG1 channels enhanced the activation of FAK downstream of β1 integrin and promoted metastasis in breast cancer cells injected into mice. Thus, whether hERG1 promotes proliferation or metastasis in cancer cells depends on the conformation of the channel and suggests that hERG1 inhibitors that are tailored to the channel conformation could be used to prevent different aspects of tumor progression. Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell–extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-à-go-go–related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in tumors, where it modulates integrin-mediated signaling. We found that hERG1 interacted with the β1 integrin subunit at the plasma membrane of human cancer cells. This interaction was not detected in cardiomyocytes because of the presence of the hERG1 auxiliary subunit KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1), which blocked the β1 integrin–hERG1 interaction. Although open hERG1 channels did not interact as strongly with β1 integrins as did closed channels, current flow through hERG1 channels was necessary to activate the integrin-dependent phosphorylation of Tyr397 in focal adhesion kinase (FAK) in both normal and cancer cells. In immunodeficient mice, proliferation was inhibited in breast cancer cells expressing forms of hERG1 with impaired K+ flow, whereas metastasis of breast cancer cells was reduced when the hERG1/β1 integrin interaction was disrupted. We conclude that the interaction of β1 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression.

Depleting MET-Expressing Tumor Cells by ADCC Provides a Therapeutic Advantage over Inhibiting HGF/MET Signaling

Hepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development, generating an antibody (ARGX-111) with improved pharmacologic properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity, downregulated cell surface expression of MET, curbing HGF-independent MET activity, and engaged natural killer cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade and generates proof-of-concept for ARGX-111 clinical testing in MET-positive oncologic malignancies.

Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.

Stroma-derived HGF drives metabolic adaptation of colorectal cancer to angiogenesis inhibitors

The role of paracrine Hepatocyte Growth Factor (HGF) in the resistance to angiogenesis inhibitors (AIs) is hidden in xenograft models because mouse HGF fails to fully activate human MET. To uncover it, we compared the efficacy of AIs in wild-type and human HGF knock-in SCID mice bearing orthotopic human colorectal tumors. Species-specific HGF/MET signaling dramatically impaired the response to anti-angiogenic agents and boosted metastatic dissemination. In cell-based assays mimicking the consequences of anti-angiogenic therapy, colorectal cancer cells were completely resistant to hypoxia but extremely sensitive to nutrient deprivation. Starvation-induced apoptosis could be prevented by HGF, which promoted GLUT1-mediated glucose uptake, sustained glycolysis and activated autophagy. Pharmacological inhibition of GLUT1 in the presence of glucose killed tumor cells as effectively as glucose deprivation, and this effect was antagonized by HGF. Concomitant targeting of GLUT1 and HGF potently suppressed growth and dissemination of AI-resistant human tumors in human HGF knock-in SCID mice without exacerbating tumor hypoxia. These data suggest that stroma-derived HGF protects CRC cells against glucose starvation-induced apoptosis, promoting resistance to both AIs and anti-glycolytic agents. Combined inhibition of glucose metabolism and HGF/MET signaling (‘anti-METabolic therapy’) may represent a more effective CRC treatment compared to utterly blocking tumor blood supply.

Abstract P2-05-17: ARGX-111 depletes MET-expressing circulating tumor cells via enhanced ADCC, resulting in inhibition of metastasis

Several lines of experimental evidence suggest that Hepatocyte Growth Factor (HGF) and its receptor MET play an important role in breast cancer progression and drug resistance. To date, targeted MET inhibitors in clinical development have primarily shown cytostatic rather than cytotoxic effects. Development of a cytotoxic MET inhibitor would serve to complement standard breast cancer therapy, especially when administered in the adjuvant/neo-adjuvant setting. We have developed ARGX-111, a human antibody antagonist of MET function. ARGX-111 blocks both HGF-dependent and -independent signaling, down-regulates tumor cell surface expression of MET and kills MET-overexpressing cells by enhanced antibody-dependent cellular cytotoxicity (ADCC). ARGX-111 was shown to be more efficacious than an ADCC-inactive control antibody in both HGF-dependent and -independent tumor xenograft models. ADCC reporter assays confirmed the cytotoxic effects of ARGX-111 in patient-derived primary tumor specimens, including MET-expressing breast cancer stem-like cells. In an orthotopic mouse model of metastatic mammary carcinoma (MDA-MB-231), adjuvant or neo-adjuvant treatment with ARGX-111 was significantly more effective in depleting circulating tumor cells (CTCs) and suppressing the development of bone and lung metastases than the ADCC-inactive control. Taken together, these results provide a rationale for clinical investigation of ARGX-111 in the early breast cancer setting. An ongoing Phase 1 study (NCT02055066) is examining the effects of ARGX-111 on CTCs, alongside the assessment of its safety and efficacy. Citation Format: Morello V, Hultberg A, De Jonge N, Huyghe L, Hanssens V, Brouckaert P, Saunders M, Dreier T, Thibault A, Rolfo C, Aftimos P, Awada A, Michieli P, de Haard H. ARGX-111 depletes MET-expressing circulating tumor cells via enhanced ADCC, resulting in inhibition of metastasis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-17.

Abstract LB-330: Four individually druggable Met hotspots mediate HGF-driven tumor progression

Activation of Met by HGF plays a key role in tumor progression. Using a newly developed llama platform generating human-like immunoglobulins, we selected 68 different antibodies that competed with HGF for binding to Met. HGF-competing antibodies recognized four hotspots localized in different domains of Met. One hotspot coincides with the previously identified HGF β-chain binding site on blades 2-3 of the SEMA β-propeller. Two hotspots lie within SEMA blade 5 and IPT domains 2-3, respectively, where the HGF α-chain is thought to bind. The last hotspot identifies a new region across the PSI-IPT 1 domains. Individual or combined targeting of these hotspots effectively interrupted HGF-Met signaling in multiple biochemical and biological assays. Selected antibodies directed against SEMA blades 2-3 and the PSI-IPT 1 region inhibited brain invasion and prolonged survival in a glioblastoma multiforme model, prevented metastatic disease following neo-adjuvant therapy in a triple-negative mammary carcinoma model, and suppressed cancer cell dissemination to the liver in a KRAS-mutant metastatic colorectal cancer model. These results identify multiple regions of Met responsible for HGF-mediated tumor progression, unraveling the complexity of HGF-Met interaction and providing new molecular tools for targeting Met activity in cancer. Citation Format: Anna Hultberg, Cristina Basilico, Cristophe Blanchetot, Natalie De Jonge, Valerie Hanssens, Gitte De Boeck, Alessia Mira, Manuela Cazzanti, Virginia Morello, Torsten Dreier, Michael Saunders, Hans De Haard, Paolo Michieli. Four individually druggable Met hotspots mediate HGF-driven tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-330. doi:10.1158/1538-7445.AM2014-LB-330