Colleen L. Doçi

Semaphorin signaling in angiogenesis, lymphangiogenesis and cancer

Angiogenesis, the formation of new blood vessels from preexisting vasculature, is essential for many physiological processes, and aberrant angiogenesis contributes to some of the most prevalent human diseases, including cancer. Angiogenesis is controlled by delicate balance between pro- and anti-angiogenic signals. While pro-angiogenic signaling has been extensively investigated, how developmentally regulated, naturally occurring anti-angiogenic molecules prevent the excessive growth of vascular and lymphatic vessels is still poorly understood. In this review, we summarize the current knowledge on how semaphorins and their receptors, plexins and neuropilins, control normal and pathological angiogenesis, with an emphasis on semaphorin-regulated anti-angiogenic signaling circuitries in vascular and lymphatic endothelial cells. This emerging body of information may afford the opportunity to develop novel anti-angiogenic therapeutic strategies.

SDF-1/CXCL12 induces directional cell migration and spontaneous metastasis via a CXCR4/Gαi/mTORC1 axis

Multiple human malignancies rely on C‐X‐C motif chemokine receptor type 4 (CXCR4) and its ligand, SDF‐1/CXCL12 (stroma cell‐derived factor 1/C‐X‐C motif chemokine 12), to metastasize. CXCR4 inhibitors promote the mobilization of bone marrow stem cells, limiting their clinical application for metastasis prevention. We investigated the CXCR4‐initiated signaling circuitry to identify new potential therapeutic targets. We used HeLa human cancer cells expressing high levels of CXCR4 endogenously. We found that CXCL12 promotes their migration in Boyden chamber assays and single cell tracking. CXCL12 activated mTOR (mechanistic target of rapamycin) potently in a pertussis‐sensitive fashion. Inhibition of mTOR complex 1 (mTORC1) by rapamycin [drug concentration causing 50% inhibition (lC50) = 5 nM] and mTORC1/mTORC2 by Torin2 (IC50 = 6 nM), or by knocking down key mTORC1/2 components, Raptor and Rictor, respectively, decreased directional cell migration toward CXCL12. We developed a CXCR4‐mediated spontaneous metastasis model by implanting HeLa cells in the tongue of SCIDNOD mice, in which 80% of the animals develop lymph node metastasis. It is surprising that mTORC1 disruption by Raptor knockdown was sufficient to reduce tumor growth by 60% and spontaneous metastasis by 72%, which were nearly abolished by rapamycin. In contrast, disrupting mTORC2 had no effect in tumor growth or metastasis compared with control short hairpin RNAs. These data suggest that mTORC 1 may represent a suitable therapeutic target in human malignancies using CXCR4 for their meta‐static spread.—Dillenburg‐Pilla, P., Patel, V., Mikelis, C. M., Zárate‐Bladés, C. R., Doçi, C. L., Amornphimoltham, P., Wang, Z., Martin, D., Leelahavanichkul, K., Dorsam, R. T., Masedunskas, A., Weigert, R., Molinolo, A. A., Gutkind, J. S., SDF‐1/CXCL12 induces directional cell migration and spontaneous metastasis via a CXCR4/Gαi/mTORC1 axis. FASEB J. 29, 1056–1068 (2015). www.fasebj.org

Genetic Identification of SEMA3F as an Antilymphangiogenic Metastasis Suppressor Gene in Head and Neck Squamous Carcinoma

Head and neck squamous cell carcinomas (HNSCC) often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. HNSCCs are remarkably lymphangiogenic and represent a clear example of a cancer that utilizes the lymphatic vasculature for malignant dissemination; however, the molecular mechanisms underlying lymphangiogenesis in HNSCC is still poorly understood. Of interest, we found that an axon guidance molecule, Semaphorin 3F (SEMA3F), is among the top 1% underexpressed genes in HNSCC, and that genomic loss of SEMA3F correlates with increased metastasis and decreased survival. SEMA3F acts on its coreceptors, plexins and neuropilins, among which neuropilin-2 (NRP2) is highly expressed in lymphatic endothelial cells (LEC) but not in oral epithelium and most HNSCCs. We show that recombinant SEMA3F promotes LEC collapse and potently inhibits lymphangiogenesis in vivo. By reconstituting all possible plexin and neuropilin combinations, we found that SEMA3F acts through multiple receptors, but predominantly requires NRP2 to signal in LECs. Using orthotopic HNSCC metastasis mouse models, we provide direct evidence that SEMA3F re-expression diminishes lymphangiogenesis and lymph node metastasis. Furthermore, analysis of a large tissue collection revealed that SEMA3F is progressively lost during HNSCC progression, concomitant with increased tumor lymphangiogenesis. SEMA3F is localized to 3p21, an early and frequently deleted locus in HNSCC and many other prevalent human malignancies. Thus, SEMA3F may represent an antilymphangiogenic metastasis suppressor gene widely lost during cancer progression, hence serving as a prognostic biomarker and an attractive target for therapeutic intervention to halt metastasis.

Transcriptional signature primes human oral mucosa for rapid wound healing

Transcriptional profiling of human cutaneous and oral wound healing reveals pathways involved in rapid wound resolution. Rapid repair Wounds in the mouth heal faster and with less scarring than wounds in other locations on the body. To understand differences in healing, Iglesias-Bartolome et al. performed transcriptional analysis on sequential, paired oral and skin biopsies from healthy human subjects. Compared to baseline, skin samples showed a larger number of up-regulated genes on subsequent biopsies than oral samples, indicating that healing was unresolved. Oral wounds healed faster than skin wounds, and certain transcription factors were consistently up-regulated in the oral wounds but not in skin wounds. Overexpressing some of these transcription factors in a mouse model of skin wounding enhanced healing. The authors suggest that the molecular signature of the oral mucosa could be used to develop therapies for wound healing. Oral mucosal wound healing has long been regarded as an ideal system of wound resolution. However, the intrinsic characteristics that mediate optimal healing at mucosal surfaces are poorly understood, particularly in humans. We present a unique comparative analysis between human oral and cutaneous wound healing using paired and sequential biopsies during the repair process. Using molecular profiling, we determined that wound-activated transcriptional networks are present at basal state in the oral mucosa, priming the epithelium for wound repair. We show that oral mucosal wound–related networks control epithelial cell differentiation and regulate inflammatory responses, highlighting fundamental global mechanisms of repair and inflammatory responses in humans. The paired comparative analysis allowed for the identification of differentially expressed SOX2 (sex-determining region Y-box 2) and PITX1 (paired-like homeodomain 1) transcriptional regulators in oral versus skin keratinocytes, conferring a unique identity to oral keratinocytes. We show that SOX2 and PITX1 transcriptional function has the potential to reprogram skin keratinocytes to increase cell migration and improve wound resolution in vivo. Our data provide insights into therapeutic targeting of chronic and nonhealing wounds based on greater understanding of the biology of healing in human mucosal and cutaneous environments.

Epidermal loss of Gαq confers a migratory and differentiation defect in keratinocytes

G-protein coupled receptors (GPCRs), which activate heterotrimeric G proteins, are an essential class of transmembrane receptors that are responsible for a myriad of signaling events in normal and pathologic conditions. Two members of the G protein family, Gαq and Gα11, activate one of the main GPCR pathways and function as oncogenes by integrating mitogen-stimulated signaling cascades that are active under malignant conditions. Recently, it has been shown that targeted deletion of Gα11 and Gαq from endothelial cells impairs the Rho-mediated formation of focal adherens junctions, suggesting that Gα11/q signaling may also play a significant role in cytoskeletal-mediated cellular responses in epithelial cells. Indeed, combined deletion of Gα11 and Gαq confers a significant migratory defect in keratinocytes that delays cutaneous wound healing in an in vivo setting. This delay can be attributed to a defect during the reepithelialization phase due to significantly attenuated migratory capacity of Gαq-null keratinocytes under combined Gα11 deficiency. In fact, cells lacking Gα11/q demonstrate a severely reduced ability to respond to mitogenic and migratory signals in the microenvironment, leading to inappropriate and premature terminal differentiation. These results suggest that Gα11/q signaling pathways may be critical for integrating mitogenic signals and cytoskeletal function to achieve normal physiological responses. Emergence of a malignant phenotype may therefore arise from both under- and overexpression of Gα11/q signaling, implicating its upstream regulation as a potential therapeutic target in a host of pathologic conditions.

Using Heterologous COS-7 Cells to Identify Semaphorin-Signaling Components

Semaphorins are a family of membrane-bound and secreted type of proteins which were initially identified as chemorepulsive axon guidance molecules. Plexins and neuropilins are two major receptor families of semaphorins, and their common downstream targets are the actin cytoskeleton and cell-to-extracellular matrix adhesions. Semaphorins promote the collapse of growth cones by inducing rapid changes in the cytoskeleton and disassembly of focal adhesion structures. When transfected with appropriate receptors, non-neuronal COS-7 cells exhibit a similar cell collapse phenotype upon semaphorin stimulation. This heterologous system using COS-7 cells has been developed and widely used to investigate semaphorin-signaling pathways. In this chapter, we describe a COS-7 collapse assay protocol used to identify semaphorin-signaling components and a method to produce recombinant class 3 semaphorin proteins.

Targeting the PI3 K-mTOR Signaling Circuitry in HPV-Associated Oral Malignancies: Novel Precision Molecular Therapies

HPV is the most common sexually transmitted infection and it is predicted that up to 80 % of Americans will have HPV infections in their lifetime (Centers for Disease Control and Prevention 2015). The etiology of cervical cancer has long been linked with persistent HPV infection, but the correlation between HPV and other cancer types, including head and neck squamous cell carcinomas (HNSCC), oropharyngeal carcinomas (OPCs), anal carcinomas, and cancers of the genital tract is emerging (Forman et al. 2012). Compared to alcohol- and tobacco-related HNSCC, HPV-associated head and neck cancer involves activation of specific molecular mechanisms, making HPV(+) HNSCCs diagnostically and therapeutically distinct. Here, we will review the current understanding of the molecular mechanisms in HPV(+) HNSCC with emphasis on the signaling events that drive the growth of HPV-associated HNSCC and the emerging opportunities for the development of novel precision molecular-targeted therapies for this disease.

Abstract 3249: Recurrent 3p21 deletion in head and neck squamous cell carcinoma identifies SEMA3F as an anti-lymphangiogenic metastasis suppressor gene

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Head and neck squamous cell carcinomas (HNSCC) often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. HNSCC are remarkably lymphangiogenic and represent a clear example of a cancer that utilizes the lymphatic vasculature for malignant dissemination; however, the molecular mechanisms underlying lymphangiogenesis in HNSCC is still poorly understood. Of interest, we found that an axon guidance molecule, Semaphorin 3F (SEMA3F), is among the top 1% underexpressed genes in HNSCC, and that genomic loss of SEMA3F correlates with increased metastasis and decreased survival. SEMA3F acts on its co-receptors, plexins and neuropilins, among which neuropilin-2 (NRP2) is highly expressed in lymphatic endothelial cells (LECs) but not in oral epithelium and most HNSCCs. We show that recombinant SEMA3F promotes LEC collapse and potently inhibits lymphangiogenesis in vivo. By reconstituting all possible plexin and neuropilin combinations, we found that SEMA3F acts through multiple receptors, but predominantly requires NRP2 to signal in LECs. Using orthotopic HNSCC metastasis mouse models, we provide direct evidence that SEMA3F re-expression diminishes lymphangiogenesis and lymph node metastasis. Furthermore, analysis of a large tissue collection revealed that SEMA3F is progressively lost during HNSCC progression, concomitant with increased tumor lymphangiogenesis. SEMA3F is localized to 3p21, an early and frequently deleted locus in HNSCC and many other prevalent human malignancies. Thus, SEMA3F may represent an antilymphangiogenic metastasis suppressor gene widely lost during cancer progression, hence serving as a prognostic biomarker and an attractive target for therapeutic intervention to halt metastasis. Citation Format: Colleen L. Doci, Constantinos M. Mikelis, Michail S. Lionakis, Alfredo A. Molinolo, J Silvio Gutkind. Recurrent 3p21 deletion in head and neck squamous cell carcinoma identifies SEMA3F as an anti-lymphangiogenic metastasis suppressor gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3249. doi:10.1158/1538-7445.AM2015-3249

Abstract 4050: A central role for mTORC1 in CXCR4-mediated directional migration and metastasis

Tumor cells can co-opt the promigratory activity of chemokines and their cognate G protein-coupled receptors to disseminate to regional lymph nodes and distant organs. Indeed, the migration to SDF-1 of tumor cells expressing its receptor, CXCR4, is implicated in lymphatic and organ-specific metastasis in multiple human malignancies. However, the critical role of CXCR4 in bone marrow stem cell retention has hampered the development of CXCR4 inhibitors for metastasis prevention. Therefore the study of CXCR4 downstream signaling circuitry may provide new opportunities for the treatment of many human malignancies that depend on CXCR4 for their metastatic spread. The PI3K/mTOR pathway represents a major player in normal and aberrant cell growth. Consistently, mTOR inhibitors targeting its complex 1 (mTORC1) alone or mTORC1/mTORC2 have already shown promising therapeutic responses in many tumor types. Our data show that mTOR is activated upon SDF-1 stimulation of CXCR4 on epithelial-derived cancer cells, and that the disruption of mTORC1 or mTORC2 abrogates CXCR4-mediated directional migration. To study the CXCR4/mTOR axis in vivo we developed a simple and robust model for CXCR4-mediated spontaneous metastasis. Surprisingly, disruption of mTORC1 alone was sufficient to decrease tumor cell proliferation, angiogenesis, lymphangiogenesis, and CXCR4-mediated metastasis, whereas mTORC2 impairment had no effect on tumor dissemination or growth in vivo. Taken together, our data suggest that mTORC1 blockade could inhibit the migration of CXCR4 expressing cancer cells to their secondary sites, while disrupting the establishment of a permissive tumor microenvironment thereby halting the spread of highly aggressive tumors that require CXCR4 to metastasize. Citation Format: Patricia Dillenburg-Pilla, Vyomesh Patel, Constantinos M. Mikelis, Carlos Rodrigo Zarate-Blades, Panomwat Amornphimoltham, Zhiyong Wang, Daniel Martin, Kantima Leelahavanichkul, Colleen L. Doci, Robert T. Dorsam, Andrius Masedunskas, Nijiro Nohata , Roberto Weigert, Alfredo A. Molinolo, J. Silvio Gutkind . A central role for mTORC1 in CXCR4-mediated directional migration and metastasis. [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 4050. doi:10.1158/1538-7445.AM2014-4050