Federica Maione

Semaphorin 3A is an endogenous angiogenesis inhibitor that blocks tumor growth and normalizes tumor vasculature in transgenic mouse models

Tumor growth and progression rely upon angiogenesis, which is regulated by pro- and antiangiogenic factors, including members of the semaphorin family. By analyzing 3 different mouse models of multistep carcinogenesis, we show here that during angiogenesis, semaphorin 3A (Sema3A) is expressed in ECs, where it serves as an endogenous inhibitor of angiogenesis that is present in premalignant lesions and lost during tumor progression. Pharmacologic inhibition of endogenous Sema3A during the angiogenic switch, the point when pretumoral lesions initiate an angiogenic phase that persists throughout tumor growth, enhanced angiogenesis and accelerated tumor progression. By contrast, when, during the later stages of carcinogenesis following endogenous Sema3A downmodulation, Sema3A was ectopically reintroduced into islet cell tumors by somatic gene transfer, successive waves of apoptosis ensued, first in ECs and then in tumor cells, resulting in reduced vascular density and branching and inhibition of tumor growth and substantially extended survival. Further, long-term reexpression of Sema3A markedly improved pericyte coverage of tumor blood vessels, something that is thought to be a key property of tumor vessel normalization, and restored tissue normoxia. We conclude, therefore, that Sema3A is an endogenous and effective antiangiogenic agent that stably normalizes the tumor vasculature.

Semaphorin 3A overcomes cancer hypoxia and metastatic dissemination induced by antiangiogenic treatment in mice

Cancer development, progression, and metastasis are highly dependent on angiogenesis. The use of antiangiogenic drugs has been proposed as a novel strategy to interfere with tumor growth, but cancer cells respond by developing strategies to escape these treatments. In particular, animal models show that antiangiogenic drugs currently used in clinical settings reduce tumor tissue oxygenation and trigger molecular events that foster cancer resistance to therapy. Here, we show that semaphorin 3A (Sema3A) expression overcomes the proinvasive and prometastatic resistance observed upon angiogenesis reduction by the small-molecule tyrosine inhibitor sunitinib in both pancreatic neuroendocrine tumors (PNETs) in RIP-Tag2 mice and cervical carcinomas in HPV16/E2 mice. By improving cancer tissue oxygenation and extending the normalization window, Sema3A counteracted sunitinib-induced activation of HIF-1α, Met tyrosine kinase receptor, epithelial-mesenchymal transition (EMT), and other hypoxia-dependent signaling pathways. Sema3A also reduced tumor hypoxia and halted cancer dissemination induced by DC101, a specific inhibitor of the VEGF pathway. As a result, reexpressing Sema3A in cancer cells converts metastatic PNETs and cervical carcinomas into benign lesions. We therefore suggest that this strategy could be developed to safely harnesses the therapeutic potential of the antiangiogenic treatment.

AP-2α and AP-2γ regulate tumor progression via specific genetic programs

The events occurring during tumor formation and progression display similarities to some of the steps in embryonic morphogenesis. The family of AP‐2 proteins consists of five different transcription factors (α, β, γ, δ, and ε) that play relevant roles in embryonic development, as demonstrated by the phenotypes of the corresponding knockout mice. Here, we show that AP‐2α and AP‐2γ proteins play an essential role in tumorigenesis. Down‐modulation of AP‐2 expression in tumor cells by RNA interference (RNAi) led to enhanced tumor growth and reduced chemotherapy‐induced cell death, as well as migration and invasion. Most of these biological modulations were rescued by AP‐2 overexpression. We observed that increased xeno‐transplant growth was mostly due to highly enhanced proliferation of the tumor cells together with reduced innate immune cell recruitment. Moreover, we showed that migration impairment was mediated, at least in part, by secreted factors. To identify the genetic programs involved in tumorigenesis, we performed whole genome microarray analysis of AP‐2α knockdown cells and observed that AP‐2α regulates specific genes involved in cell cycle, cell death, adhesion, and migration. In particular, we showed that ESDN, EREG, and CXCL2 play a major role in AP‐2 controlled migration, as ablation of any of these genes severely altered migration.—Orso, F., Penna, E., Cimino, D., Astanina, E., Maione, F., Valdembri, D., Giraudo, E., Serini, G., Sismondi, P., De Bortoli, M., Taverna, D. AP‐2α and AP‐2γ regulate tumor progression via specific genetic programs. FASEB J. 22, 2702‐2714 (2008)

Tumour growth inhibition and anti‐metastatic activity of a mutated furin‐resistant Semaphorin 3E isoform

Secreted Semaphorin 3E (Sema3E) promotes cancer cell invasiveness and metastatic spreading. The pro‐metastatic activity of Sema3E is due to its proteolytic fragment p61, capable of transactivating the oncogenic tyrosine kinase ErbB2 that associates with the Sema3E receptor PlexinD1 in cancer cells. Here, we show that a mutated, uncleavable variant of Sema3E (Uncl‐Sema3E) binds to PlexinD1 like p61‐Sema3E, but does not promote the association of PlexinD1 with ErbB2 nor activates the ensuing signalling cascade leading to metastatic spreading. Furthermore, Uncl‐Sema3E competes with endogenous p61‐Sema3E produced by tumour cells, thereby hampering their metastatic ability. Uncl‐Sema3E also acts independently as a potent anti‐angiogenic factor. It activates a PlexinD1‐mediated signalling cascade in endothelial cells that leads to the inhibition of adhesion to extracellular matrix, directional migration and cell survival. The putative therapeutic potential of Uncl‐Sema3E was validated in multiple orthotopic or spontaneous tumour models in vivo, where either local or systemic delivery of Uncl‐Sema3E‐reduced angiogenesis, growth and metastasis, even in the case of tumours refractory to treatment with a soluble vascular endothelial growth factor trap. In summary, we conclude that Uncl‐Sema3E is a novel inhibitor of tumour angiogenesis and growth that concomitantly hampers metastatic spreading.

Semaphorins and tumor angiogenesis

Semaphorins belong to a large family of proteins well-conserved along evolution from viruses to mammalians. Secreted and membrane-bound semaphorins participate in a wide range of biological phenomena including development and regeneration of nervous system, cardiovascular development, and immune system activities. Different classes of semaphorins are bifunctional and often exert opposite effects (i.e., repellent or attractive) by acting through the plexin receptor family. However, some classes use other membrane receptors and the same plexin-mediated signals may be modulated by co-receptors, in particular neuropilins or some tyrosine kinase receptors. In cancer, semaphorins have both tumor-suppressor and tumor-promoting functions, by acting on both tumor and stromal components. Here, we review the role of semaphorins in tumor angiogenesis and propose that an unbalance between autocrine loops respectively involving angiogenic inducers and class 3 semaphorin is instrumental for structural and functional abnormalities observed in tumor vasculature.

Semaphorin 4A Exerts a Proangiogenic Effect by Enhancing Vascular Endothelial Growth Factor-A Expression in Macrophages

The axon guidance cues semaphorins (Semas) and their receptors plexins have been shown to regulate both physiological and pathological angiogenesis. Sema4A plays an important role in the immune system by inducing T cell activation, but to date, the role of Sema4A in regulating the function of macrophages during the angiogenic and inflammatory processes remains unclear. In this study, we show that macrophage activation by TLR ligands LPS and polyinosinic-polycytidylic acid induced a time-dependent increase of Sema4A and its receptors PlexinB2 and PlexinD1. Moreover, in a thioglycollate-induced peritonitis mouse model, Sema4A was detected in circulating Ly6Chigh inflammatory monocytes and peritoneal macrophages. Acting via PlexinD1, exogenous Sema4A strongly increased macrophage migration. Of note, Sema4A-activated PlexinD1 enhanced the expression of vascular endothelial growth factor-A, but not of inflammatory chemokines. Sema4A-stimulated macrophages were able to activate vascular endothelial growth factor receptor-2 and the PI3K/serine/threonine kinase Akt pathway in endothelial cells and to sustain their migration and in vivo angiogenesis. Remarkably, in an in vivo cardiac ischemia/reperfusion mouse model, Sema4A was highly expressed in macrophages recruited at the injured area. We conclude that Sema4A activates a specialized and restricted genetic program in macrophages able to sustain angiogenesis and participates in their recruitment and activation in inflammatory injuries.

Inactivation of DNA repair triggers neoantigen generation and impairs tumour growth

Molecular alterations in genes involved in DNA mismatch repair (MMR) promote cancer initiation and foster tumour progression. Cancers deficient in MMR frequently show favourable prognosis and indolent progression. The functional basis of the clinical outcome of patients with tumours that are deficient in MMR is not clear. Here we genetically inactivate MutL homologue 1 (MLH1) in colorectal, breast and pancreatic mouse cancer cells. The growth of MMR-deficient cells was comparable to their proficient counterparts in vitro and on transplantation in immunocompromised mice. By contrast, MMR-deficient cancer cells grew poorly when transplanted in syngeneic mice. The inactivation of MMR increased the mutational burden and led to dynamic mutational profiles, which resulted in the persistent renewal of neoantigens in vitro and in vivo, whereas MMR-proficient cells exhibited stable mutational load and neoantigen profiles over time. Immune surveillance improved when cancer cells, in which MLH1 had been inactivated, accumulated neoantigens for several generations. When restricted to a clonal population, the dynamic generation of neoantigens driven by MMR further increased immune surveillance. Inactivation of MMR, driven by acquired resistance to the clinical agent temozolomide, increased mutational load, promoted continuous renewal of neoantigens in human colorectal cancers and triggered immune surveillance in mouse models. These results suggest that targeting DNA repair processes can increase the burden of neoantigens in tumour cells; this has the potential to be exploited in therapeutic approaches.

Proteomics-Based Metabolic Modeling Reveals That Fatty Acid Oxidation (FAO) Controls Endothelial Cell (EC) Permeability

Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability.

SILAC-based proteomics of human primary endothelial cell morphogenesis unveils tumor angiogenic markers

Proteomics has been successfully used for cell culture on dishes, but more complex cellular systems have proven to be challenging and so far poorly approached with proteomics. Because of the complexity of the angiogenic program, we still do not have a complete understanding of the molecular mechanisms involved in this process, and there have been no in depth quantitative proteomic studies. Plating endothelial cells on matrigel recapitulates aspects of vessel growth, and here we investigate this mechanism by using a spike-in SILAC quantitative proteomic approach. By comparing proteomic changes in primary human endothelial cells morphogenesis on matrigel to general adhesion mechanisms in cells spreading on culture dish, we pinpoint pathways and proteins modulated by endothelial cells. The cell–extracellular matrix adhesion proteome depends on the adhesion substrate, and a detailed proteomic profile of the extracellular matrix secreted by endothelial cells identified CLEC14A as a matrix component, which binds to MMRN2. We verify deregulated levels of these proteins during tumor angiogenesis in models of multistage carcinogenesis. This is the most in depth quantitative proteomic study of endothelial cell morphogenesis, which shows the potential of applying high accuracy quantitative proteomics to in vitro models of vessel growth to shed new light on mechanisms that accompany pathological angiogenesis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000359.

Targeting oncogenic serine/threonine-protein kinase BRAF in cancer cells inhibits angiogenesis and abrogates hypoxia

Carcinomas are comprised of transformed epithelial cells that are supported in their growth by a dedicated neovasculature. How the genetic milieu of the epithelial compartment influences tumor angiogenesis is largely unexplored. Drugs targeted to mutant cancer genes may act not only on tumor cells but also, directly or indirectly, on the surrounding stroma. We investigated the role of the BRAFV600E oncogene in tumor/vessel crosstalk and analyzed the effect of the BRAF inhibitor PLX4720 on tumor angiogenesis. Knock-in of the BRAFV600E allele into the genome of human epithelial cells triggered their angiogenic response. In cancer cells harboring oncogenic BRAF, the inhibitor PLX4720 switches off the ERK pathway and inhibits the expression of proangiogenic molecules. In tumor xenografts harboring the BRAFV600E, PLX4720 extensively modifies the vascular network causing abrogation of hypoxia. Overall, our results provide a functional link between oncogenic BRAF and angiogenesis. Furthermore, they indicate how the tumor vasculature can be “indirectly” besieged through targeting of a genetic lesion to which the cancer cells are addicted.