Andrea Casazza

Impeding Macrophage Entry into Hypoxic Tumor Areas by Sema3A/Nrp1 Signaling Blockade Inhibits Angiogenesis and Restores Antitumor Immunity

Recruitment of tumor-associated macrophages (TAMs) into avascular areas sustains tumor progression; however, the underlying guidance mechanisms are unknown. Here, we report that hypoxia-induced Semaphorin 3A (Sema3A) acts as an attractant for TAMs by triggering vascular endothelial growth factor receptor 1 phosphorylation through the associated holoreceptor, composed of Neuropilin-1 (Nrp1) and PlexinA1/PlexinA4. Importantly, whereas Nrp1 levels are downregulated in the hypoxic environment, Sema3A continues to regulate TAMs in an Nrp1-independent manner by eliciting PlexinA1/PlexinA4-mediated stop signals, which retain them inside the hypoxic niche. Consistently, gene deletion of Nrp1 in macrophages favors TAMs entrapment in normoxic tumor regions, which abates their pro-angiogenic and immunosuppressive functions, hence inhibiting tumor growth and metastasis. This study shows that TAMs heterogeneity depends on their localization, which is tightly controlled by Sema3A/Nrp1 signaling.

miR-511-3p Modulates Genetic Programs of Tumor-Associated Macrophages

Expression of the mannose receptor (MRC1/CD206) identifies macrophage subtypes, such as alternatively activated macrophages (AAMs) and M2-polarized tumor-associated macrophages (TAMs), which are endowed with tissue-remodeling, proangiogenic, and protumoral activity. However, the significance of MRC1 expression for TAMs protumoral activity is unclear. Here, we describe and characterize miR-511-3p, an intronic microRNA (miRNA) encoded by both mouse and human MRC1 genes. By using sensitive miRNA reporter vectors, we demonstrate robust expression and bioactivity of miR-511-3p in MRC1(+) AAMs and TAMs. Unexpectedly, enforced expression of miR-511-3p tuned down the protumoral gene signature of MRC1(+) TAMs and inhibited tumor growth. Our findings suggest that transcriptional activation of Mrc1 in TAMs evokes a genetic program orchestrated by miR-511-3p, which limits rather than enhances their protumoral functions. Besides uncovering a role for MRC1 as gatekeeper of TAMs protumoral genetic programs, these observations suggest that endogenous miRNAs may operate to establish thresholds for inflammatory cell activation in tumors.

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.

Identification of a chronic non-neurodegenerative microglia activation state in a mouse model of peroxisomal β-oxidation deficiency.

The functional diversity and molecular adaptations of reactive microglia in the chronically inflamed central nervous system (CNS) are poorly understood. We previously showed that mice lacking multifunctional protein 2 (MFP2), a pivotal enzyme in peroxisomal β‐oxidation, persistently accumulate reactive myeloid cells in the gray matter of the CNS. Here, we show that the increased numbers of myeloid cells solely derive from the proliferation of resident microglia and not from infiltrating monocytes. We defined the signature of Mfp2−/− microglia by gene expression profiling after acute isolation, which was validated by quantitative polymerase reaction (qPCR), immunohistochemical, and flow cytometric analysis. The features of Mfp2−/− microglia were compared with those from SOD1G93A mice, an amyotrophic lateral sclerosis model. In contrast to the neurodegenerative milieu of SOD1G93A spinal cord, neurons were intact in Mfp2−/− brain and Mfp2−/− microglia lacked signs of phagocytic and neurotoxic activity. The chronically reactive state of Mfp2−/− microglia was accompanied by the downregulation of markers that specify the unique microglial signature in homeostatic conditions. In contrast, mammalian target of rapamycin (mTOR) and downstream glycolytic and protein translation pathways were induced, indicative of metabolic adaptations. Mfp2−/− microglia were immunologically activated but not polarized to a pro‐ or anti‐inflammatory phenotype. A peripheral lipopolysaccharide challenge provoked an exaggerated inflammatory response in Mfp2−/− brain, consistent with a primed state. Taken together, we demonstrate that chronic activation of resident microglia does not necessarily lead to phagocytosis nor overt neurotoxicity. GLIA 2015;63:1606–1620

In vivo antitumoral efficacy of PhAc-ALGP-doxorubicin, an enzyme-activated doxorubicin prodrug, in patient-derived soft tissue sarcoma xenograft models

Given the very limited efficacy of doxorubicin (doxo) in soft tissue sarcoma, there is a clear need for more active and less toxic treatments for this family of diseases. However, due to the rarity of these malignancies and lack of reliable preclinical models, development of new therapies has lagged behind. We evaluated the efficacy of PhAc-ALGP-doxorubicin (ALGP-doxo), a prodrug metabolized to doxo by peptidases present in tumor cells and/or tumor microenvironment, in a synovial sarcoma (SynSa) and two dedifferentiated liposarcoma (DDLPS) patient-derived xenograft models. Sixty-eight mice were engrafted bilaterally with human DDLPS or SynSa and randomized to control, doxo, or ALGP-doxo treatment, which were administered using an intraperitoneal minipump. Tumor volume measurement, histopathology, and Western blotting were used to assess treatment efficacy. Tumor regrowth was evaluated in a subset of mice over a period of 2 weeks after treatment cessation. Although tumor volume in the control and doxo groups increased steadily, ALGP-doxo caused tumor volume stabilization in the DDLPS xenografts and significant tumor shrinkage in the SynSa model, continuing after treatment cessation. A significant decrease in proliferation and increase in apoptosis compared with control and doxo was observed during and after treatment with ALGP-doxo in all models. In conclusion, ALGP-doxo shows considerably higher antitumoral efficacy compared with doxo in all patient-derived xenograft models tested. Administration of a 30- to 40-fold higher dose of ALGP-doxo than doxo is tolerated without significant adverse events. These results warrant further testing of this prodrug in anthracycline-sensitive and -resistant models of soft tissue sarcoma. Mol Cancer Ther; 16(8); 1566–75. ©2017 AACR.

Sustained Placental Growth Factor-2 Treatment Does Not Aggravate Advanced Atherosclerosis in Ischemic Cardiomyopathy

Angiogenic growth factor therapy for ischemic cardiovascular disease carries a risk of stimulating atherosclerotic plaque growth. We evaluated risk benefit ratio of sustained administration of recombinant human placental growth factor (rhPlGF)-2 in mice with advanced atherosclerosis and chronic ischemic cardiomyopathy. We maintained apolipoprotein E-deficient mice on a high cholesterol diet and induced myocardial infarction by transient ligation at 4xa0weeks. At 8xa0weeks, we assessed left ventricular (LV) function and randomized mice to receive rhPlGF-2 or vehicle (VEH) subcutaneously for 28xa0days. Administration of rhPlGF-2 significantly increased PlGF plasma levels without adverse hemodynamic or systemic inflammatory effects. RhPlGF-2 did not increase plaque area, composition, or vulnerability in the aortic arch. RhPlGF-2 significantly improved contractile function and reduced LV end-systolic and end-diastolic volume indices with a concomitant increase in capillary and arteriolar density in ischemic myocardium. RhPlGF-2 may represent a promising therapeutic strategy in chronic ischemic cardiomyopathy.

Abstract 180: PhAc-ALGP-Dox is a new tumor selective peptide prodrug of doxorubicin that shows improved efficacy and systemic tolerance in triple negative breast cancer models

Use of traditional chemotherapeutic drugs is restricted by severe side effects and lack of tumor specificity of these cytotoxic agents. Less toxic prodrug that can be selectively activated in tumor tissue have been explored in attempts to improve the therapeutic index. Some approaches to the development of tumor activating prodrugs take advantage of inherent properties of the tumor, for example, selective enzyme expression, hypoxia, or low extracellular pH in the vicinity of the tumor. CoBioRes makes use of capped, tetrapeptidic prodrug of existing chemotherapeutics. Doxorubicin (Dox) was the first chemotherapeutic that was linked to the tetrapeptide sequence and tested experimentally (PhAc-ALGP-Dox). This approach makes the prodrug impermeable to cell membranes of both normal and tumor cells. Furthermore, it remains stable in blood by the use of the capping group on the amino-terminal group of the peptide that prevents aspecific activation by circulating esopeptidases. The prodrug as such is thus not active. The peptide sequences have been developed to be sensitive to the hydrolytic action of a selected group of peptidases that are released within the tumor microenvironment. These peptidases, which accumulate in the tumors, also play an important role in cancer cell invasion and metastasis. What makes this approach unique is that it is not targeting a single enzyme, but requires, for its selectivity, a two-step activation based on enzymes with an increasing selectivity. In vivo activation starts when the tetrapeptide prodrug is exposed to endonucleases (CD10 and THOP1) leading to the formation of intermediates that are still inactive and poorly permeable through cell membranes. This leads to a tissue distribution that is determined by the expression of the endonucleases. Since these are known to be overexpressed in several tumors, and only present at lower level in some normal tissues, the prodrug will be preferentially located and activated in the vicinity of tumors. The second step of the activation is the cleavage at the prolyl by dipeptidases FAP and DPPIV, releasing the free doxorubicin. The simultaneous expression of all the enzymes involved in the prodrug activation was confirmed both in human triple negative breast cancer biopsies and in experimental triple negative breast cancer models. In vitro analysis of PhAc-ALGP-Dox reveals higher tumor cells tropism and higher cytotoxicity in cancer cells than in normal cells. In vivo PhAc-ALGP-Dox gives promising preliminary results in terms of systemic tolerance even at high concentration, and in vivo efficacy in xenograft mouse models compared to free doxorubicin. Taken together, these results provide a strong rationale for further investigation aimed at unleashing the potential clinical value of this compound. Citation Format: Andrea Casazza, Massimiliano Mazzone, Peter Pokreisz. PhAc-ALGP-Dox is a new tumor selective peptide prodrug of doxorubicin that shows improved efficacy and systemic tolerance in triple negative breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 180. doi:10.1158/1538-7445.AM2017-180

Abstract PR01: CLIC3 is secreted by CAFs and enhances angiogenesis and tumor cell invasion by cooperating with TGM2

Cancer-associated fibroblasts (CAFs) play a central role in tumor progression through the mechanical remodeling of the stroma. Indeed CAFs secrete a plethora of extracellular matrix (ECM) components and ECM modifiers which contribute to generate stiff and dense tumors. Increased tumor stiffness induces endothelial cell (EC) sprouting and tumor cell invasion. Moreover, excessive stiffness represents a critical barrier to therapy because it blocks perfusion thus preventing diffusion of drugs and favoring hypoxia. Tuning tumor stiffness has therefore the potential to contribute to improve the efficacy of conventional anti-cancer therapies. Our study aims at using unbiased proteomics approach to identify CAF proteins which alter the tumor stroma, and investigating their functional role. We have established mass spectrometry-based proteomics approach to accurately and in-depth analyze secretomes of cells in culture, and used it to compare cell lines of human mammary normal (iNF) and myofibroblasts-like cancer-associated (iCAF) fibroblasts. Unexpectedly, we detected the chloride intracellular channel protein 3 (CLIC3), which so far was known as an intracellular regulator of receptor trafficking in tumor cells, amongst the most up-regulated proteins in the ECM generated by iCAF. Highlighting the relevance of CLIC3 in a clinical context, high levels of CLIC3 were also measured in CAFs isolated from patient samples. Notably, CLIC3 silencing reduces the pro-angiogenic and pro-invasive activity of CAFs in vitro. To investigate the role of secreted CLIC3, we have generated the recombinant protein (rCLIC3) and the mutant for the active site (rCLIC3mut) and show that rCLIC3 induces angiogenesis in vitro and in vivo, and tumor cell invasion in vitro and that its active site is required for this activity. Furthermore, we show that rCLIC3 is used by iCAF to increase the stiffness of the ECM that they produce. Using gene silencing and specific inhibitors in combination with functional assays in vitro and in vivo, we show that rCLIC3 exerts its functions through the cooperation with the ECM remodeling enzyme transglutaminase 2 (TGM2). Importantly, TGM2 is highly abundant in many tumors. In a clinical context, CLIC3 staining of various TMAs shows that this protein is expressed in the stroma of some tumors and that it is particularly abundant in the stroma of ovarian cancers. Preliminary analysis of ovarian TMAs shows that the stroma of aggressive and highly vascularized tumors strongly stains for CLIC3. In conclusion, our work has discovered a completely unexpected function for CLIC3, which we have now characterized as a stromal protein which is secreted and alters the ECM stiffness cooperating with TGM2, thus enhancing the pro-angiogenic and pro-invasive properties of the CAFs. CLIC3 is therefore a promising candidate to be further investigated for its role in tumor progression and as a target to improve current anti-cancer therapy. Citation Format: Juan Ramon Hernandez-Fernaud, Elena Ruengeler, Andrea Casazza, Lisa Neilson, Ellie Pulleine, Iain MacPherson, Karen Blyth, Huabing Yin, Massimiliano Mazzone, Jim Norman, Sara Zanivan. CLIC3 is secreted by CAFs and enhances angiogenesis and tumor cell invasion by cooperating with TGM2. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr PR01.