Jay Harper

Cartilage to bone—Angiogenesis leads the way

Vascular endothelial growth factor plays an important part in longitudinal bone growth by stimulating angiogenesis into the epiphyseal growth plate (pages 623–628).

VEGF-A induces angiogenesis by perturbing the cathepsin-cysteine protease inhibitor balance in venules, causing basement membrane degradation and mother vessel formation

Tumors initiate angiogenesis primarily by secreting vascular endothelial growth factor (VEGF-A(164)). The first new vessels to form are greatly enlarged, pericyte-poor sinusoids, called mother vessels (MV), that originate from preexisting venules. We postulated that the venular enlargement necessary to form MV would require a selective degradation of their basement membranes, rigid structures that resist vascular expansion. To identify the specific proteases responsible for MV formation, we induced angiogenesis in mouse tissues with an adenoviral vector expressing VEGF-A(164) (Ad-VEGF-A(164)) or with VEGF-A-secreting TA3/St mammary tumors. We found that MV formation resulted from greatly increased activity of cathepsins (B>S>L) in venules transitioning into MV, as well as from a reciprocal decrease in the expression of several cysteine protease inhibitors (CPI), stefin A and cystatins B and C, by these same venules. Using a fluorescence probe that selectively binds cellular sites of cathepsin protease activity in vivo, we showed that increased cathepsin activity was localized exclusively to perivenular cells, not to venule endothelial cells. CPI strikingly inhibited angiogenesis in the Matrigel assay, and Ad-VEGF-A(164)-induced angiogenesis was reduced by approximately 50% in cathepsin B-null mice. Thus, VEGF-A, whether expressed by interstitial cells infected with an adenoviral vector or by tumor cells, upsets the normal cathepsin-CPI balance in nearby venules, leading to degradation of their basement membranes, an important first step in angiogenesis.

Neuropilin-1 expression in osteogenic cells: down-regulation during differentiation of osteoblasts into osteocytes.

The expression of neuropilin‐1 (NRP1), a recently described VEGF and semaphorin receptor expressed by endothelial cells (EC) but some non‐EC types as well, was analyzed in osteoblasts in vitro and in vivo. Cultured MC3T3‐E1 osteoblasts expressed NRP1 mRNA and bound VEGF165 but not VEGF121, characteristic of the VEGF isoform‐specific binding of NRP1. These cells did not express VEGFR‐1 or VEGFR‐2 so that VEGF binding to osteoblasts was strictly NRP1‐dependent. In a chick osteocyte differentiation system, NRP1 was expressed by osteoblasts but its expression was absent as the cells matured into osteocytes. Immunohistochemical localization of NRP1 within the developing bones of 36‐day‐old mice and embryonic Day 17 chicks demonstrated that NRP1 was expressed by osteoblasts migrating alongside invading blood vessels within the metaphysis of the growth plate, as well as by osteoblasts at the developing edge of trabeculae within the marrow cavity. On the other hand, NRP1 was not expressed by osteocytes in either species, consistent with the in vitro results. In addition to osteogenic cells, NRP1 expression by EC was observed throughout the bone. Together these results suggest that NRP1 might have a dual function in bone by mediating osteoblast function directly as well as angiogenesis. J. Cell. Biochem. 81:82–92, 2001.

Molecular regulation of tumor angiogenesis: mechanisms and therapeutic implications

Angiogenesis, the process of new capillary formation from a pre-existing vessel plays an essential role in both embryonic and postnatal development, in the remodeling of various organ systems, and in several pathologies, particularly cancer. In the last 20 years of angiogenesis research, a variety of angiogenic regulators, both positive and negative, have been identified. The discovery of several anti-angiogenic factors has led to the development of novel cancer therapies based on targeting a tumors vascular supply. A number of these new therapies are currently being tested in clinical trials in the U.S.A. and elsewhere. A major advance in the field of anti-angiogenic therapy occurred recently when the FDA approved Avastin (bevacizumab), the first solely anti-angiogenesis therapy approved for treatment of human cancer. While it has long been appreciated that tumor growth and progression are dependent on angiogenesis, it is only recently that progress has been made in elucidating the molecular mechanisms that regulate the earliest stage in the angiogenic program, the angiogenic switch. This checkpoint is characterized by the transition of a dormant, avascular tumor into an active, vascular one. Anti-angiogenic therapies to date have essentially been designed to suppress the neovasculature in established tumors. However, identifying the mechanisms that cause a tumor to acquire an angiogenic phenotype may lead to the discovery of new therapeutic modalities and complementary diagnostics that could be used to block the angiogenic switch, thereby preventing subsequent tumor progression. In this chapter on the role of angiogenesis in cancer, we (1) provide an overview of the process of angiogenesis with special regard to the molecules and physiological conditions that regulate this process, (2) review recent studies describing the use of anti-angiogenic approaches in the treatment of a variety of human cancers, and (3) discuss the recent literature focused on the study of the molecules and molecular mechanisms that may be regulating the initiation of the angiogenic phenotype in tumors, and the clinical impact that this knowledge may have in the future.

Repression of Vascular Endothelial Growth Factor Expression by the Zinc Finger Transcription Factor ZNF24

Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis. Although many positive regulators of VEGF have been identified, relatively little is known regarding the negative regulation of VEGF expression. We identified a zinc finger transcription factor, ZNF24, that may repress VEGF transcription. An inverse correlation between expression of VEGF and ZNF24 was observed in a series of independent studies. ZNF24 was up-regulated in angiogenic tumor nodules where VEGF expression is significantly decreased compared with preangiogenic nodules. In human breast carcinoma cells cultured under normoxic conditions, ZNF24 levels were significantly up-regulated whereas VEGF levels were low. In contrast, VEGF was significantly increased in hypoxic cells whereas ZNF24 was down-regulated. The same inverse correlation between ZNF24 and VEGF was also observed in 70% of matched cDNA pairs of normal and malignant tissues from human colon and breast biopsies. Overexpression of ZNF24 resulted in a significant down-regulation of VEGF, whereas silencing of ZNF24 with small interfering RNA led to increased VEGF expression. Cotransfection of ZNF24 and a VEGF promoter luciferase reporter construct in MDA-MB-231 cells resulted in a significant decrease in VEGF promoter activity. Taken together, these data suggest that ZNF24 is involved in negative regulation of VEGF and may represent a novel repressor of VEGF transcription.

Matrilin-1 is an inhibitor of neovascularization

Background: The relative avascularity of cartilage has made it a promising source of angiogenesis inhibitors. Results: MATN-1, identified by mass spectrometry, suppresses capillary endothelial cell proliferation and migration. Conclusion: MATN-1 is a novel inhibitor of neovascularization in vivo and in vitro. Significance: This is the first demonstration that MATN-1 is an inhibitor of both normal and pathological neovascularization. In the course of conducting a series of studies whose goal was to discover novel endogenous angiogenesis inhibitors, we have purified matrilin-1 (MATN-1) and have demonstrated, for the first time, that it inhibits neovascularization both in vitro and in vivo. Proteins were extracted from cartilage using a 2 m NaCl, 0.01 m HEPES buffer at 4 °C, followed by concentration of the extract. The concentrate was fractionated by size exclusion chromatography, and fractions were then screened for their ability to inhibit capillary endothelial cell (EC) proliferation in vitro. Fractions containing EC inhibitory activity were pooled and further purified by cation exchange chromatography. The resulting fractions from this step were then screened to isolate the antiangiogenic activity in vitro. This activity was identified by tandem mass spectrometry as being MATN-1. Human MATN-1 was cloned and expressed in Pichia pastoris and purified to homogeneity. Purified recombinant MATN-1, along with purified native protein, was shown to inhibit angiogenesis in vivo using the chick chorioallantoic membrane assay by the inhibition of capillary EC proliferation and migration. Finally, using a MATN-1-deficient mouse, we showed that angiogenesis during fracture healing was significantly higher in MATN-1−/− mice compared with the wild type mice as demonstrated by in vivo imaging and by elevated expression of angiogenesis markers including PECAM1, VEGFR, and VE-cadherin.

Abstract 76: Synthetic lethal targeting ofBRCAmutant tumors with antibody linked pyrrolobenzodiazepine dimers

Pyrrolbenzodiazepine dimers (PBDs) are amongst the most potent DNA alkylating agents, with activity against a broad spectrum of tumors. PBDs form cross-links within the minor groove of DNA causing double strand breaks (DSB). DNA repair genes such as BRCA1 and BRCA2 play important roles in homologous recombination repair (HRR) of DSB. Cells defective in BRCA1 or BRCA2 are known to be sensitive to DNA interstrand crosslinks. Accordingly, it is possible that PBD-based ADCs will have enhanced killing of cells (synthetic lethality) in which HR processes are defective by inactivation of BRCA1 or BRCA2 genes in breast, ovarian and other cancers. To determine anti-tumor activity of PBD dimers, we have used MEDI0641, PBD-dimer conjugated to anti-5T4 antibody, against BRCA wild type and mutant xenograft tumor models. MEDI0641 was >3-fold more potent in BRCA1 or BRCA2 mutant models than in wild-type xenografts. Similar observations were seen in 25 patient-derived xenograft (PDX) models (19 breast and 6 ovarian) bearing mutations in BRCA1 or BRCA2 (blinded to 5T4 expression) treated with MEDI0641. Out of a total of 25 PDX models, 17 models had tumor regression with a single administration of MEDI0641 at 0.3 mg/kg (response rate = 68%), and 14 models showed response to 0.1 mg/kg of MEDI0641 (response rate = 56%). In BRCA wild-type PDX models, a higher dose of 1 mg/kg was required to achieve full anti-tumor efficacy. Retrospective analysis of 5T4 expression in PDX tumors demonstrated no correlation between efficacy and target expression in BRCA mutant PDX models. To further delineate the role of BRCA1/2 mutations in determining sensitivity to PBD, we used siRNA knock-down of both BRCA1 and BRCA2 in the DNA repair wild type HeLa cells. Knockdown of BRCA genes sensitized Hela cells to PBD payload and MEDI0641 in vitro. Anti-tumor activity of MEDI0641 was further examined in isogenic BRCA2 knockout xenograft models. Genetic deletion of BRCA2 markedly increased anti-tumor activity of MEDI0641. In conclusion, PBD based ADCs may have improved therapeutic window in cancer patients with somatic BRCA mutations. Citation Format: Haihong Zhong, Ravinder Tammali, Cui Chen, Christine Fazenbaker, Kennedy Maureen, Noel Monks, Jay Harper, Ronald Herbst, Dave Tice. Synthetic lethal targeting of BRCA mutant tumors with antibody linked pyrrolobenzodiazepine dimers [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 76. doi:10.1158/1538-7445.AM2017-76