RATIONALE:Vascular endothelial growth factor (VEGF) is the main driver of angiogenesis and vascular permeability via VEGF receptor 2 (VEGFR2), whereas lymphangiogenesis signals are transduced by ...VEGFC/D via VEGFR3. VEGFR3 also regulates sprouting angiogenesis and blood vessel growth, but to what extent VEGFR3 signaling controls blood vessel permeability remains unknown.
OBJECTIVE:To investigate the role of VEGFR3 in the regulation of VEGF-induced vascular permeability.
METHODS AND RESULTS:Long-term global Vegfr3 gene deletion in adult mice resulted in increased fibrinogen deposition in lungs and kidneys, indicating enhanced vascular leakage at the steady state. Short-term deletion of Vegfr3 in blood vascular endothelial cells increased baseline leakage in various tissues, as well as in tumors, and exacerbated vascular permeability in response to VEGF, administered via intradermal adenoviral delivery or through systemic injection of recombinant protein. VEGFR3 gene silencing upregulated VEGFR2 protein levels and phosphorylation in cultured endothelial cells. Consistent with elevated VEGFR2 activity, vascular endothelial cadherin showed reduced localization at endothelial cell–cell junctions in postnatal retinas after Vegfr3 deletion, or after VEGFR3 silencing in cultured endothelial cells. Furthermore, concurrent deletion of Vegfr2 prevented VEGF-induced excessive vascular leakage in mice lacking Vegfr3.
CONCLUSIONS:VEGFR3 limits VEGFR2 expression and VEGF/VEGFR2 pathway activity in quiescent and angiogenic blood vascular endothelial cells, thereby preventing excessive vascular permeability.
Angiogenesis, the formation of new blood vessels, is regulated by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). VEGFR2 is abundant in the tip cells of angiogenic sprouts, ...where VEGF/VEGFR2 functions upstream of the delta-like ligand 4 (DLL4)/Notch signal transduction pathway. VEGFR3 is expressed in all endothelia and is indispensable for angiogenesis during early embryonic development. In adults, VEGFR3 is expressed in angiogenic blood vessels and some fenestrated endothelia. VEGFR3 is abundant in endothelial tip cells, where it activates Notch signaling, facilitating the conversion of tip cells to stalk cells during the stabilization of vascular branches. Subsequently, Notch activation suppresses VEGFR3 expression in a negative feedback loop. Here we used conditional deletions and a Notch pathway inhibitor to investigate the cross-talk between VEGFR2, VEGFR3, and Notch in vivo. We show that postnatal angiogenesis requires VEGFR2 signaling also in the absence of Notch or VEGFR3, and that even small amounts of VEGFR2 are able to sustain angiogenesis to some extent. We found that VEGFR2 is required independently of VEGFR3 for endothelial DLL4 up-regulation and angiogenic sprouting, and for VEGFR3 functions in angiogenesis. In contrast, VEGFR2 deletion had no effect, whereas VEGFR3 was essential for postnatal lymphangiogenesis, and even for lymphatic vessel maintenance in adult skin. Knowledge of these interactions and the signaling functions of VEGFRs in blood vessels and lymphatic vessels is essential for the therapeutic manipulation of the vascular system, especially when considering multitargeted antiangiogenic treatments.
Significance VEGF/VEGFR and Notch signaling pathways are major molecular regulators of sprouting angiogenesis. Here we investigated the cross-talk between VEGFR and Notch signaling by using state-of-the-art inducible genetic mouse models. We found that VEGFR2 is absolutely required for the sprouting of endothelial cells that display low Notch activity. VEGFR3 cannot rescue the angiogenic sprouting associated with loss of Notch signaling. On the other hand, postnatal lymphangiogenesis required VEGFR3, but not VEGFR2. Because several new VEGF/VEGFR and Notch modulators are in clinical trials for the treatment of angiogenesis-related diseases, it is of the utmost importance to understand the signaling relationships of VEGFRs. This should also facilitate the design of more effective multitargeted treatments.
Vascular endothelial growth factor C (VEGF-C) is a major driver of lymphangiogenesis in embryos and adults. Vegfc gene deletion in mouse embryos results in failure of lymphangiogenesis, fluid ...accumulation in tissues, and lethality. The VEGF-C receptors VEGFR3 and VEGFR2 are required for embryonic blood vessel formation. The related VEGF is essential for both blood vessel formation and embryonic hematopoiesis, whereas the possible involvement of VEGF-C in hematopoiesis is unknown. Here we unveil a novel hematopoietic function of VEGF-C in fetal erythropoiesis. Deletion of Vegfc in embryonic day 7.5 (E7.5) embryos in the C57BL6 mouse genetic background led to defective fetal erythropoiesis, characterized by anemia and lack of enucleated red blood cells in blood circulation. Macrophages and erythroid cells in the fetal liver (FL) were also decreased after midgestation because of decreased cell proliferation and increased apoptosis. However, the Lin−Sca-1+c-Kit+ stem cell compartment in E14.5 FL was not affected by Vegfc deletion. VEGF-C loss did not disrupt the generation of primitive erythroid cells or erythro-myeloid progenitors (EMPs) in the yolk sac, but it decreased the expression of α4-integrin on EMPs and compromised EMP colonization of the FL. The distribution, maturation, and enucleation of primitive erythroblasts were also impaired by Vegfc deletion. In contrast, Vegfc deletion from E10.5 onward did not compromise definitive hematopoiesis in the liver, and Vegfc deletion in adult mice did not cause anemia. These results reveal an unexpected role for VEGF-C, a major lymphangiogenic growth factor, in the transition to FL erythropoiesis.
•Vegfc is essential for mobilization, maturation, and enucleation of primitive erythroblasts.•Vegfc deletion compromises liver colonization by erythro-myeloid progenitors and subsequent macrophage/erythroid expansion.
Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are quintessential for the development and maintenance of blood and lymphatic vessels. However, genetic interactions between ...the VEGFRs are poorly understood. VEGFR2 is the dominant receptor that is required for the growth and survival of the endothelium, whereas deletion of VEGFR1 or VEGFR3 was reported to induce vasculature overgrowth. Here we show that vascular regression induced by VEGFR2 deletion in postnatal and adult mice is aggravated by additional deletion of VEGFR1 or VEGFR3 in the intestine, kidney, and pancreas, but not in the liver or kidney glomeruli. In the adult mice, hepatic and intestinal vessels regressed within a few days after gene deletion, whereas vessels in skin and retina remained stable for at least four weeks. Our results show changes in endothelial transcriptomes and organ-specific vessel maintenance mechanisms that are dependent on VEGFR signaling pathways and reveal previously unknown functions of VEGFR1 and VEGFR3 in endothelial cells.
Abstract
Background: Several new medicinal products have been introduced for the treatment of advanced breast cancer (aBC) in recent years, many of which are indicated for a specific patient ...population. One of these compounds is alpelisib, a phosphatidylinositol 3-kinase (PI3K) inhibitor, which has shown efficacy in the treatment of hormone receptor positive and human epidermal growth factor receptor 2 negative (HR+/HER2-) advanced breast cancer (aBC) harboring phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) “hotspot” mutations, i.e. mutations affecting the helical (E542K and E545K) and kinase (H1047R) domains. In this retrospective register-based study, the frequency of PIK3CA gene mutations as well as survival among HR+/HER2- aBC patients in Finland was analyzed.
Methods: This study utilized retrospective register-based data from the Hospital District of Southwest Finland (Auria Biobank), which covers approximately 20% of the population in Finland. Patients diagnosed with aBC between 2004─2013 were identified using ICD-10 code C50* (breast cancer) together with customized text mining algorithms to extract metastatic patients. Tumor biomarker data including estrogen receptor (ER), progesterone receptor (PR), and HER2 status were used to identify HR+/HER2- patients. The formalin fixed paraffin embedded (FFPE) tumor tissue samples available for these patients in the Auria Biobank’s tissue archives were screened for PIK3CA mutations with next generation sequencing. Study follow-up period was defined to start from the date of aBC diagnosis and to continue until death or end of 2016, whichever occurred first. Clinical pathology and survival data were collected from the Auria Biobank and electronic medical records of the Hospital District of Southwest Finland. Overall survival (OS) was estimated using the Kaplan-Meier method.
Results: Altogether 444 adult female patients with aBC were identified. HR and HER2 status were available for 377 patients (85%), out of which 274 (73%) were HR+/HER2-. Representative FFPE tumor samples were available for 187 patients and PIK3CA was successfully screened in 161 patients. Out of the sequenced HR+/HER2- samples, 53.4% showed mutation in the PIK3CA gene and 46.6% wild type (wt) PIK3CA gene. Approximately one third (32.3% n=52) of the samples represented PIK3CA hotspot mutations and 18% (n=29) of these displayed more than one PIK3CA variant. The most common PIK3CA hotspot mutation was H1047R. HR+/HER2- patients with the wt PIK3CA gene showed slightly shorter OS compared to patients showing PIK3CA hotspot mutations (18.9 months (95% CI: 14.1-25.1) vs. 22.3 months (95% CI: 17.0-26.3). Higher portion of the wt patients also developed metastases within 1 year from the primary diagnosis compared to the patients with PIK3CA hotspot mutations (41.3% vs. 25.0%). At the time of diagnosis, 92.0% of the wt and 84.6% of the PIK3CA hotspot mutated population was 50 years or older, respectively.
Conclusion: Approximately one third of the HR+/HER2- aBC patient cohort in Finland had at least one variant of PIK3CA hotspot mutations in line with the SOLAR-1 clinical trial population. The OS did not differ markedly between the patients with wt and hotspot mutated PIK3CA gene. The short OS of the patients is probably due to the fact that the patients were treated before the availability of CDK4/6is as a treatment option.
Citation Format: Krista Heinolainen, Silva Saarinen, Antti Ellonen, Antti Karlsson, Meri Utriainen, Simona Vertuani, Barbro Holm. Pik3ca mutations among hormone receptor positive and HER-2 negative advanced breast cancer patients in Finland abstract. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-44.
Purpose
In recent years, several new targeted therapies have emerged for advanced breast cancer (aBC). However, real-life data specific to aBC and different breast cancer subtypes are scarce. This ...retrospective cohort study was designed to describe the distribution of aBC subtypes, incidence, treatment patterns, survival, and
PIK3CA
hotspot mutation frequency.
Methods
The study included all patients in the Hospital District of Southwest Finland diagnosed with aBC between 2004 and 2013 and with a sample available in Auria Biobank. In addition to registry-based data collection, 161 HR+/HER2− aBCs were screened for
PIK3CA
mutations.
Results
Altogether, 54.7% of the 444 patients included in the study had luminal B subtype. The smallest representations were in HR−/HER2+ (4.5%) and triple-negative (5.6%) subgroups. The percentage of aBC among all diagnosed breast cancers increased until 2010, after which it remained stable. The triple-negative cancers were associated with shorter median overall survival (5.5 months) compared to other subgroups (16.5–24.6 months). Most (84%) triple-negative cancers also metastasized during the first two years, whereas this was more evenly distributed over time in other subgroups. Of the HR+/HER2− tumors, 32.3% harbored a
PIK3CA
hotspot mutation. These patients, however, did not have inferior survival compared to patients with
PIK3CA
wild-type cancers.
Conclusion
This study described real-world aBC subgroups and indicated that the clinical outcomes of subgroups vary. Although
PIK3CA
hotspot mutations did not lead to inferior survival, they are relevant as possible treatment targets. Overall, these data could be utilized to further evaluate the subgroup-specific medical needs in breast cancer.
Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth ...factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.
PDF
