The human papillomavirus type 18 E6 oncoprotein induces Vascular Endothelial Growth Factor 121 (VEGF121) transcription from the promoter through a p53-independent mechanism

Altered angiogenic response is associated with high-grade cervical dysplasia and with invasive squamous carcinoma of the cervix. Vascular Endothelial Growth Factor (VEGF) is one of the most potent inducers of angiogenesis and is up-regulated in carcinoma of the cervix. Infection by high-risk human papillomavirus and persistent expression of viral oncogene E6 are etiologically linked to the development of cervical cancer. E6 is able to immortalize cells and induce malignant transformation by inactivating p53. In cervical cancer, regulation of VEGF expression is poorly described. Thus, we investigated whether E6 oncoprotein could regulate VEGF expression in HPV18-positive cervical cancer-derived HeLa cells harboring a wild-type p53. The alternative splicing of vegf mRNA renders three major isoforms of 121, 165 and 189 amino-acids in humans. We have designed isoform specific real time QRT-PCR assays to quantitate vegf transcripts and VEGF121 was the predominant isoform. Silencing HPV18 E6 mRNA with specific siRNA reduced VEGF121 expression by at least 50% whereas silencing of p53 did not alter its expression. Treatment with cycloheximide did not inhibit E6-induced VEGF121 expression. Collectively, these results suggest that HPV18 E6 oncoprotein contributes to tumor angiogenesis by inducing VEGF transcription from the promoter in a p53-independent manner

GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment

The G protein estrogen receptor GPER/GPR30 mediates estrogen action in breast cancer cells as well as in breast cancer-associated fibroblasts (CAFs), which are key components of microenvironment driving tumor progression. GPER is a transcriptional target of hypoxia inducible factor 1 alpha (HIF-1α) and activates VEGF expression and angiogenesis in hypoxic breast tumor microenvironment. Furthermore, IGF1/IGF1R signaling, which has angiogenic effects, has been shown to activate GPER in breast cancer cells. We analyzed gene expression data from published studies representing almost 5000 breast cancer patients to investigate whether GPER and IGF1 signaling establish an angiocrine gene signature in breast cancer patients. Next, we used GPER-positive but estrogen receptor (ER)-negative primary CAF cells derived from patient breast tumours and SKBR3 breast cancer cells to investigate the role of GPER in the regulation of VEGF expression and angiogenesis triggered by IGF1. We performed gene expression and promoter studies, western blotting and immunofluorescence analysis, gene silencing strategies and endothelial tube formation assays to evaluate the involvement of the HIF-1α/GPER/VEGF signaling in the biological responses to IGF1. We first determined that GPER is co-expressed with IGF1R and with the vessel marker CD34 in human breast tumors (n = 4972). Next, we determined that IGF1/IGF1R signaling engages the ERK1/2 and AKT transduction pathways to induce the expression of HIF-1α and its targets GPER and VEGF. We found that a functional cooperation between HIF-1α and GPER is essential for the transcriptional activation of VEGF induced by IGF1. Finally, using conditioned medium from CAFs and SKBR3 cells stimulated with IGF1, we established that HIF-1α and GPER are both required for VEGF-induced human vascular endothelial cell tube formation. These findings shed new light on the essential role played by GPER in IGF1/IGF1R signaling that induces breast tumor angiogenesis. Targeting the multifaceted interactions between cancer cells and tumor microenvironment involving both GPCRs and growth factor receptors has potential in future combination anticancer therapies

Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation

Background: Estrogen receptors alpha (ERa) and beta (ERb) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ERb being able to modulate the effects of ERa on gene transcription and cell proliferation. ERb is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERb in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology. Results: Expression of full-length ERb in hormone-responsive, ERa-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERb and 6024 ERa binding sites in estrogen-stimulated cells, comprising sites occupied by either ERb, ERa or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ERb+ vs ERb- cells, 424 showed one or more ERb site within 10 kb. These putative primary ERb target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ERb binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions. Conclusions: Results indicate that the vast majority of the genomic targets of ERb can bind also ERa, suggesting that the overall action of ERb on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell

Hypertonic Stress Induces VEGF Production in Human Colon Cancer Cell Line Caco-2: Inhibitory Role of Autocrine PGE2

Vascular Endothelial Growth Factor (VEGF) is a major regulator of angiogenesis. VEGF expression is up regulated in response to micro-environmental cues related to poor blood supply such as hypoxia. However, regulation of VEGF expression in cancer cells is not limited to the stress response due to increased volume of the tumor mass. Lipid mediators in particular arachidonic acid-derived prostaglandin (PG)E2 are regulators of VEGF expression and angiogenesis in colon cancer. In addition, increased osmolarity that is generated during colonic water absorption and feces consolidation seems to activate colon cancer cells and promote PGE2 generation. Such physiological stimulation may provide signaling for cancer promotion. Here we investigated the effect of exposure to a hypertonic medium, to emulate colonic environment, on VEGF production by colon cancer cells. The role of concomitant PGE2 generation and MAPK activation was addressed by specific pharmacological inhibition. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked VEGF and PGE2 production. VEGF production was inhibited by selective inhibitors of ERK 1/2 and p38 MAPK pathways. To address the regulatory role of PGE2 on VEGF production, Caco-2 cells were treated with cPLA2 (ATK) and COX-2 (NS-398) inhibitors, that completely block PGE2 generation. The Caco-2 cells were also treated with a non selective PGE2 receptor antagonist. Each treatment significantly increased the hypertonic stress-induced VEGF production. Moreover, addition of PGE2 or selective EP2 receptor agonist to activated Caco-2 cells inhibited VEGF production. The autocrine inhibitory role for PGE2 appears to be selective to hypertonic environment since VEGF production induced by exposure to CoCl2 was decreased by inhibition of concomitant PGE2 generation. Our results indicated that hypertonicity stimulates VEGF production in colon cancer cell lines. Also PGE2 plays an inhibitory role on VEGF production by Caco-2 cells exposed to hyperosmotic stress through EP2 activation

Integrated Relaxation Pressure (IRP) Distinguishes between Reflux-Predominant and Dysphagia-Predominant Phenotypes of Esophageal “Absent Contractility”

Background: Patients with absent contractility (AC) often suffer from either reflux or dysphagia. It remains unclear what factors determine which phenotype patients present with. We sought to evaluate if high-resolution manometry metrics, especially integrated relaxation pressure (IRP), could explain this. Methods: Cases of AC from three medical centers were reviewed for demographic, clinical, and manometric data. Cases with an IRP between 10–15 mmHg or subsequent diagnosis of achalasia were excluded. Results: 69 subjects were included (mean age 56.1; 71% female). A total of 41 (59.4%) were reflux-predominant. The reflux-predominant group was younger (51.1 vs. 63.5, p = 0.002) and had lower median LES basal pressures (7.5 vs. 12.5 mmHg, p = 0.014) and IRP values (1.5 vs. 5.6 mmHg, p < 0.001) compared to the dysphagia group. When divided into tertiles, the trend in symptoms between LES basal pressure tertiles was not significant. However, the trend for IRP was significant (p < 0.001). For example, in the lowest IRP tertile, 91.3% of subjects were reflux-predominant compared to only 26.1% in the highest tertile, while the dysphagia-predominant group increased from 8.7% to 73.9%. In a regression model controlling for age and using IRP tertile 1 as the reference, having an IRP in tertile 2 increased the likelihood of having dysphagia-predominant disease by 7, while being in tertile 3 increased the likelihood by 22. Conclusions: IRP helps distinguish between the reflux-predominant and dysphagia-predominant phenotypes of AC. This may have therapeutic clinical consequences as procedures such as fundoplication to tighten the LES may benefit patients with reflux and a low IRP, while procedures like peroral endoscopic myotomy (POEM) to disrupt the LES may benefit patients with dysphagia and a relatively high IRP