High grade serous ovarian cancer (HGSC) is the most common and deadly type of ovarian cancer, largely due to difficulties in early diagnosis and rapid metastasis throughout the peritoneal cavity. Previous studies have shown that expression of Notch3 correlates with worse prognosis and increased tumorigenic cell behaviors in HGSC. They investigated the mechanistic role of Notch3 in a model of metastatic ovarian cancer using the murine ovarian surface epithelial cell line, ID8 IP2. Notch3 was activated in ID8 IP2 cells via expression of the Notch3 intracellular domain (Notch3IC). Notch3IC ID8 IP2 cells injected intraperitoneally caused accelerated ascites and reduced survival compared to control ID8 IP2, particularly in the early stages of the disease. They interrogated downstream targets of Notch3IC in ID8 IP2 cells by RNA sequencing and found significant induction of genes that encode adhesion and extracellular matrix proteins. Notch3IC ID8 IP2 showed increased expression of ITGA1 mRNA and cell-surface protein. Notch3IC-mediated increase of ITGA1 was also seen in two human ovarian cancer cells. Notch3IC ID8 IP2 cells showed increased adhesion to collagens I and IV in vitro. They propose that Notch3 activation in ovarian cancer cells causes increased adherence to collagen-rich peritoneal surfaces. Thus, the correlation between increased Notch3 signaling and poor prognosis may be influenced by increased metastasis of HGSC via increased adherence of disseminating cells to new metastatic sites in the peritoneum.
Ectopic expression of Notch3IC activates Notch 3 signaling in ID8 IP2 cells.
(A) No Notch3 transcripts are detected in ID8 IP2 by semiquantitative RT-PCR (box). Notch receptors 1, 2, and 4 as well as ligands Jagged1 and Delta-like1 are detected in ID8 IP2 cells. (B) ID8 IP2 cells have undetectable levels of Notch3 protein when compared to cell lines previously characterized by detectable levels of Notch3 (OVCAR3, A2780, Caov3, and PA-1, Western blot). (C) Representative Western blots show that expression of Notch3 intracellular domain is upregulated in Notch3IC lentivirally infected ID8 IP2 cell lines. Numbers indicate set number. (D) qRT-PCR indicates that Notch target genes are upregulated in Notch3IC Sets #1-#5 compared to matched Controls (error bars = S.E.M in all figures). This panel shows evidence of Notch activation across all 5 matched Sets, however, some variability was observed between sets. (E) Selected established Notch target genes are upregulated in RNA-Seq data, demonstrating upregulation of active Notch3 signaling.
Ovarian cancer is the deadliest gynecologic malignancy and expected to be the 5th leading cause of cancer death in women in the United States in 2017. The most common subtype of ovarian cancer is high-grade serous ovarian cancer (HGSC). Standard treatment for HGSC includes surgical cytoreduction and combination chemotherapy with platinum and taxane. HGSC recurs in over 80% of cases that were initially disseminated beyond the pelvis. Due to rapid dissemination and few or non-specific symptoms, approximately 70–75% of ovarian cancers are discovered after the dissemination of the disease.
Notch3 activation correlates with reduced survival in early tumorigenesis.
(A) Kaplan Meier survival curve demonstrates a significant decrease in survival time for mice implanted with Notch3IC cells using a Gehan-Breslow-Wilcoxon test (p = 0.0183), which is more sensitive to disproportionate early differences than the Mantel-Cox test (p = 0.0592). (B-C) Survival analysis at different stages of serous ovarian cancers using kmplot shows highly significant correlation between high Notch3 expression and reduced survival in early stage ovarian cancers (B), but not late stage ovarian cancers (C).
HGSC metastasizes either by direct invasion of adjacent organs or by dissemination in the peritoneal fluid to new sites on the peritoneal lining. The peritoneal lining consists of a single layer of mesothelial cells over the collagen-rich extracellular matrix and other stroma, and ovarian cancer is thought to preferentially attach to sites where the mesothelial layer is disrupted and extracellular matrix is exposed. Once tumor cells adhere, they further reduce the integrity of the peritoneal mesothelial layer by triggering mesothelial contraction, metalloprotease expression, and increased cytokine and growth factor signaling, leading to increased invasion, motility, and further growth of metastases.
Notch3IC tumor bearing mice have increased ascites accumulation.
(A) Representative images of the exposed peritoneal wall (top) and bioluminescent IVIS imaging (bottom) of Control and Notchıc tumor-bearing mice. (B-C) Notch3IC tumor-bearing mice show non-significant increases in tumor burden in the peritoneal wall (B) and right ovary (C) as measured by IVIS signal at 8 weeks post-implantation (p = 0.1686 and p = 0.2015, respectively, Welch’s t-test). (D) Representative photographs of Control and Notch3IC implanted mice at 8 weeks post-implantation, with indicated circumference percent changes relative to the circumference at the start of the experiment. (E) Notch3IC tumor-bearing mice show a significant increase in circumference as a result of ascites accumulation at the 8-week time point (p = 0.0423 Welch’s t-test).
Expression of specific members of the collagen and integrin families are upregulated in Notch3IC cells.
(A) Heat map of RNA-seq results of collagen gene family. Collagens marked by asterisks are significantly regulated (p ≤ 0.1, average log2 fold change ≥ 1) in Notch3IC cells. (B) Heat map of integrin gene family. Multiple integrins are upregulated in Notch3IC cells, while Itga3 is significantly downregulated. (C) ITGA1 is significantly upregulated on the surface of Notch3IC cells as assessed by flow cytometry (p = 0.0195, Student’s t-test, each Set assessed twice and results averaged).
Ovarian cancers often cause dramatic increases in the amount of peritoneal fluid, called ascites, which can result from tumor cell blockage of lymph nodes and/or an increase in vascular leakage in the tumors or adjacent tissues of the peritoneum. Ascites accumulation can increase the peritoneal fluid from less than 20mL to volumes over 500mL and can cause considerable patient distress. Ascites accumulation is correlated with worse prognosis, possibly because ascites contain growth factors, extracellular matrix proteins, proteolytic enzymes, and inflammatory signals that support tumor growth and dissemination. Increasing intraperitoneal pressure due to ascites may also directly induce metastatic epithelial to mesenchymal transition.
Ovarian tumors frequently show high levels of Notch3 signaling, particularly in HGSC cases and HGSC-derived cell lines. Physiologic Notch3 signaling depends on Notch ligand interactions (Jagged or Delta-like) which leads to the release of the Notch3 intracellular domain (N3IC) and subsequent formation of a transcriptionally active N3IC complex that includes both MAML and RBPJκ . Unbiased screening of almost 500 HGSC tumors demonstrated that the Notch3 pathway was altered in 22% of HGSC samples assessed, and patients with NOTCH3 alterations demonstrated poor survival. Most of the identified alterations were predicted to activate signaling, including copy number amplification, predicted activating mutations, and up-regulation of Notch3 mRNA expression.
Notch3IC cells show increased adherence to collagens and decreased collective migration on ECM substrate, but unaltered invasion through ECM.
(A) The ratio of Notch3IC cells to Control cells adhered to plates coated with indicated extracellular matrix. Dotted line indicates no change. Notch3IC cells display a significant increase in adherence to collagen I (p = 0.0118, Welch’s t-test) and collagen IV (p = 0.0163) and a small but significant reduction in adhesion to laminin (p = 0.0474). There is no significant difference in adhesion to fibronectin, vitronectin, or uncoated tissue culture dishes (p = 0.6237, p = 0.3075, p = 0.4206, Welch’s t-test). Sets #3–5 of ID8 IP2 were assessed in triplicate on 3 separate occasions. The Notch3IC mean was compared to the Control mean for each line. (B) Representative images of ID8 IP2 and Control cells adhered to collagen I-coated plates. (C) The ability of Notch3IC cells to collectively migrate into an open wound area is significantly reduced on collagen I (p = 0.0432, p = 0.0181 for 6 and 12 hours respectively) and fibronectin (p = 0.0004, p = 0.0020 for 6 and 12 hours respectively), but not on uncoated tissue culture dishes (p = 0.5422, p = 0.9637 for 6 and 12 hours respectively). Sets #3-#5 were assessed in quadruplicate wells twice. Graphs display the average of the duplicate experiments for each set. (D) The ability of Notch3IC cells to invade through insert pores coated with Collagen I, coated with Matrigel GFR, or uncoated was not significantly changed relative to Controls (p = 0.5634, p = 0.6076, and p = 0.1205, respectively). Sets #3-#5 of ID8 IP2 were assessed in triplicate wells and each set was averaged.
The most frequent alterations of the Notch pathway were in NOTCH3 itself, but other components of the Notch pathway were altered at a lower frequency, including the JAGGED1 and JAGGED2 ligand genes and Notch activation complex genes MAML1,MAML2, MAML3, and RBPJκ (CSL). Re-analysis of this dataset with additional tumors showed that over half of HGSC samples harbor deletions of WWP2, which targets NOTCH3 (but no other Notch genes) for endocytosis and degradation, and these WWP2 mutations correlate with increased Notch3 expression. Studies on independent sets of serous ovarian tumor samples have confirmed increased NOTCH3 copy number, increased NOTCH3 transcript levels, and increased NOTCH3 protein levels. Patients with high levels of NOTCH3 showed shorter overall survival, higher grade and stage tumors, increased ascites accumulation, and increased recurrence.
Notch3 expression levels also correlate with tumorigenic phenotypes, including proliferation, viability, cell cycle arrest, and apoptosis, in ovarian cancer cell lines in vitro, indicating a role in tumor growth. Notch3 inhibition similarly reduced proliferation and induced apoptosis, indicating that Notch3 doesn’t just induce growth, but is a critical cell survival factor in some ovarian cancers. Notch3 signaling up-regulates other tumorigenic behaviors, such as epithelial to mesenchymal cell transition and resistance to anoikis.
Despite the strong evidence implicating NOTCH3 in ovarian cancer development, the role of Notch3 signaling in HGSC dissemination and progression is not well understood. Here they demonstrate that in an in a vivo mouse model of disseminated ovarian cancer, Notch3 activity decreases survival, upregulates expression of adhesion genes, and increases tumor cell affinity for extracellular matrix in the peritoneal wall.
Price JC, Azizi E, Naiche LA, Parvani JG, Shukla P, Kim S, et al. (2020) Notch3 signaling promotes tumor cell adhesion and progression in a murine epithelial ovarian cancer model. PLoS ONE 15(6): e0233962. https://doi.org/10.1371/journal.pone.0233962