Inhibition of Hedgehog signaling antagonizes serous ovarian cancer growth in a primary xenograft model

Abstract

Background: Recent evidence links aberrant activation of Hedgehog (Hh) signaling with the pathogenesis of several cancers including medulloblastoma, basal cell, small cell lung, pancreatic, prostate and ovarian. This investigation was designed to determine if inhibition of this pathway could inhibit serous ovarian cancer growth.

Methodology: We utilized an in vivo pre-clinical model of serous ovarian cancer to characterize the anti-tumor activity of Hh pathway inhibitors cyclopamine and a clinically applicable derivative, IPI-926. Primary human serous ovarian tumor tissue was used to generate tumor xenografts in mice that were subsequently treated with cyclopamine or IPI-926.

Principal findings: Both compounds demonstrated significant anti-tumor activity as single agents. When IPI-926 was used in combination with paclitaxel and carboplatinum (T/C), no synergistic effect was observed, though sustained treatment with IPI-926 after cessation of T/C continued to suppress tumor growth. Hh pathway activity was analyzed by RT-PCR to assess changes in Gli1 transcript levels. A single dose of IPI-926 inhibited mouse stromal Gli1 transcript levels at 24 hours with unchanged human intra-tumor Gli1 levels. Chronic IPI-926 therapy for 21 days, however, inhibited Hh signaling in both mouse stromal and human tumor cells. Expression data from the micro-dissected stroma in human serous ovarian tumors confirmed the presence of Gli1 transcript and a significant association between elevated Gli1 transcript levels and worsened survival.

Conclusions/significance: IPI-926 treatment inhibits serous tumor growth suggesting the Hh signaling pathway contributes to the pathogenesis of ovarian cancer and may hold promise as a novel therapeutic target, especially in the maintenance setting.

Figure 1. Sonic hedgehog (Shh) and Gli1 are variably expressed in human ovarian tumors.

A. Immunohistochemical analysis of Shh expression was carried out on a tissue microarray carrying 64 individual primary ovarian tumors to assess both the frequency and level of Shh expression in human ovarian cancer. The Shh staining in a poorly differentiated serous cystadenocarcinoma (upper left panel), a poorly differentiated mucinous cystadenocarcinoma (upper right panel), a well-differentiated cystadenocarcinoma (lower left panel), and a poorly differentiated adenocarcinoma (lower right panel) is shown. The table summarizes the frequency of Shh staining in the benign tumors and non-epithelial and epithelial malignancies present in the ovarian tumor microarray. B. Real-time Q-PCR was performed for analysis of Gli1 mRNA levels in 10 primary human papillary serous ovarian cancer tumors. Primers specific for human Gli1 and GAPDH were used. For each sample, the level of Gli1 mRNA relative to the level of GAPDH mRNA is shown.

Figure 2. Hedgehog pathway components are differentially expressed in primary human ovarian tumors.

The expression of Gli1, Ptch1, SHH and SMO was analyzed by RT-qPCR in 4 primary human serous ovarian tumors (SOC11-SOC14) established as xenografts in NOD/SCID mice. Primers specific for each human gene and human GADPH were used. For each tumor, the levels of each gene relative to GADPH mRNA are shown.

Figure 3. Cyclopamine alone impedes ovarian tumor growth.

Mice bearing human papillary serous ovarian tumor xenografts from sample SOC11 were randomized into 2 cohorts with matched tumor volumes and treated with either vehicle or cyclopamine. Tumor volumes were assessed regularly. Change in tumor volume is shown relative to the tumor volume at the beginning of the experiment. Error bars represent the standard error of the mean (S.E.M). The significance was determined using a Wilcoxon rank sum test.

Figure 4. IPI-926 is an effective maintenance therapy.

Mice bearing tumors derived from patients diagnosed with serous ovarian cancer were randomized into 4 cohorts (n = 4–6 animals per treatment cohort) with matched tumor volumes and subjected to the indicated treatment regimens. Three independent papillary serous ovarian cancer samples, SOC12 (A), SOC13 (B) and SOC14 (C) were used as biological replicates. Cohorts were initially treated with vehicle alone (Control), paclitaxel and carboplatinum (T/C) alone, IPI-926 alone or T/C+IPI-926 as indicated. The last T/C dose was delivered on day 14 (indicated by the dotted line). Percent change in tumor volume represents the change in tumor volume relative to the tumor volume at the beginning of the experiment. Error bars represent the standard error of the mean (S.E.M). Significance was determined using a Wilcoxon rank sum test.

Figure 5. IPI-926 blocks Hh pathway activity in mouse stromal and human tumor cells.

Gli1 mRNA expression levels in xenograft tumors derived from SOC12, SOC13, and SOC14 were analyzed by real time quantitative PCR following treatment of mice with IPI-926 for 24 hours (upper panel) or 21 days (lower panel). The relative levels of human (left panel) and mouse (right panel) Gli1 mRNA expression were analyzed using species-specific primers. Error bars represent the standard error of the mean (S.E.M). The significance (#p≤0.03, *p = 0.05, ** p≤0.001) was determined using Student's t –test.

Figure 6. Stromal levels of Gli1 mRNA correlate with survival.

Survival analysis data obtained for Gli1 mRNA expression on microdissected stroma samples of 19 patients using qRT-PCR validation. Higher Gli1 mRNA expression correlated with decreased survival (Log-rank Test, p<0.015) with a median survival of 26.50 months for patients with low Gli1 mRNA expression versus 16 months for the patients that had tumors with higher Gli1 mRNA expression in stroma.