Pathway-specific analysis of gene expression data identifies the PI3K/Akt pathway as a novel therapeutic target in cervical cancer

Abstract

Purpose: Cervical tumor response on posttherapy 2[(18)F]fluoro-2-deoxy-d-glucose-positron emission tomography (FDG-PET) is predictive of survival outcome. The purpose of this study was to use gene expression profiling to identify pathways associated with tumor metabolic response.

Experimental design: This was a prospective tissue collection study for gene expression profiling of 62 pretreatment biopsies from patients with advanced cervical cancer. Patients were treated with definitive radiation. Fifty-three patients received concurrent chemotherapy. All patients underwent a pretreatment and a 3-month posttherapy FDG-PET/computed tomography (CT). Tumor RNA was harvested from fresh frozen tissue and hybridized to Affymetrix U133Plus2 GeneChips. Gene set enrichment analysis (GSEA) was used to identify signaling pathways associated with tumor metabolic response. Immunohistochemistry and in vitro FDG uptake assays were used to confirm our results.

Results: There were 40 biopsies from patients with a complete metabolic response (PET-negative group) and 22 biopsies from patients with incomplete metabolic response (PET-positive group). The 3-year cause-specific survival estimates were 98% for the PET-negative group and 39% for the PET-positive group (P < 0.0001). GSEA identified alterations in expression of genes associated with the PI3K/Akt signaling pathway in patients with a positive follow-up PET. Immunohistochemistry using a tissue microarray of 174 pretreatment biopsies confirmed p-Akt as a biomarker for poor prognosis in cervical cancer. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 inhibited FDG uptake in vitro in cervical cancer cell lines.

Conclusions: Activation of the PI3K/Akt pathway is associated with incomplete metabolic response in cervical cancer. Targeted inhibition of PI3K/Akt may improve response to chemoradiation.

Figure 1

A, Kaplan–Meier curves for cause-specific survival for the 62 patient data set. There were 40 pretreatment biopsies from patients with no evidence of disease on the 3-month posttherapy PET (PET-negative group) and 22 pretreatment biopsies from patients with residual or progressive disease on the 3-month posttherapy PET (PET-positive group). B, Kaplan–Meier curves for progression-free survival for the 62 patient data set.

Figure 2

GSEA enrichment plot of KEGG endometrial cancer pathway genes in PET⁺ versus PET⁻ tumors. Genes in the KEGG endometrial cancer signaling pathway showed significant enrichment in PET⁺ versus PET⁻ tumors (FDR q value = 0.006). The top portion of the figure plots the enrichment scores (ES) for each gene, whereas the bottom portion of the plot shows the value of the ranking metric moving down the list of ranked genes. The table enumerates the genes in the pathway for which a majority of probe sets were significantly enriched and upregulated in PET-positive versus PET-negative tumors. FDR q value (FDR, false discovery rate); FWER p value (FWER, family wise-error rate).

Figure 3

Immunohistochemical staining of the TMA from human cervical cancers. Top, example of p-AKT437 staining from different cases: negative (case number: 1,15,618), weak (1,15,621), medium (1,15,630), and strong (1,15,584). Bottom, hematoxylin and eosin (H&E) staining.

Figure 4

Kaplan–Meier curves for progression-free survival in patients with squamous cell carcinoma and high Akt expression versus low/no Akt expression. Pretreatment biopsies positive for squamous cell carcinoma were tested for p-Akt by immunohistochemistry as described in Fig. 2. Results are shown for 149 patients with squamous cell histology and high p-Akt immunohistochemistry (n = 6) versus squamous cell histology and low/no Akt expression (n = 143; P = 0.53).

Figure 5

A, Western blotting for total Akt and p-Akt from 8 cervical cancer cell lines. Cervix cell lines SW756, Caski, C33A, C41, Me180, HeLa, HT-3, and SiHa were grown in standard media supplemented with FBS to 70% to 80% confluence. Cells were washed and extracted in lysis buffer containing 1% NaF, 0.5% Na₃VO₄, and protease inhibitor. Blots were probed with primary antibodies against p-Akt^Ser473 (1:1,000; Cell Signaling Technology), total Akt (1:1,000, Cell Signaling Technology), or β-actin (1:10,000, Sigma). β-Actin was used as the internal control. B, glucose uptake assays for cervical cancer cell lines. Cervix cell lines SiHa, ME180, and C33A were grown in media supplemented with FBS to 70% to 80% confluence. Thirty minutes before adding radiolabeled glucose, media were changed to glucose-free DMEM + 10% FBS. Cells were incubated for 1 hour at 37°C in each of the following conditions: 20 μCi FDG alone, 20 μCi FDG + 5 mmol/L glucose and 50 μmol/L cytochalasin B, 20 μCi FDG and 100 μmol/L LY294002 (LY; Cell Signaling Technology). Cells were rinsed in cold PBS, harvested in 500 μL of 1% SDS + 10 mmol/L Na borate and counted on a gamma counter.