Poor prognosis in carcinoma is associated with a gene expression signature of aberrant PTEN tumor suppressor pathway activity

Abstract

Pathway-specific therapy is the future of cancer management. The oncogenic phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in solid tumors; however, currently, no reliable test for PI3K pathway activation exists for human tumors. Taking advantage of the observation that loss of PTEN, the negative regulator of PI3K, results in robust activation of this pathway, we developed and validated a microarray gene expression signature for immunohistochemistry (IHC)-detectable PTEN loss in breast cancer (BC). The most significant signature gene was PTEN itself, indicating that PTEN mRNA levels are the primary determinant of PTEN protein levels in BC. Some PTEN IHC-positive BCs exhibited the signature of PTEN loss, which was associated to moderately reduced PTEN mRNA levels cooperating with specific types of PIK3CA mutations and/or amplification of HER2. This demonstrates that the signature is more sensitive than PTEN IHC for identifying tumors with pathway activation. In independent data sets of breast, prostate, and bladder carcinoma, prediction of pathway activity by the signature correlated significantly to poor patient outcome. Stathmin, encoded by the signature gene STMN1, was an accurate IHC marker of the signature and had prognostic significance in BC. Stathmin was also pathway-pharmacodynamic in vitro and in vivo. Thus, the signature or its components such as stathmin may be clinically useful tests for stratification of patients for anti-PI3K pathway therapy and monitoring therapeutic efficacy. This study indicates that aberrant PI3K pathway signaling is strongly associated with metastasis and poor survival across carcinoma types, highlighting the enormous potential impact on patient survival that pathway inhibition could achieve.

Fig. 1.

The PTEN/PI3K microarray gene expression signature in breast carcinoma. (A) Gene set enrichment analysis (17) verifies that the PTEN signature faithfully captures known biological outputs of the pathway. For each gene set tested (see SI Methods), the normalized enrichment score (black bars; positive and negative values indicate higher expression in the PTEN^IHC− or PTEN^IHC+ group, respectively) and the corresponding nominal P value (green bars) are plotted. The number of matched genes per set is within parentheses. (B) Hierarchical clustering of the 105 breast tumor samples (columns) by using the top 246 signature genes (rows) with an APV < 0.02. The two major tumor dendrogram clusters, “Signature Absent and Signature Present,” are indicated by blue- and red-colored branches, respectively. PTEN IHC status is indicated by filled (positive) or white (negative) boxes. In the heat map, fold change is relative to the median for each gene according to the color scale shown (red, overexpression; blue, underexpression; yellow, missing values), and selected gene symbols are displayed to the right. Clustering was performed by using the 1-Pearson correlation metric and centroid linkage. (C) Microarray PTEN mRNA expression levels from B illustrate PTEN to be a better marker of the SP group than PTEN IHC status. HER2 amplification, PIK3CA KD or CD/HD mutation, and the ER status for each case are indicated by filled (positive) boxes, open (negative) boxes, or a diagonal line (missing data).

Fig. 2.

KM survival estimates for tumors classified as SP (red curves) or SA (blue curves) indicates the PTEN/PI3K signature to predict patient survival across independent carcinoma microarray data sets. Log-rank P value comparing these classes at complete follow-up and at 5-year follow-up are given. Cox regression survival analyses are presented in SI Table 5. (A) DDFS and (B) OS for the classification of 295 Dutch BC (3). (C) Relapse-free survival and (D) BCS for the classification of 99 BCs (4). (E) DDFS for the classification of 79 prostate cancers (25). (F) OS for 86 lung cancers (1). (G) OS for 80 bladder cancers (26). Here, a three-group classification was used (see SI Methods), with a black curve for unclassified cases. For all KM graphs, the corresponding three- or two-group analyses are presented in SI Fig. 9.

Fig. 3.

Stathmin is a marker of the PTEN/PI3K signature. (A) Immunoblotting analysis of relative protein levels for four selected signature genes across a panel of eight BC cell lines. (B) Box plots illustrating stathmin IHC scores to be significantly higher in PTEN^IHC− vs. PTEN^IHC+ breast tumors among all 181 cases analyzed for both proteins (Left) and within the subgroup of 62 ER-negative cases (Right). P values were calculated by using the Mann–Whitney test. (C) Stathmin IHC levels are highly correlated to the presence of the PTEN/PI3K signature (ROCarea = 0.809, P < 0.0001). The hierarchical clustering tumor dendrogram, marker annotations, and STMN1 (represented by two independent reporters) and PTEN message levels are from Fig. 1 B and C. Stathmin IHC scores are centered to the median score, 6 (white boxes), with higher and lower scores colored in red and blue, respectively (key to right). (D) Scatterplot analyses show stathmin IHC levels to closely track STMN1 mRNA levels (average of the two reporters) and to be significantly inversely related to PTEN mRNA levels. Linear regression and the Pearson correlation r and P values are presented. (E) KM analysis indicates stathmin-high (score >10) tumors have a significantly higher rate of distant disease recurrence compared with stathmin-low (score 0–10) tumors among 191 BCs analyzed. Log-rank P values for complete follow up (top right corner) and the 2- and 5-year intervals are shown. Cox regression analysis is presented in SI Table 5. Representative stathmin IHC examples are presented in SI Fig. 10.

Fig. 4.

The PTEN/PI3K pathway regulates stathmin protein levels. (A) Immunoblotting analysis of stathmin down-regulation after adenovirus-mediated PTEN expression (ad-PTEN) in MDA-MB-468 PTEN-null cells compared with control (ad-lacZ). (B) Stathmin is down-regulated in BC lines by treatment with the PI3K-inhibitor LY294002 (LY; 20 μM) for 4 days as detected by immunoblotting. (C) Immunoblotting analyses show that, in vivo, stathmin is down-regulated upon PTEN induction with doxycyclin (Dox) in MDA-468TR-PTEN xenograft tumors and (D) after treatment of mice with MDA-MB-468 tumors with the PI3K-inhibitor PWT-458 (100 mg/kg). For C and D, independent xenografts from separate animals were analyzed in each sample lane.