Tumour cell survival mechanisms in lethal metastatic prostate cancer differ between bone and soft tissue metastases

Abstract

The complexity of survival mechanisms in cancer cells from patients remains poorly understood. To obtain a comprehensive picture of tumour cell survival in lethal prostate cancer metastases, we examined five survival proteins that operate within three survival pathways in a cohort of 185 lethal metastatic prostate metastases obtained from 44 patients. The expression levels of BCL-2, BCL-XL, MCL-1, cytoplasmic survivin, nuclear survivin, and stathmin were measured by immunohistochemistry in a tissue microarray. Simultaneous expression of three or more proteins occurred in 81% of lethal prostate cancer metastases and BCL-2, cytoplasmic survivin and MCL-1 were co-expressed in 71% of metastatic sites. An unsupervised cluster analysis separated bone and soft tissue metastases according to patterns of survival protein expression. BCL-2, cytoplasmic survivin and MCL-1 had significantly higher expression in bone metastases (p < 10(-5)), while nuclear survivin was significantly higher in soft tissue metastases (p = 3 × 10(-14)). BCL-XL overexpression in soft tissue metastases almost reached significance (p = 0.09), while stathmin expression did not (p = 0.28). In addition, the expression of MCL-1 was significantly higher in AR-positive tumours. Neuroendocrine differentiation was not associated with specific survival pathways. These studies show that bone and soft tissue metastases from the same patient differ significantly in expression of a panel of survival proteins and that with regard to survival protein expression, expression is associated with the metastatic site and not the patient. Altogether, this suggests that optimal therapeutic inhibition may require combinations of drugs that target both bone and soft tissue-specific survival pathways.

Figure 1. Expression of survival proteins in prostate cancer metastases

A) Data collection from the tissue microarray (TMA). A tissue microarray of 185 prostate cancer metastases from 44 patients was analyzed by immunohistochemistry. The numbers of bone and soft tissue metastases that were scored are listed for each antibody. B) Proportion of staining intensities for each antibody. Percentages of groups with high (>150), low (≤150) and negative staining intensities are represented within each bar. C) Evaluation of simultaneous expression of survival proteins in prostate cancer metastases. The co-expression of 6 proteins was determined across metastatic sites. The bars depict the percentages of metastasis co-expressing 1 to 6 of survival proteins (BCL-XL, BCL-2, MCL-1, cytoplasmic Survivin, nuclear Survivin and Stathmin). D) Co-expression of cytoplasmic Survivin, BCL-2 and MCL-1 in prostate cancer metastases.

Figure 2. Cluster analysis of prostate cancer metastasis by protein expression

The average expression of each marker was calculated separately across bone and soft tissue metastases from 44 patients. A) Supervised cluster analysis of patient metastases. For each patient bone and soft tissue metastases are aligned in consecutive columns. Above each column, bone metastases are indicated in yellow and soft tissue metastases in purple. In addition, patients are indicated by marks at the top. Please note that some patients have only bone or only soft tissue metastases. B) Unsupervised cluster analysis of patient metastases. Bone metastases are marked in yellow, while soft tissue metastases are marked in purple. Note segregation of groups according to metastatic sites in the unsupervised, but not the supervised cluster analysis. White color identifies cores that did not yield data.

Figure 3. Protein expression in bone and soft tissue metastases

Each panel shows box-plots of the distribution of expression values for the indicated protein in bone and soft tissue metastasis in a box and whisker diagram. The boxes represent the 25th–75th percentiles of biomarker expression values in each group. The whisker bars represent the lowest and highest datum within the 1.5 interquartile range. The mean values are indicated by the dark, horizontal line. A double asterisk demarks statistically significant differences between bone and soft tissue expression levels. The p-values are - BCL-2: 1.1×10⁻⁶, BCL-XL: 0.0875, MCL-1: 1.5×10⁻⁸, Stathmin: 0.288, cytoplasmic Survivin: 3.9×10⁻¹⁶ and nuclear Survivin: 3.2×10⁻¹⁴.

Figure 4. Average expression of survival proteins in bone and soft tissue metastases from each patients

To visualize the difference in expression of metastases within patients, the average expression in metastatic bone and soft tissue sites is connected by a bar. Note the increased expression in bone metastases (closed circles) for BCL-2, MCL-1 and Survivin-C and increased expression in soft tissue metastases (open triangles) for Survivn-N and marginally also for BCL-XL.