alpha-Methylacyl-CoA racemase: expression levels of this novel cancer biomarker depend on tumor differentiation

Abstract

alpha-Methylacyl-CoA racemase (AMACR) has previously been shown to be a highly sensitive marker for colorectal and clinically localized prostate cancer (PCa). However, AMACR expression was down-regulated at the transcript and protein level in hormone-refractory metastatic PCa, suggesting a hormone-dependent expression of AMACR. To further explore the hypothesis that AMACR is hormone regulated and plays a role in PCa progression AMACR protein expression was characterized in a broad range of PCa samples treated with variable amounts and lengths of exogenous anti-androgens. Analysis included standard slides and high-density tissue microarrays. AMACR protein expression was significantly increased in localized hormone-naive PCa as compared to benign (P < 0.001). Mean AMACR expression was lower in tissue samples from patients who had received neoadjuvant hormone treatment but still higher compared to hormone-refractory metastases. The hormone-sensitive tumor cell line, LNCaP, demonstrated stronger AMACR expression by Western blot analysis than the poorly differentiated cell lines DU-145 and PC-3. AMACR protein expression in cells after exposure to anti-androgen treatment was unchanged, whereas prostate-specific antigen, known to be androgen-regulated, demonstrated decreased protein expression. Surprisingly, this data suggests that AMACR expression is not regulated by androgens. Examination of colorectal cancer, which is not hormone regulated, demonstrated high levels of AMACR expression in well to moderately differentiated tumors and weak expression in anaplastic colorectal cancers. Taken together, these data suggest that AMACR expression is not hormone-dependent but may in fact be a marker of tumor differentiation.

Figure 1.

Seventy-six prostate tissue samples including benign prostate, benign prostatic hyperplasia, localized PCa, and metastatic PCa, were analyzed using cDNA expression array analysis. Hierarchical clustering was performed, filtering for only those genes with at least a 1.5-fold expression difference compared to a pool of normal prostate. AMACR was one of the genes that was found to be significantly overexpressed in PCa. Data given are a measure of relative gene expression of AMACR in each sample. Red and green represent up- and down-regulation, respectively. Lighter shading correlates with increased expression.

Figure 2.

A: AMACR protein expression in localized hormone-naive PCa. The specificity of AMACR is demonstrated by the near complete absence of AMACR expression in benign prostate glands (center). Error bars (right) representing the 95% CI of the mean expression level of AMACR for the four tissue types: benign prostate (benign), localized hormone-naive PCa (PCa), hormone-treated localized PCa (hPCa), and hormone-refractory distant PCa metastases (HR mets). B: Strong AMACR expression in a naive lymph node metastasis. Error bars (right) representing the 95% CI of the mean expression of the primary naive PCa and corresponding lymph node metastases. Original magnifications, ×400.

Figure 3.

A: PCa demonstrating strong hormonal effect (right) because of anti-androgen treatment (H&E). Weak AMACR expression (middle) seen in the corresponding immunostained section. Comparison of the mean AMACR expression between PCa demonstrating minimal and strong hormonal effects by H&E examination (right, 95% CI). B: Left: Western blot analysis representing the baseline AMACR expression in different prostate cell lines. Internal loading control with β-tubulin. Right: Western blot analysis of LNCaP cells for AMACR and PSA expression after treatment with an androgen or an anti-androgen for 24 hours and 48 hours. Results of the nontreated control samples (C) and the hormone-treated (T) cells are shown. Original magnification, ×200 (A).

Figure 4.

Top: AMACR expression in well-differentiated colon cancer. Cancer specificity demonstrated by the lack of staining in benign colonic glands (*). Middle: Weak AMACR expression in an anaplastic colon cancer case. Bottom: Weak AMACR expression in representative hormone-refractory PCa metastases. Original magnifications: ×400 (A, C); ×1000 (B); ×100 (D); ×200 (E and F).