Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis

Abstract

The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, protein synthesis, mitochondrial function and stem cell renewal. A region on chromosome 8q24 encompassing the MYC locus is amplified in prostate cancer, but this occurs mostly in advanced disease suggesting that MYC alterations occur late in prostate cancer. In contrast, MYC mRNA is elevated in most prostate cancers, even those of relatively low stage and grade (eg Gleason score 6) suggesting that MYC plays a role in initiation. However, since MYC protein levels are tightly regulated, elevated MYC mRNA does not necessarily imply elevated MYC protein. Thus, it is critical to determine whether MYC protein is elevated in human prostate cancer, and if so, at what stage of the disease this elevation occurs. Prior studies of MYC protein localization have been hampered by lack of suitable antibodies and controls. We utilized a new anti-MYC antibody coupled with genetically defined control experiments to localize MYC protein within human tissue microarrays consisting of normal, atrophy, PIN, primary adenocarcinoma, and metastatic adenocarcinoma. Nuclear overexpression of MYC protein occurred frequently in luminal cells of PIN, as well as in most primary carcinomas and metastatic disease. MYC protein did not correlate with gain of 8q24, suggesting alternative mechanisms for MYC overexpression. These results provide evidence that upregulation of nuclear MYC protein expression is a highly prevalent and early change in prostate cancer and suggest that increased nuclear MYC may be a critical oncogenic event driving human prostate cancer initiation and progression.

Figure 1. Box Plots of MYC mRNA Expression Values in BPH (n=9), normal (n=25), and cancer tissues (n=25)

The expression values were normalized to the common BPH reference denominator as described in detail. Each box is lined at lower quartile, median, and upper quartile score values for each group, and extended to 95 percentiles by the whiskers. The “+” symbols mark data values beyond the ends of the whiskers.

Figure 2. Differential Expression of MYC mRNA in the Prostate

The results from 7 experiments from 5 different studies^– obtained through the Oncomine database are represented as box plots as output from the Oncomine website built in features. NP: Normal Prostate, PCa: Primary Prostatic Carcinoma, NAP: Normal Adult Prostate, NPP: Normal Pre-pubertal Prostate, HRM: Hormone-Refractory Metastatic Prostate Carcinoma, AT: Atrophic Epithelium, PIN: Prostatic Intraepithelial Neoplasia, MET: Metastatic Prostate Carcinoma, CaLN: Metastatic Prostatic Carcinoma in Lymph Node.

Figure 3. Verification of the Specificity of the Anti-MYC Antibody

A. Western blot of LnCaP, CWR22Rv1 and p493 cell lines, the latter without (−Tet) or with (+Tet) application of tetracycline, stained with anti-MYC antibody. B. Wild type FVB mouse prostate tissue stained with anti-MYC antibody showing negative staining C. Lo-MYC mouse prostate tissue, expressing the human MYC protein, stained with anti-MYC antibody showing strong staining in epithelial cells, primarily within nuclei. The Lo-MYC mice are on the FVB background.

Figure 4. Human Prostate Tissues Stained with Anti-MYC Antibody

A. Normal prostate tissue. B. Focal prostate atrophy (simple atrophy). Note the nuclear staining in luminal epithelial cells (arrows). C. High grade PIN. D. PIN, higher power view. Widespread nuclear staining in the luminal epithelial cells can be seen (arrows).

Figure 5. MYC Staining in Primary and Metastatic Prostatic Adenocarcinoma

A. Low-power view of tissue microarray spot containing Gleason pattern 3 adenocarcinoma and mixed stroma and benign glands. Note the pronounced difference in staining between benign and malignant glands (arrows). B. Higher power view. MYC staining in the benign prostate glands is weaker compared to the malignant glands, and is confined to the basal epithelial cell layer (arrowhead). A few weakly staining endothelial cells can be seen (arrows). C. Primary prostatic carcinoma, Gleason pattern 4. D. Metastatic prostate carcinoma in a lymph node.

Figure 6

(A–B) Combined Dot Plot And Box-And-Whisker Graph of The MYC Nuclear Area Ratio And The MYC Scores of Normal Prostate Glands, Atrophy, PIN, Primary And Metastatic Carcinoma.