GOLPH2 and MYO6: putative prostate cancer markers localized to the Golgi apparatus - PubMed (original) (raw)

GOLPH2 and MYO6: putative prostate cancer markers localized to the Golgi apparatus

Shuanzeng Wei et al. Prostate. 2008.

Abstract

Background: Malignant transformation is often accompanied by morphological and functional alterations in subcellular organelles. The Golgi apparatus is a subcellular structure primarily involved in modification and sorting of macromolecules for secretion and transport to other cellular destinations. Molecular alterations associated with the Golgi apparatus may take place during prostate carcinogenesis but such alterations have not been documented.

Methods: To demonstrate that the Golgi apparatus undergoes alterations during prostate carcinogenesis, we examined the expression and localization of two candidate molecules, Golgi phosphoprotein 2 (GOLPH2) and myosin VI (MYO6), both overexpressed in prostate cancer as initially identified by expression microarray analysis.

Results: Elevated GOLPH2 expression in prostate cancers was validated through real-time RT-PCR, Western blot, and tissue microarray analysis, and its Golgi localization in surgical prostate cancer tissues confirmed using two-color immunofluorescence. In addition, distinctive juxtanuclear MYO6 staining pattern consistent with Golgi localization was observed in surgical prostate cancer tissues. Two-color immunofluorescence revealed intensive Golgi-specific staining for both GOLPH2 and myosin VI in prostate cancer cells but not in the adjacent normal prostate epithelium.

Conclusions: We show that the Golgi apparatus in prostate cancer cells differs from the normal Golgi by elevated levels of two molecules, GOLPH2 and MYO6. These results for the first time demonstrated consistent cancer cell-specific alterations in the molecular composition of the Golgi apparatus. Such alterations can be explored for discovery of novel prostate cancer biomarkers through targeted organellar approaches.

(c) 2008 Wiley-Liss, Inc.

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Figures

Fig. 1

Fig. 1

Validation of GOLPH2 mRNA and protein overexpression in human prostate cancer. A: Real-time RT-PCR analysis of GOLPH2 mRNA in prostate cancer tissues (n =30) and paired normal prostate tissues (n =30) from surgical prostate cancer specimens (_X_-axis). Normalized (against GAPDH) Ct values were converted to fold expression changes (_Y_-axis) relative to the median value of the normal samples. B: Western blot analysis of GOLPH2 protein in five paired normal/cancer prostate tissue samples collected fresh from five surgical cases. β-Actin is a loading control. LNCaP cell lysate was used as a positive control. N1–N5: normal prostate epithelial tissues, T1–T5: prostate cancer tissues.

Fig. 2

Fig. 2

Tissue microarray (TMA) analysis of GOLPH2 expression in human prostate cancer. A: TMA spot containing predominantly Gleason 4 (red arrow) prostate cancer tissue. B: TMA spot containing mixed Gleason 3 (blue arrow) and Gleason 5 (red arrow) prostate cancer tissues and normal prostate epithelium (black arrow). Note that only cancer cells are positive for GOLPH2 and the staining pattern is consistent with Golgi localization. C,D: Box plots showing automated TMA image analysis results for GOLPH2 staining in normal and prostate cancer tissues. Altogether 132 normal and 93 cancer lesions were evaluated excluding array spots with poor quality and ambiguous diagnoses. Box plots showed differences in positively stained areas (by pixel ratios, C) and intensity scores (D) between the normal and cancerous prostate epithelium. Note that pixel ratios (see Materials and Methods Section) cannot reach high percentages due to Golgi-confined staining for GOLPH2. Each box is lined at lower quartile, median, and upper quartile values for each group. The whiskers show the 1.5× inter-quartile range of the data. The circles and asterisks indicate outliers and extreme outliers beyond the whiskers.

Fig. 3

Fig. 3

Immunohistochemical analysis of GOLPH2 and TGN46 in human prostate cancer tissues. A: Two adjacent serial sections of surgical prostate cancer tissues stained with GOLPH2 and TGN46, respectively, as indicated. Black arrows indicate cancer tissues, blue arrows indicate a normal prostate gland. Insets show high power view of the boxed areas. Note strong GOLPH2 staining in cancer cells and weak or negative staining in the normal prostate epithelium, and no difference for TGN46 staining in normal and cancer cells. B: Immunofluorescence double staining for TGN46 and GOLPH2 in the same formalin fixed paraffin embedded surgical prostate cancer section. Prostate cancer tissues were stained for nuclei (blue, by DAPI staining), TGN46 (green), GOLPH2 (red), and the images merged, as indicated. Note the nearly identical, relatively weak TGN46 staining in both normal and cancer cells, and cancer cell-specific overexpression of GOLPH2. White arrow indicates a normal duct and yellow arrows indicate cancerous acini.

Fig. 4

Fig. 4

Myosin VI and GOLPH2 both localize to the Golgi apparatus. A: Prostate cancer tissues of varying histological grade (as indicated) stained with the myosin VI polyclonal antibody. Higher power views of a restricted area in top panels were presented immediately below. Note the distinctive perinuclear staining pattern present in the cancer epithelium (red arrows) regardless of the Gleason grades, and weak or absent staining in the normal prostate epithelium (black arrows). G3: Gleason 3; G4: predominantly Gleason 4. B: Myosin VI and GOLPH2 Immunofluorescence double labeling in formalin fixed paraffin embedded surgical prostate cancer tissues. Prostate cancer tissues were stained for nuclei (blue, by DAPI staining), MYO6 (green), GOLPH2 (red), and the images merged, as indicated. Note the distinctive Golgi staining pattern for GOLPH2 and MYO6 specifically in the cancer epithelium (yellow arrow), and the negative or weak signals for both proteins in the normal appearing prostate epithelium (white arrow).

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