Chemotherapy-induced monoamine oxidase expression in prostate carcinoma functions as a cytoprotective resistance enzyme and associates with clinical outcomes

Abstract

To identify molecular alterations in prostate cancers associating with relapse following neoadjuvant chemotherapy and radical prostatectomy patients with high-risk localized prostate cancer were enrolled into a phase I-II clinical trial of neoadjuvant chemotherapy with docetaxel and mitoxantrone followed by prostatectomy. Pre-treatment prostate tissue was acquired by needle biopsy and post-treatment tissue was acquired by prostatectomy. Prostate cancer gene expression measurements were determined in 31 patients who completed 4 cycles of neoadjuvant chemotherapy. We identified 141 genes with significant transcript level alterations following chemotherapy that associated with subsequent biochemical relapse. This group included the transcript encoding monoamine oxidase A (MAOA). In vitro, cytotoxic chemotherapy induced the expression of MAOA and elevated MAOA levels enhanced cell survival following docetaxel exposure. MAOA activity increased the levels of reactive oxygen species and increased the expression and nuclear translocation of HIF1α. The suppression of MAOA activity using the irreversible inhibitor clorgyline augmented the apoptotic responses induced by docetaxel. In summary, we determined that the expression of MAOA is induced by exposure to cytotoxic chemotherapy, increases HIF1α, and contributes to docetaxel resistance. As MAOA inhibitors have been approved for human use, regimens combining MAOA inhibitors with docetaxel may improve clinical outcomes.

Figure 1. MAOA expression is induced in vivo following exposure to docetaxel and mitoxantrone.

(A) Schema of the neoadjuvant chemotherapy and prostatectomy trial. (B) Heat-map of gene expression changes in prostate carcinoma cells that associate with biochemical relapse following radical prostatectomy. Columns are 31 patients and rows are 141 genes. Yellow indicates increased expression following chemotherapy. Blue indicates decreased expression and black indicates no change. Grey indicates absent or poor quality data. R is relapse and NR is no relapse. (C) MAOA transcript alterations shown for 31 study participants. Ratios are intra-individual post-treatment versus pre-treatment MAOA transcript abundance measurements determined by qRT-PCR from microdissected neoplastic epithelium. (D) MAOA protein expression determined by immunohistochemistry. Representative images of neoplastic prostate epithelium acquired before (D1) and after (D2) chemotherapy exposure. D1′ and D2′ indicate higher magnification images. Brown pigment indicates presence of MAOA protein. (E) MAOA protein expression by immunohistochemistry in prostate cancer metastasis from 44 patients. Each metastasis is indicated by a datapoint with multiple metastasis from the same individual located on the y-axis corresponding to each patient number. Grey and black datapoints alternate for ease of visualization. The horizontal line indicates the mean expression of MAOA across all metastasis for a given individual.

Figure 2. MAOA expression is induced by chemotherapy in vitro and promotes cell proliferation.

(A) Treatment of LNCaP prostate cancer cells with docetaxel increases MAOA enzyme activity. (B) Over-expression of MAOA increases cell proliferation in PC3 prostate cancer cells. *p<0.05. (C) The irreversible MAOA inhibitor clorgyline reduces MAOA activity in LNCaP cells (*p<0.05). (D) Expression of MAOA in the PC3 prostate cancer cell line (PC3-MAOA) increases cell growth which is inhibited by clorgyline (MAOI). (E) LNCaP cell growth is inhibited by the MAOI clorgyline (*p<0.05).

Figure 3. MAOA expression inhibits docetaxel cytotoxicity.

(A) PC3 cells expressing MAOA exhibit enhanced cell survival following 48 and 72 hours of exposure to docetaxel (*p<0.05) (B) Elevated MAOA expression reduces cellular apoptosis following exposure to docetaxel. The addition of clorgyline enhances the cytotoxicity of docetaxel toward LNCaP (C) and VCaP (D) prostate cancer cells (*p<0.05).

Figure 4. MAOA expression increases ROS and the expression of HIF1α and HIF1α pathway genes.

(A) Deamination reaction catalyzed by monoamine oxidase (MAO) enzymes produces H₂0₂ as a reactive oxygen species (ROS) byproduct. (B) ROS levels are increased in PC3 cells expressing MAOA (*p<0.05). (C) Expression of MAOA in PC3 cells results in elevated nuclear HIF1α and NFκB protein. (D) Expression of MAOA in PC3 cells results in increased levels of transcripts encoding known HIF1A target genes. (E) Association of MAOA and HIF1 transcript level changes following chemotherapy. Plotted are the Log2 post-chemotherapy versus pre-chemotherapy transcript abundance ratios for each of 31 patients. The Pearson correlation value is 0.42 (p = 0.02). (F) Treatment of VCaP cells with the MAOA inhibitor clorgyline suppresses HIF1A expression. A four-fold reduction of HIF1A mRNA was quantitated by qRT-PCR at 48 hours relative to vehicle control (p<0.01).

Figure 5. MAOA expression enhances in-vivo tumor growth and ROS production.

(A) Animals harboring xenograft tumors overexpressing MAOA developed significantly larger tumor burdens over the four week observation period as compared to animals carrying the vector control tumors (*p<0.01 at the 28 week timepoint). (B) ROS levels are increased in xenograft tumor cells expressing MAOA (*p<0.05). (C) Heatmap of transcripts differentially expressed between PC3 vector control xenografts and PC3-MAOA expressing xenografts. Shown are transcripts increased (yellow) or decreased (blue) between the PC3-control versus PC3-MAOA tumors. (D) Quantitation of transcripts encoding MAOA, TACSD2, S100A8 and TRPA1 by qRT-PCR in PC3-MAOA versus PC3-control xenografts (*p<0.05).