AMACR Expression Research Articles

The helix-loop-helix transcriptional regulator Id4 is required for terminal differentiation of luminal epithelial cells in the prostate.

Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. In this study we investigated the effect of loss of Id4 (Id4-/-) on mouse prostate development. Histological analysis was performed on prostates from 25 days, 3 months and 6 months old Id4-/- mice. Expression of Amacr, Ck8, Ck18, Fkbp51, Fkbp52, androgen receptor, Pten, sca-1 and Nkx3.1 was investigated by immunohistochemistry. Results were compared to the prostates from Nkx3.1-/- mice. Id4-/- mice had smaller prostates with fewer and smaller tubules. Subtle PIN like lesions were observed at 6mo. Decreased Nkx3.1 and Pten and increased stem cell marker sca-1, PIN marker Amacr and basal cell marker p63 was observed at all ages. Persistent Ck8 and Ck18 expression suggested that loss of Id4 results in epithelial commitment but not terminal differentiation in spite of active Ar. Loss of Id4 attenuates normal prostate development and promotes hyperplasia/ dysplasia with PIN like lesions. The results suggest that loss of Id4 maintains stem cell phenotype of “luminal committed basal cells”, identifying a unique prostate developmental pathway regulated by Id4.

Endometrial Tumor Classification by Histomorphology and Biomarkers in the Nurses' Health Study.

Objective Endometrial cancers have historically been classified by histomorphologic appearance, which is subject to interobserver disagreement. As molecular and biomarker testing has become increasingly available, the prognostic significance and accuracy of histomorphologic diagnoses have been questioned. To address these issues for a large, prospective cohort study, we provide the results of a centralized pathology review and biomarker analysis of all incidental endometrial carcinomas occurring between 1976 and 2012 in the Nurses' Health Study. Methods Routine histology of all (n = 360) cases was reviewed for histomorphologic diagnosis. Cases were subsequently planted in a tissue microarray to explore expression of a variety of biomarkers (e.g., ER, PR, p53, PTEN, PAX2, AMACR, HNF1β, Napsin A, p16, PAX8, and GATA3). Results Histologic subtypes included endometrioid (87.2%), serous (5.6%), carcinosarcoma (3.9%), clear cell (1.7%), and mixed type (1.7%). Biomarker results within histologic subtypes were consistent with existing literature: abnormal p53 was frequent in serous cases (74%), and HNF1β (67%), Napsin A (67%), and AMACR (83%) expression was frequent in clear cell carcinomas. Our dataset also allowed for examination of biomarker expression across non-preselected histologies. The results demonstrated that (1) HNF1β was not specific for clear cell carcinoma, (2) TP53 mutations occurred across many histologies, and (3) GATA3 was expressed across multiple histotypes, with 75% of positive cases demonstrating high-grade features. Conclusions Our findings establish the subtypes of endometrial cancer occurring in the Nurses' Health Study, corroborate the sensitivity of certain well-established biomarkers, and call into question previously identified associations between certain biomarkers (e.g., HNF1B) and particular histotypes.

Diagnostic Utility of AMCAR and p63 Cocktail Antibody in the Benign and Malignant Lesions of Prostate

Background: Histopathological examination of prostatic specimen is gold standard for the diagnosis of prostate cancer. Current study evaluates the expression of AMACR and p63 in the prostate lesions using AMACR and p63 cocktail.
 Materials and Method: Total of 180 cases were collected and Haematoxylin and Eosin staining performed followed by immunohistochemical analysis using AMACR and p63 antibody.
 Result: Out of 180 cases, Benign Prostatic Hyperplasia is the most common lesion noted in about 120 cases. In this study, the predominant population was in the 6th to 7th decade of age. Most of the patients presented with difficulty in micturition. Immunohistochemistry revealed that p63 expression is positive in all normal basal cells, 118 cases (98.33%) were negative for AMACR and only 2 cases were showing focal and weak AMACR immunoreactivity. AMACR was positive in all the 6 HGPIN cases with variable intensity. Out of 5 LGPIN cases AMACR was positive in 3 cases with low intensity, remaining 2 cases shows AMACR negative. p63 is positive in all 11 PIN cases (LGPIN & HGPIN) showing discontinuous staining pattern. All 22 cases of Prostatic adenocarcinomas were negative for p63 and all cases expressed positive immunostaining with AMACR. A diagnosis of adenocarcinoma was made in 48% of atypical cases. Cases which were negative for both AMACR and p63 were diagnosed as Atypical Small Acinar Proliferation, for which further follow-up is required.
 
 Conclusion: AMACR/p63 Cocktail antibody is very much useful as it saves time, tissue and is cost-effective.

Alpha-Methylacyl-CoA Racemase (AMACR), a Potential New Biomarker for Glioblastoma.

Alpha-Methylacyl-CoA racemase (AMACR), which was initially discovered as a prostate cancer marker, is critical for the chiral inversion mechanism of branched-chain fatty acids. However, the function of AMACR in brain tumors has not been investigated. In this study, AMACR appeared to be involved in glioblastoma. The protein and mRNA levels of AMACR were highly elevated in glioblastoma. Downregulation of AMACR inhibited cell proliferation. Comprehensive analysis of the public REMBRANDT GBM dataset also confirmed that the level of AMACR expression was correlated with the clinical prognosis of glioma patients. In summary, these findings indicate that AMACR expression is increased in a glioblastoma cell line and glioma patients, suggesting that AMACR might be a potential diagnostic marker and therapeutic target for cancer, including glioma.

The Percentage of Free PSA and Urinary Markers Distinguish Prostate Cancer from Benign Hyperplasia and Contribute to a More Accurate Indication for Prostate Biopsy.

The main advantage of urinary biomarkers is their noninvasive character and the ability to detect multifocal prostate cancer (CaP). We have previously implemented a quadruplex assay of urinary markers into clinical practice (PCA3, AMACR, TRPM8 and MSMB with KLK3 normalization). In this study, we aimed to validate it in a larger cohort with serum PSA 2.5–10 ng/mL and test other selected transcripts and clinical parameters, including the percentage of free prostate-specific antigen (PSA) (% free PSA) and inflammation. In the main cohort of 299 men, we tested the quadruplex transcripts. In a subset of 146 men, we analyzed additional transcripts (CD45, EPCAM, EZH2, Ki67, PA2G4, PSGR, RHOA and TBP). After a prostate massage, the urine was collected, RNA isolated from a cell sediment and qRT-PCR performed. Ct values of KLK3 (i.e., PSA) were strongly correlated with Ct values of other genes which play a role in CaP (i.e., PCA3, AMACR, TRPM8, MSMB and PSGR). AMACR, PCA3, TRPM8 and EZH2 mRNA expression, as well as % free PSA, were significantly different for BPH and CaP. The best combined model (% free PSA plus PCA3 and AMACR) achieved an AUC of 0.728 in the main cohort. In the subset of patients, the best AUC 0.753 was achieved for the combination of PCA3, % free PSA, EPCAM and PSGR. PCA3 mRNA was increased in patients with inflammation, however, this did not affect the stratification of patients indicated for prostate biopsy. In conclusion, the percentage of free PSA and urinary markers contribute to a more accurate indication for prostate biopsy.

Dynamic characterization of intestinal metaplasia in the gastric corpus mucosa of Atp4a-deficient mice.

Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system that harbors gastric H+, K+-adenosine triphosphatase (ATPase), the enzyme primarily responsible for gastric lumen acidification. Here, we describe the characterization of mice deficient in the H+, K+-ATPase α subunit (Atp4a−/−) to determine the role of this protein in the biosynthesis of this membrane system and the biology of the gastric mucosa. Atp4a−/− mice were produced by gene targeting. Wild-type (WT) and Atp4a−/− mice, paired for age, were examined at 10, 12, 14 and 16 weeks for histopathology, and the expression of mucin 2 (MUC2), α-methylacyl-CoA racemase (AMACR), Ki-67 and p53 proteins was analyzed by immunohistochemistry. For further information, phosphoinositide 3-kinase (PI3K), phosphorylated-protein kinase B (p-AKT), mechanistic target of rapamycin (mTOR), hypoxia-inducible factor 1α (HIF-1α), lactate dehydrogenase A (LDHA) and sirtuin 6 (SIRT6) were detected by Western blotting. Compared with the WT mice, hypochlorhydric Atp4a−/− mice developed parietal cell atrophy and significant antral inflammation (lymphocyte infiltration) and intestinal metaplasia (IM) with elevated MUC2 expression. Areas of dysplasia in the Atp4a−/− mouse stomach showed increased AMACR and Ki-67 expression. Consistent with elevated antral proliferation, tissue isolated from Atp4a−/− mice showed elevated p53 expression. Next, we examined the mechanism by which the deficiency of the H+, K+-ATPase α subunit has an effect on the gastric mucosa. We found that the expression of phosphorylated-PI3K, p-AKT, phosphorylated-mTOR, HIF-1α, LDHA and SIRT6 was significantly higher in tissue from the Atp4a−/− mice compared with the WT mice (P<0.05). The H+, K+-ATPase α subunit is required for acid-secretory activity of parietal cells in vivo, the normal development and cellular homeostasis of the gastric mucosa, and attainment of the normal structure of the secretory membranes. Chronic achlorhydria and hypergastrinemia in aged Atp4a−/− mice produced progressive hyperplasia and mucolytic and IM, and activated the Warburg effect via PI3K/AKT/mTOR signaling.

The Role of AMACR, CD10, TMPRSS2-ERG, and p27 Protein Expression Among Different Gleason Grades of Prostatic Adenocarcinoma on Needle Biopsy.

Objective:We examined the immunohistochemical expression of α-methyl acyl coenzyme A racemase (AMACR), CD10, TMPRSS2-ERG, and p27 in prostate adenocarcinoma tumors with different Gleason growth patterns and nonneoplastic prostate tissues to elucidate their roles in prostate carcinogenesis and histological aggressiveness.Material and Methods:In total, 80 archival core biopsy tissues diagnosed as prostate carcinoma, benign prostate hyperplasia, and atrophy cases were included. Immunoreactivity was evaluated by calculating the percentage of positively stained cells and the staining intensity. The mean values and test of significance were obtained using the Kruskal-Wallis test.Results:We obtained mostly intense immunoreactivity for AMACR, CD10, and ERG in adenocarcinomas. Although no significant differences were noted regarding AMACR and ERG expression, samples with Gleason growth patterns 3 and 5 tended to be strongly positive for ERG. Pattern 3 tumors exhibited the weakest positivity for CD10. The p27 expression was strong and diffuse in nonneoplastic prostate tissues. The loss of p27 expression was more frequent for pattern 5 tumors.Conclusion:ERG and AMACR were powerful markers to detect cancer. Especially, ERG is evident in early tumors may reflect its interaction with functional androgen receptors in cancer initiation. Pattern 5 tumors associated with stroma may have been exposed to more stromal substrates and upregulate their CD10 content as a protein degrader. We suggest that CD10 expression is associated with an increasing tumor grade. Decreased concentrations of p27 protein might be implicated in prostate carcinogenesis and may be a useful immunohistochemical adjunct in predicting histological aggressiveness.

Suppression of α-methylacyl-coenzyme A racemase by miR200c inhibits prostate adenocarcinoma cell proliferation and migration.

Overexpression of α-methylacyl-coenzyme A racemase (AMACR/P504S) is a major abnormality that has been observed in prostate cancer, whereas microRNA (miRNA/miR) 200c, is downregulated. The aim of the present study was to explore whether miR200c was able to exert any regulatory effects on AMACR. To meet this aim, bioinformatics analysis was performed to identify potential binding sites for miR200c in the 3′-untranslated region (3′-UTR) of AMACR. Recombinant adenoviral and dual reporter gene assays were designed to examine the binding of miR200c to the potential seed sequences in the AMACR 3′-UTR. Conventional reverse transcription (RT)-PCR, RT-quantitative (q)PCR and western blotting were also used to examine the regulatory effects of miR200c on AMACR at the mRNA and protein levels. Furthermore, Cell Counting Kit-8, wound healing and Transwell assays were performed to investigate the biological effects of miR200c-AMACR deregulation on prostate cancer cell proliferation, migration and invasion. It was revealed that miR200c post-transcriptionally suppressed AMACR expression by interacting with the 90–97 nucleotide sequence of the AMACR mRNA 3′-UTR. Artificial overexpression of miR200c significantly downregulated the mRNA and protein levels of AMACR in DU145 and PC-3 prostate cancer cells. Knockdown of AMACR by RNA interference, or overexpression of miR200c by recombinant adenoviral Ad-miR200c, inhibited prostate cancer cell proliferation, migration and invasiveness. Taken together, the results of the present study revealed that miR200c may suppress the AMACR expression level post-transcriptionally. The results also indicate that perturbation of the miR200c-AMACR regulatory mechanism may be involved in prostate carcinogenesis and that this may be exploited in future therapeutic approaches to prostate cancer.

Study of Serum PSA, AMACR, P63 And PTEN in Prostatic Adenocarcinoma

Background: Prostatic adenocarcinoma is the most common malignancy in older males. The present study was planned with an aim to evaluate serum PSA, expression of P63, AMACR and PTEN in prostatic carcinoma and to correlate them clinico-pathologically. It is a descriptive analytical study which includes patients of prostatic adenocarcinoma reporting to a large tertiary care hospital in Lucknow during the period of 2015-to 2017. Methods: Clinical details and complete hematological workup was done in 50 cases of prostatic adenocarcinoma during our study period of two years. Routine histopathology with hematoxylin and eosin (H & E) staining was done. Gleason grading was performed. Relevant sections were taken for Immunohistochemistry (IHC) to study PTEN, AMACR and P63. Result: Serum PSA levels ranged from 7.5 to 60 ng/ml. Half the patients had S. PSA in range 10-20 ng/ml (50%) followed by 20 ng/ml (18%) respectively. Mean S. PSA levels were 17.25+14.01 ng/ml. p63 assessment was conclusive in 48 cases. Expression of p63 was seen in 20 (41.7%) cases only (Score 1 or above). Maximum number of cases showing p63 expression had score 1 (35.4%) followed by score 2 (n=2; 4.2%) and score 3 (2.1%) respectively. AMACR assessment was conclusive in 48 cases. Expression of AMACR was seen in 47/48 (97.9%) cases. Maximum had score 2+ (n=22; 45.8%) followed by Score 3+ (n=18; 37.5%), score 1+ (n=7; 14.6%) and score 0 (n=1; 2.1%) respectively. PTEN assessment was conclusive in 46 cases. There were 21 (45.7%) cases with score 0, 16 (34.8%) with score 1 and 9 (19.6%) with score 2. Conclusion: In present study, serum PSA levels of patients ranged from 7.5 to 60 ng/ml with half having S. PSA in 10-20 ng/ml range (50%). According to AJCC criteria, S. PSA levels <10 ng/ml are in general indicators of lower severity of prostate cancer while those in 10-20 ng/ml range indicate an intermediate severity of prostate cancer.

Does the addition of AMACR to CK20 help to diagnose challenging cases of urothelial carcinoma in situ?

BackgroundUrothelial carcinoma in situ (CIS) in the bladder can be difficult to diagnose due to factors including procedural artifact, minimal tissue sampled, therapy-related changes, and various CIS growth patterns. Prior data has demonstrated an increase in alpha-methylacyl-CoA-racemase (AMACR) in urothelial CIS, but there is no information on its utility for diagnosing difficult cases. The aim of this investigation was to assess the expression of AMACR that was ordered on equivocal bladder cases during clinical practice.MethodsTransurethral resections of the bladder in which AMACR and CK20 were performed during diagnostic workup were identified and cases with a final diagnosis of CIS (n = 22) or non-neoplastic urothelium (n = 30) were selected. Additionally, cases in which a diagnosis of CIS was rendered without IHC (n = 20) were selected and tested for AMACR expression.ResultsSensitivity of AMACR for CIS diagnosed with IHC during clinical practice was 73% and specificity was 97%, while CK20 was 95% sensitive and 80% specific. Sensitivity of AMACR in CIS diagnosed without IHC was 100%. In all groups, AMACR had inconsistent intensity, compared to CK20 which had consistent, strong intensity.ConclusionsAMACR was usually positive in urothelial CIS and negative in non-neoplastic urothelium. However, it is important to note that AMACR was less sensitive in difficult cases, while CK20 was more sensitive with more consistent, strong staining compared to AMACR.

Diagnostic utility of Alpha Methylacyl CoA Racemase in prostatic adenocarcinoma: An institutional experience

P504S is a prostate cancer specific gene that encodes for ?-methylacyl CoA racemase (AMACR). AMACR has been shown to be selectively over expressed in prostatic adenocarcinoma with minimal to undetectable expression in benign prostatic tissue. We studied the expression of AMACR in 30 cases of prostatic adenocarcinoma using polyclonal anti-AMACR antibody and correlated it with Gleason score. Extent of staining/proportion score (0: no positive cells; 1+:1-10% positive cells; 2+11-50% positive cells; 3+>50% positive cells) was also recorded and correlated with Gleason score. All the 30 cases showed strong, cytoplasmic granular AMACR staining irrespective of their Gleason score with 26 (86.7%) cases showing immunostaining in more than 50% tumor cells (3+ proportion score). Benign prostate tissue adjacent to adenocarcinoma showed negative AMACR staining. No correlation was seen between Gleason score and AMACR proportion score. We concluded that AMACR is a highly sensitive positive marker of prostatic adenocarcinoma. Keywords: Prostate adenocarcinoma, Gleason score, AMACR.

Napsin-A and AMACR are Superior to HNF-1β in Distinguishing Between Mesonephric Carcinomas and Clear Cell Carcinomas of the Gynecologic Tract.

Mesonephric carcinoma is a rare gynecologic neoplasm commonly mistaken for clear cell carcinoma, because of their overlapping morphologic features. Both tumors are negative for estrogen receptor and p16, magnifying this diagnostic dilemma. Recently, hepatocyte nuclear factor-1 beta (HNF-1β), a marker for clear cell carcinoma, has also been shown to be positive in mesonephric carcinomas. Other more recent markers for clear cell carcinoma, however, such as Napsin-A and alpha-methylacyl-CoA racemase (AMACR), have not yet been studied in mesonephric carcinomas. Here we examine HNF-1β, AMACR, and Napsin-A immunohistochemistry in 18 mesonephric and 55 endometrial/cervical clear cell carcinomas. HNF-1β was considered positive if nuclear staining was present in ≥70% of cells and at least moderate intensity; for Napsin-A and AMACR, any cytoplasmic staining was considered positive (≥1%). H-scores were determined by multiplying the intensity score by proportion score. HNF-1β was positive in a substantial portion of mesonephric carcinomas (9/18, 50%; H-score 98) and clear cell carcinomas (34/55, 62%; H-score 163) and did not distinguish between the 2 entities (specificity, 50%; P-value of H-score=0.08). Napsin-A and AMACR expression was significantly higher in clear cell [43/55 (78%) and 41/55 (75%), respectively] than mesonephric carcinomas [4/18 (22%) and 4/18 (22%) respectively], and helpful in this differential (specificity: 78% and 78%; P<0.05 for both). When Napsin-A and AMACR staining were seen in mesonephric carcinomas, staining was focal (≤5%), whereas staining in clear cell carcinomas was patchy/diffuse. In summary, Napsin-A and AMACR are helpful in distinguishing mesonephric carcinomas from clear cell carcinomas of the female genital tract, but HNF-1β is not.

Differential immunohistochemical expression of AMACR and ERG in prostatic lesions

Introduction Alpha-methylacyl-CoA (AMACR) is an enzyme of the mitochondria and perixosomes. It was found that it showed great overexpression in prostatic adenocarcinoma, even with minimal foci. TMPRSS2/ERG fusion is one of the common genetic anomalies affecting PCa. A great correlation between positive immunohistochemical (IHC) expression for ERG and the state of fusion has been found, but the diagnostic and prognostic importance of ERG IHC remains undetected. This study aimed to evaluate the differential diagnostic and prognostic significance of both AMACR and ERG in PCa with correlation to the clinicopathological variables.Materials and methods TThe authors reviewed 70 prostatic lesions. Prostatic adenocarcinoma (50 cases), prostatic intraepithelial neoplasia (PIN) (15 with high-grade PIN, five with low-grade PIN), and benign prostatic hyperplasia (10 cases) that were used as a control. Immunohistochemical staining for AMACR and ERG was done. Follow-up of the cases for 18 months was also done.Results A statistically significant difference in the expression of ERG among the examined cases was found. It was negative in control benign prostatic hyperplasia and low-grade PIN cases), positive in 20% of high-grade PIN, and 74% of prostatic adenocarcinoma cases (P<0.01). No statistically significant correlation was found between ERG expression and tumor volume, Gleason sum score, stage, surgical margin, presence of lymphovascular invasion, perinural invasion, distant metastasis, and survival (P>0.05). Regarding AMACR expression, a statistically significant difference in the expression of AMACR among the examined cases was found (negative in benign prostatic hyperplasia, and low PIN cases) to be positive in 33.3% of high-grade PIN and in 62% of prostatic adenocarcinoma cases (P<0.01). A statistically significant correlation was found between AMACR expression and tumor volume, Gleason sum score, surgical margin, presence of lymphovascular invasion, and survival (P<0.05).Conclusion The study provides an additional evidence that IHC expression of AMACR has a role in prostatic carcinoma development. IHC expression of AMACR was absent in benign prostatic lesions. AMACR is an essential tumor marker for the diagnosis and prognosis of prostatic lesions. IHC expression of ERG may have a role in the diagnosis and biological stratification of prostate cancer.

Diagnostic utility of triple antibody (AMACR, HMWCK and P63) stain in prostate neoplasm.

Aims and Objective:In recent period, basal cell markers high molecular weight cytokeratin (HMWCK), P63 and prostate biomarker AMACR have been used as adjuvant to morphology in diagnostically challenging cases with a very high sensitivity and specificity.Materials and Methods:In this prospective study, total of 80 cases including 40 cases of malignant lesions and 40 cases of benign lesions of the prostate were taken. Tumor grade was determined according to Gleason's grading system. AMACR, HMWCK, P63 expressions were determined by immunohistochemical staining.Results:This study showed AMACR had a sensitivity of 90%, specificity of 100%. AMACR was not expressed in any of the 40 cases of benign lesions of the prostate while in malignant lesions of prostate it was expressed in 36 of 40 (90%) cases; 15 of 16 (93%) of well-differentiated carcinoma were positive for AMACR expression; 17 of 19 (89.47%) moderately differentiated and 4 of 5 (80%) cases of poorly differentiated tumors were positive for AMACR. There was statistically significant difference in expression of AMACR between benign and malignant lesions of the prostate (P = 0.001). In benign lesions, HMWCK and P63 were expressed in all the 40 (100%) cases, while in malignant lesions of prostate it was not expressed in any of the (0%) case. AMACR expression was not seen in any of the benign lesion. Out of 40 malignant cases, 4 cases were negative for AMACR, HMWCK and P63, 36 cases were positive only for AMACR, but no case was positive for HMWCK and P63.Conclusions:As an adjunct to biopsy, AMACR, HMWCK and P63 have potential for combating diagnostically challenging cases.

Exploiting the transcriptional specificity of the alpha-methylacyl-CoA racemase AMACR promoter for the molecular imaging of prostate cancer.

The metabolic protein alpha-methylacyl-CoA racemase (AMACR) is significantly overexpressed in prostate cancer compared to the normal prostate and other non-malignant tissue. Though an attractive target, there are no reports in the literature on leveraging the expression of AMACR for the molecular imaging of prostate cancer. Here, we used a molecular-genetic imaging strategy to exploit the transcriptional specificity of the AMACR promoter for the in vivo detection of prostate cancer using the reporter gene luciferase. We performed a stepwise truncation of the promoter and identified a 565 base pair minimal promoter for AMACR that retained both high activity and specificity. Following identification of the minimal promoter for AMACR, we used an advanced two-step transcriptional amplification system to maximize the promoter output. We showed that our optimized AMACR promoter can drive expression of luciferase for molecular imaging in subcutaneous xenograft models of androgen receptor-positive and androgen receptor-negative prostate cancer using a non-replicative adenovirus for gene delivery. Our results provide evidence that the AMACR promoter can be exploited to drive the cancer-specific expression of reporter genes and potentially even be incorporated into conditionally replicative adenoviruses for oncolytic therapy and other applications.

RNA-based markers in biopsy cores with atypical small acinar proliferation: Predictive effect of T2E fusion positivity and MMP-2 upregulation for a subsequent prostate cancer diagnosis.

Atypical small acinar proliferation (ASAP) is a precursor lesion of prostate cancer (PC), and PC develops from this suspicious focus or an unsampled malignant gland nearby. However, PC-related molecular alterations that could guide the timing of repeat biopsies and help monitor PC risk in ASAP-diagnosed patients have not been investigated. The purpose of this study was to first investigate the expression of seven different PC-related RNAs that included serine 2 (TMPRSS2): erythroblastosis virus E26 oncogene homolog (ERG) gene (TMPRSS2-ERG, T2E) fusion, alpha-methylacyl-CoA racemase (AMACR), kallikrein related peptidase 3 (KLK3), androgen receptor (AR), prostate cancer specific antigen 3 (PCA3), and matrix metalloproteinases (MMP)-2 and 9. PC-related RNAs were evaluated using a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) system in pathologically ASAP-diagnosed prostate biopsy cores from 55 patients presenting with a normal digital rectal examination and a PSA level of 4-10 ng/mL. We detected that positive T2E fusion status (P = 0.013) and the expression of AMACR (P = 0.016), AR (P = 0.016) and MMP-2 (P = 0.013) were independently and significantly associated with PC risk in ASAP patients. There were also several statistically significant correlations between expression levels. Additionally, we demonstrated that T2E fusion positive ASAP patients with higher MMP-2 expression were more likely to be diagnosed with PC at a subsequent biopsy during the follow-up period (P = 0.003). Although, more clinical validations are needed for the stratification of PC risk in ASAP-diagnosed biopsy cores, our current results indicate that the coexistence of T2E fusion positivity with MMP-2 upregulation may help clinicians adjust their biopsy timetable and/or assessment of PC risk in ASAP-diagnosed patients with a PSA level of 4-10 ng/mL.

Abstract A013: Prostate cancer gene expression panel to address racial differences of molecular alterations in prostate cancer

Abstract Introduction and Objectives: Prostate cancer (CaP) affects 1 in 7 men in their lifetime. African American (AA) men have significantly higher incidence and mortality from CaP compared to Caucasian American (CA) men. Emerging data, including ours, have described significantly lower frequencies of alterations in common CaP driver genes (ERG and PTEN) in AA men as compared to CA men. We have also noted that genes commonly overexpressed in CaP (ERG, AMACR, PCA3), and currently used as diagnostic markers, exhibit much lower frequency and more heterogeneity in AA men. The goal of this study was to define a CaP marker panel that is overexpressed equally well in AA and CA CaPs. Methods: Three platforms (RNA-Seq, NanoString, and qRT-PCR) were used for evaluating CaP-associated gene expression in CA and AA patients (N=144). Candidate genes with robust tumor overexpression (over 4-fold) in CaP in paired normal and tumor specimens from AA and CA patients were selected from NanoString and RNA-Seq data for validation by qRT-PCR (TaqMan) in laser microdissected (LCM) tumor and benign cells of frozen tissue sections (50 CA and 35 AA). An assay protocol (gene specific RT and preamplification followed by TaqMan PCR) was developed for noninvasive early detection of candidate genes in regular urine (non-DRE) using urinary exosomal RNA. Results: Tumor transcriptomes of CA patients consistently revealed elevated expression of PCA3 and AMACR. However, these genes had variable overexpression in the AA cohort. The top genes that were similarly overexpressed in tumors of AA and CA patients were validated by qRT-PCR in LCM tumor and normal epithelial cells (N=85). At least one gene of a six-gene signature (DLX1, HOXC4, NKX2-3, COL10A1, HOXC6, and PSGR) was overexpressed in tumor cells of all AA and CA cases, providing a consistent ethnicity-informed tumor expression signature, which was further validated in silico in TCGA RNA-Seq data. Urinary exosome-based assay was developed and optimized for PSGR, DLX1, HOXC4, NKX2-3, as well as PCA3, PCGEM1, and ERG. All markers have been evaluated in a prospective cohort of 100 patients. In 36 AA patients a sensitivity of 78%, specificity of 68%, and AUC of 0.83 was achieved, surpassing currently used urine CaP markers of ERG and PCA3 in this cohort. Conclusions: A CaP tissue-based gene expression marker panel has been defined with potential diagnostic utility for both CA and AA men in the context of urinary exosomes. Source of Funding: This study is supported by NCI/EDRN ACN12011-001-0 and NCI RO1 CA162383-05 grants to S.S. Citation Format: Indu Kohaar, Sreedatta Banerjee, Lakshmi Ravindranath, Yongmei Chen, Amina Ali, Jacob Kagan, Sudhir Srivastava, Albert Dobi, David McLeod, Inger Rosner, Shiv Srivastava, Gyorgy Petrovics. Prostate cancer gene expression panel to address racial differences of molecular alterations in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A013.

Abstract A33: Loss of Id4 initiates PIN-like lesions by maintaining stemness in mice prostate

Abstract Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators, has emerged as a tumor suppressor in prostate cancer (PCa). Recent studies have shown that Id4 is highly expressed in the normal prostate and decreases in prostate cancer (PCa) due to epigenetic silencing. Id4 knockdown in androgen-sensitive LNCaP cells has been shown to lead to castration-resistant prostate cancer (CRPC) in vitro and in vivo. Id4-/- mice leads to underdeveloped prostate without the loss of androgen receptor (AR) expression. In this study we demonstrate that prostates from the Id4 knockout (Id4-/-) mice show PIN-like lesions, suggesting that loss of Id4 plays a major role in initiation of prostate PCa. Histology of Id4 -/- mice prostate shows more PIN lesions at 3 months and 6 months without showing clear evidence of prostate cancer. Immunohistochemical analysis demonstrated increased expression of Amacr, which is a biomarker for PIN and PCa, only in Id4-/- 3-month- and 6-month-old prostate tissue. The expression of Nkx3.1 and Pten tumor suppressor genes in Id4-/-mouse prostate was undetectable, suggesting that loss of Id4 has potential to initiate or progress to PCa. Although PTEN protein was not present in Id4-/- mice, the presence of the corresponding PTEN mRNA suggested intact transcriptional program. These results suggested that Id4 plays a role in regulating the translation of the PTEN mRNA. We are currently investigating the ID4-regulated mechanisms involved in the translational control of PTEN. The results suggested that id4-/- results in PIN lesions that are in part due to a block in Pten translation. Sca-1 is a member of the Ly6 family of glycosyl phostidylinositol (GPI)-anchored cell surface proteins used as stem cell marker in murine prostate. According to cancer stem cell theory, stem cells in basal layer of the prostate tubules give rise to transient amplifying cells and differentiate into terminally differentiated secretory luminal epithelial cells. From the same pathway it gives rise to the tumor-promoting cells, which ultimately form prostate cancer. Sca-1 is expressed in Id4-/- mice 3-month- and 6-month-old prostate glands. These data suggest that loss of Id4 can initiate PIN like lesions by maintaining stemness through regulating transcriptional, translational mechanism via altering the expression of PTEN, NKX3.1, C-MYC and other regulatory molecules. Currently we are investigating that regulatory mechanism, mainly focusing on mRNA interference mechanisms. Citation Format: Dhanushka Hewa Bostanthirige, Jugal Joshi, Shravan Kumar, Divya Patel, Jaideep Chaudhary. Loss of Id4 initiates PIN-like lesions by maintaining stemness in mice prostate [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A33.

MP70-15 TRANSCRIPTIONAL ACTIVATION OF ?-CATENIN BY MIR-138 PROMOTES UP-REGULATION OF ALPHA-METHYLACYL-COA RACEMASE IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2018MP70-15 TRANSCRIPTIONAL ACTIVATION OF ?-CATENIN BY MIR-138 PROMOTES UP-REGULATION OF ALPHA-METHYLACYL-COA RACEMASE IN PROSTATE CANCER CELLS Kati Erdmann, Knut Kaulke, Susanne Fuessel, and Manfred P. Wirth Kati ErdmannKati Erdmann More articles by this author , Knut KaulkeKnut Kaulke More articles by this author , Susanne FuesselSusanne Fuessel More articles by this author , and Manfred P. WirthManfred P. Wirth More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2259AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The tumor-suppressive microRNA miR-138 has been shown to be down-regulated in prostate cancer (PCa) and has been suggested as a direct regulator of alpha-methylacyl-CoA racemase (AMACR), which is highly over-expressed in PCa. The aim of this study was to validate the direct interaction of miR-138 with the 3′-UTR of AMACR. Furthermore, the influence of miR-138 on the expression of AMACR and selected AMACR regulators was investigated in PCa cells. METHODS DU-145, PC-3 and LNCaP PCa cells were transfected with 100 nM of miR-138 mimic or a control construct for 4 h. A specific siRNA targeting AMACR was used as a positive control for the expression knockdown. 48 h after transfection, mRNA and protein expression levels of AMACR and selected AMACR regulators were determined by quantitative PCR and Western Blot, respectively. Luciferase reporter assays were used to verify target and promoter interaction. RESULTS A direct interaction of miR-138 with the AMACR-3′-UTR was confirmed by using a luciferase reporter assay. Surprisingly, AMACR mRNA and protein expression were up-regulated by up to 125% and 100%, respectively, following transfection of PCa cells with miR-138. In contrast, a specific siRNA targeting AMACR led to a significant reduction of the AMACR mRNA and protein expression by up to 75% in all three PCa cell lines. The lack of any miR-138 binding sites within the AMACR promoter pointed to an indirect mechanism of up-regulation. Subsequently, the effect of miR-138 on selected AMACR regulators including CTNNB1/β-catenin, RELA, SMAD4, SP1 and TCF4 was evaluated. MiR-138 only promoted an up-regulation of CTNNB1 mRNA expression and β-catenin protein levels by up to 75%. Interestingly, a binding site for miR-138 was present within the CTNNB1 promoter. By using a luciferase reporter assay the direct activation of the CTNNB1 promoter by miR-138 was demonstrated, which consequently could contribute to the observed AMACR up-regulation. CONCLUSIONS The present findings suggest that miR-138 can indirectly up-regulate AMACR expression via transcriptional induction of CTNNB1 expression, at least in vitro in PCa cells. These results further fortify the function of miRNAs as transcriptional gene activators in addition to their established role as post-transcriptional silencers. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e940 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Kati Erdmann More articles by this author Knut Kaulke More articles by this author Susanne Fuessel More articles by this author Manfred P. Wirth More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

AMACR overexpression acts as a negative prognostic factor in oral squamous cell carcinoma.

Background: Alpha-methylacyl-CoA racemase (AMACR) is a key enzyme responsible for the metabolism of branched-chain fatty acids. It has been found to be an important prognostic factor in numerous types of cancers. This study was aimed to investigate the expression of AMACR and its prognostic significance in patients with oral squamous cell carcinoma (SCC).Methods: Analysis of publicly available microarray data of oral SCC revealed that AMACR was significantly upregulated in tumor tissue compared with normal mucosa. We further assessed the protein expression of AMACR in 164 patients with oral SCC by immunohistochemistry. The prognostic impact of AMACR expression and its association with various clinicopathological parameters were statistically analyzed.Results: AMACR overexpression was significantly associated with advanced tumor status (P=0.001), advanced nodal status (P=0.036), increased vascular invasion (P=0.026) and increased perineural invasion (P=0.004). Patients with high expression level of AMACR had significantly worse disease-specific survival (DSS), distant metastasis-free survival (DMFS) and local recurrence-free survival (LRFS) (all P<0.0001). In multivariate analysis, AMACR overexpression was also an independent negative prognostic factor for DSS (hazard ratio [HR]: 4.410, 95% confidence interval [CI]: 2.285-8.511, P<0.001), DMFS (HR: 5.157, 95% CI: 2.756-9.651, P<0.001) and LRFS (HR: 4.462, 95% CI: 2.429-8.198, P<0.001).Conclusions: High expression of AMACR was not only a key adverse prognostic factor but also a potential therapeutic target in oral SCC.