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UWM Hepatocellular and Prostate Carcinoma Research Paper

University Of Wisconsin Milwaukee

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The idea is to investigate the expression of a protein known as GP73 or GOLM1 in patients with hepatocellular carcinoma or prostate cancer in Saudi population.This protein could be sued as a biomarker in serum or in urine, which can be very cost-effective and have almost accurate results. Can you help me to wright the introduction, objective, and literature review. I am attaching some articles you can add more.

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Volume 10 Number 11 November 2008 pp. 1285–1294 1285 Golgi Protein GOLM1 Is a Tissue and Urine Biomarker of Prostate Cancer1,2 ,† Sooryanarayana Varambally* , ,† Bharathi Laxman*, Rohit Mehra* , Qi Cao*, * Saravana M. Dhanasekaran , Scott A. Tomlins*, Jill Granger*, Adaikkalam Vellaichamy*, ‡ ‡ Arun Sreekumar*, Jianjun Yu , Wenjuan Gu , ‡ ‡ Ronglai Shen , Debashis Ghosh , § ¶ Lorinda M. Wright , Raleigh D. Kladney , # § ** Rainer Kuefer , Mark A. Rubin , Claus J. Fimmel , ,†,†† and Arul M. Chinnaiyan* *Michigan Center for Translational Pathology, Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA; †Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI 48109, USA; ‡Department of Biostatistics, University of Michigan, Ann Arbor, MI 48109, USA; § Edward Hines VA Medical Center, Hines, IL and Division of Gastroenterology, Hepatology and Nutrition, Loyola University, Stritch School of Medicine, Maywood, IL 60153, USA; ¶Division of Urologic Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA; #Department of Urology, University of Ulm, Ulm, Germany; **Department of Pathology and Laboratory Medicine, Cornell University, New York, NY 10021, USA; ††Department of Urology, University of Michigan Medical School, Ann Arbor, MI 48109, USA Abstract Prostate cancer is the most common type of tumor found in American men and is the second leading cause of cancer death in males. To identify biomarkers that distinguish prostate cancer from normal, we compared multiple gene expression profiling studies. Through meta-analysis of expression array data from multiple prostate cancer studies, we identified GOLM1 (Golgi membrane protein 1, Golm 1) as consistently up-regulated in clinically localized prostate cancer. This observation was confirmed by reverse transcription–polymerase chain reaction (RT-PCR) and validated at the protein level by immunoblot assay and immunohistochemistry. Prostate epithelial cells were identified as the cellular source of GOLM1 expression using laser capture microdissection. Immunohistochemical staining localized the GOLM1 signal to the subapical cytoplasmic region, typical of a Golgi distribution. Surprisingly, Abbreviations: Golm1, Golgi membrane protein 1; PSA, prostate-specific antigen; PIN, prostatic intraepithelial neoplasia; BPH, benign prostatic hyperplasia; LCM, laser capture microdissection Address all correspondence to: Arul M. Chinnaiyan, MD, PhD, Department of Pathology, University of Michigan Medical School, 1400 E. Medical Center Dr. 5316 CCGC, Ann Arbor, MI 48109-0602. E-mail:; and Sooryanarayana Varambally, PhD, Department of Pathology, University of Michigan Medical School, 1400 E. Medical Center Dr. 5430 CCGC, Ann Arbor, MI 48109. E-mail: 1 This research was supported in part by the National Institutes of Health Prostate SPORE P50 CA69568 (to A.M.C.); Department of Defense Grants PC040517 (to R.M.), PC051081 (to A.M.C. and S.V.). C.J.F. was supported by a VA Merit Review. 2 This article refers to supplementary materials, which are designated by Tables W1 and W2 and are available online at Received 4 August 2008; Revised 2 September 2008; Accepted 2 September 2008 Copyright © 2008 Neoplasia Press, Inc. All rights reserved 1522-8002/08/$25.00 DOI 10.1593/neo.08922 1286 GOLM1 Is a Tissue Biomarker of Prostate Cancer Varambally et al. Neoplasia Vol. 10, No. 11, 2008 GOLM1 immunoreactivity was detected in the supernatants of prostate cell lines and in the urine of patients with prostate cancer. The mechanism by which intact GOLM1 might be released from cells has not yet been elucidated. GOLM1 transcript levels were measured in urine sediments using quantitative PCR on a cohort of patients presenting for biopsy or radical prostatectomy. We found that urinary GOLM1 mRNA levels were a significant predictor of prostate cancer. Further, GOLM1 outperformed serum prostate-specific antigen (PSA) in detecting prostate cancer. The area under the receiver-operating characteristic curve was 0.622 for GOLM1 (P = .0009) versus 0.495 for serum PSA (P = .902). Our data indicating the up-regulation of GOLM1 expression and its appearance in patients’ urine suggest GOLM1 as a potential novel biomarker for clinically localized prostate cancer. Neoplasia (2008) 10, 1285–1294 Introduction Prostate cancer is the most commonly diagnosed malignancy and is a leading cause of cancer-related death in the Western male population [1,2]. Early diagnosis is critical for the effective treatment of malignant tumors. High-throughput technologies such as DNA and protein microarray have enabled the identification of genes and their corresponding proteins that are differentially regulated in malignant conditions [3,4]. These high throughput studies have offered researchers a better understanding of the disease and the molecular circuitries that are dysregulated in cancer. Our previous DNA microarray studies using prostate cancer tissue RNA have identified multiple genes, including alpha methylacyl coenzyme A racemase (AMACR), Enhancer of Zeste Homolog 2 (EZH2), tumor protein TPD52, and ERG, as dysregulated in localized and metastatic prostate cancer [5–8]. Many of these findings provided new insights into the biology of prostate carcinogenesis and have identified the next generation of candidate biomarkers and potential therapeutic targets [9–12]. In the case of AMACR, a peroxisomal fatty acid– metabolizing enzyme, the up-regulated protein was detectable in patients’ urine, which indicated its potential use for noninvasive diagnostic studies [13]. Furthermore, our studies indicated an immune response to AMACR in prostate cancer patients and autoantibodies directed against AMACR that could be detected in the patient’s serum [14]. GOLM1 (Golm 1, NM_016548) is a resident cis-Golgi membrane protein of unknown function. The first evidence of its up-regulation was shown in the hepatocytes of patients with acute and chronic forms of hepatitis and hepatocellular cancer [15]. GOLM1 has a single N-terminal transmembrane domain and an extensive C-terminal, coiled-coil domain that faces the luminal surface of the Golgi apparatus. N-terminal cleavage by a furin proprotein convertase resulted in the release of the C-terminal ectomain and its appearance in serum [16]. The cleaved form of GOLM1 was detectable in the serum of patients with hepatocellular cancer, a finding that may have diagnostic value [17]. Initial gene expression array studies in our laboratory, and by others [3,18] suggested increased expression levels of GOLM1 mRNA in prostate cancer tissues. We subsequently detected GOLM1 transcripts in patient urine samples. By multiplexing with other urine markers, we demonstrated that GOLM1 mRNA levels can serve as significant predictors of prostate cancer [19]. Our study confirms the epithelial cell–specific expression of GOLM1 in prostate cancer tissues. Furthermore, we show that the GOLM1 protein is released from prostate cell lines in vitro and is detectable in the urine of pa- tients with established prostate cancer. The secretion from cell lines could be inhibited by treating cells with the protein transport inhibitor brefeldin A [20,21]. Our observation suggests that GOLM1 has potential as a noninvasive biomarker of localized prostate cancer. Materials and Methods Quantitative Real-time Polymerase Chain Reaction To validate GOLM1 overexpression observed in multiple gene expression profiling studies, we performed quantitative real-time polymerase chain reaction (qPCR) for GOLM1 expression using SYBR green [6,22]. Briefly, cDNA was made with the total RNA isolated from 11 benign prostatic hyperplasia (BPH), 27 localized prostate cancers, and 8 metastatic prostate cancer samples. The quantification of cDNA in each sample was performed by interpolating a C t value from a standard curve of C t values obtained from serially diluted, commercially prepared cDNA pooled normal prostate samples (Clontech, Palo Alto, CA). This calculated quantity of GOLM1 was then normalized against the quantity of the housekeeping gene glyceraldehyde-3 phosphate dehydrogenase (GAPDH) expressed in that sample. “No–reverse transcription” controls were included to exclude amplification of the genomic DNA. The primer sequences used for GOLM1 were: 5′-CAGCGTGAAAAGCGGAATC-3′ and 5′-TCGGCCCTGTTGTGAAATA-3′. GAPDH primers were prepared as described by Vandesompele et al. [23]. Urine samples were obtained from 333 patients with informed consent after a digital rectal examination before either needle biopsy (n = 269) or radical prostatectomy (n = 64) at the University of Michigan Health System with institutional review board approval as described earlier (Table W1) [19]. Isolation of RNA from urine and Transplex Whole Transcriptome Amplification (WTA) were as described [24]. Quantitative polymerase chain reaction was used to detect GOLM1 and the control transcripts PSA and GAPDH from WTA amplified cDNA essentially as described [24]. The primer sequences for GOLM1 [19], GAPDH [23], and PSA [25] were previously described. Quantitative polymerase chain reaction was performed on WTA cDNA from urine collected from 129 biopsynegative patients and 204 patients with prostate cancer (140 biopsypositive patients and 64 prostatectomy patients). Samples that had PSA C t values > 28 were excluded to ensure sufficient prostate cell collection, leading to 124 biopsy-negative and 195 samples from patients with prostate cancer in the analysis. This resulted in a final data Neoplasia Vol. 10, No. 11, 2008 GOLM1 Is a Tissue Biomarker of Prostate Cancer set of samples from 195 patients with prostate cancer (133 positive needle biopsy and 62 radical prostatectomy) and 124 biopsy-negative patients. Threshold levels were set during the exponential phase of the qPCR reaction using Sequence Detection Software version 1.2.2 (Applied Biosystems, Foster City, CA), with the same baseline and threshold set for each plate, to generate threshold cycle (C t) values for all genes for each sample. We adjusted GOLM1 was against its mean urine PSA and GAPDH values (2(C tPSA + C tGAPDH ) / 2 − C tGOLM1) × 1000). Statistical Analysis The receiver-operating characteristic (ROC) curve and a Box plot for GOLM1 were generated using Statistical Package for the Social Sciences 11.5 (SPSS, Inc., Chicago, IL). The area under the ROC curve (AUC-ROC) was then calculated for GOLM1 and serum PSA, respectively. Laser Capture Microdissection Normal and cancerous prostate epithelial cells were collected using laser capture microdissection (LCM) as described previously [26]. Briefly, for LCM, the SL Microtest device with μCUT software was applied (MMI GmbH, Heidelberg, Germany). A total area of 6 mm2 was cut for all of the samples and was collected with the aid of the adhesive surface lid from a specially manufactured tube (MMI GmbH). Approximately 10,000 cells collected from each sample were lysed with 30 μl of 5× SDS-reducing sample buffer and clarified by centrifugation at 12,000g for 10 minutes. The samples were then loaded onto a gel for immunoblot analysis. Immunoblot Analysis Normal and prostate cancer tissues were homogenized in NP-40 lysis buffer containing 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40 (Sigma, St. Louis, MO) and complete protease inhibitor cocktail (Roche, Indianapolis, IN). Fifteen micrograms of protein extract was mixed with SDS sample buffer and run on a 10% SDS–polyacrylamide gel under reducing conditions. The separated proteins were transferred onto polyvinylidene fluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ), which were incubated for 1 hour in blocking buffer [Tris-buffered saline with 0.1% Tween (TBS-T) and 5% nonfat dry milk]. GOLM1 rabbit polyclonal antibody (raised against amino acids 41-400) was applied at a 1:50,000 dilution in blocking buffer overnight at 4°C. After washing with TBS-T buffer, the membrane was incubated with horseradish peroxidase–linked donkey, anti–rabbit IgG antibody (GE Healthcare Life Sciences, Piscataway, NJ) at 1:5000 for 1 hour at room temperature. The signals were visualized with the ECL detection system (GE Healthcare) and autoradiography. For GAPDH and actin Western blots, the GOLM1 antibody probed membranes were stripped with western reprobe buffer (Geno-tech, St. Louis, MO), blocked in TBS-T containing 5% nonfat dry milk, and incubated with either rabbit anti-GAPDH antibodies (1:50,000 dilution; Abcam, Cambridge, MA) or rabbit anti–actin antibodies (1:5000 dilution; Sigma) for 2 hours. For laser capture microdissected prostate epithelial cell immunoblot, the cells were lysed in sample buffer and analyzed for GOLM1 protein expression by immunoblot analysis as described above. At the time of initial diagnosis, urine samples (n = 52) from biopsyproven, clinically localized prostate cancer patients (mean ± SD age, 58.1 ± 0.60 years) were collected with informed consent. Clinical Varambally et al. 1287 and pathology data from patients with biopsy-proven clinically localized prostate cancer used in this study is provided in Table W2. No treatment was administered to the subjects in the interval between biopsy and urine sample collection. As controls, urine samples from 50 male subjects (mean ± SD, 57.9 ± 0.1 years) with no known history of prostate cancer were collected from the University of Michigan Clinical Pathology Laboratories. Within 2 days of collection, the urine samples were concentrated at 4°C using a Biomax Ultrafree concentrator (5000 MW cutoff; Millipore Corporation, Bedford, MA). The protein content of each sample was estimated with Bradford reagent following the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA). The concentrated urine samples were frozen at −20°C until use. Twenty micrograms of total protein from each urine sample was electrophoresed and analyzed for GOLM1 protein expression by immunoblot analysis as described above. The intensity of the 74-kDa GOLM1 band in each sample was scored visually by a researcher who was blinded to the diagnosis of samples and the pattern of loading in the gels. A GOLM1-positive control sample (LNCaP total cell extract) was used for immunoblot experiments. While scoring, the band in the sample lanes with highest reactivity was assigned a score of 4 (highest level of reactivity), whereas absence of a band was scored 0 (non reactivity). Intermediate band intensities were assigned as follows: weak (score = 1), intermediate (score = 2), and high (score = 3). The mean values for GOLM1 reactivity were presented as population mean values with 95% confidence intervals. Student’s t test (2-sided) was used to test for statistically significant differences in GOLM1 reactivity between patients with prostate cancer and control subjects. No adjustment for multiple testing was made during the analysis. P values less than or equal to 0.05 were considered statistically significant. Receiver-operating characteristic curves were used to assess the sensitivity and specificity of urine-associated GOLM1 to detect prostate cancer. To test the secretion of the GOLM1 protein, a flag-tagged GOLM1 construct was overexpressed in HEK293 Phoenix cells. Cell culture supernatants were collected 12 and 24 hours after transfection. For the analysis, 5 μl of culture supernatant from GOLM1-transfected, vector-transfected, and control protein-transfected cells were run on SDS-PAGE. Immunoblots were probed with either flag or GOLM1 antibodies. Likewise, the presence of GOLM1 was also assessed in the culture supernatants of LNCaP (prostate cell line) and DU145 (prostate cancer cell line). Prostate cancer cell lines were cultured in RPMI medium containing 10% FBS, and the medium was changed to 5% FBS 12 hours before the collection of the supernatant to reduce the bovine serum protein content. Culture supernatants were collected at 24 hours and analyzed by SDS-PAGE and immunoblot analysis with GOLM1-specific antibody. To investigate the specificity of the secretion, the GOLM1 antibody was pretreated with 5 μg of recombinant GOLM1 protein [15]. Cell-free medium served as the negative control. In Vitro Overexpression of GOLM1 A mammalian expression construct of GOLM1 was generated by subcloning the PCR product into the pACRSVpLpA(−)loxP-SSP vector (UMICH vector core). Primers used were GOLM1-KpnI-6His-F: 3′-GCGGGTACCATGCACCACCACCACCACCACGGCTTGGGAAACGGGCGTCGCAGC-5′ and GOLM1-XbaIFLAG-R: 5′-GCGTCTAGATCAAAGCTTGTCGTCATCGTCTTTGTAGTCGAGTGTATGATTCCGCTTTTC-3′. HEK293 Phoenix cells were transfected with the flag-GOLM1 construct as 1288 GOLM1 Is a Tissue Biomarker of Prostate Cancer Varambally et al. well as vector control using FuGENE6 (Roche) transfection reagent. Culture supernatants were collected 12 and 24 hours after transfection for immunoblot analysis. Immunohistochemistry For the development of the tissue microarray (TMA), prostate cancer and normal prostate tissues were embedded in paraffin. Study pathologists (M.A.R. and R.M.) reviewed the slides of all cases and designated areas of interest. These slides were used as a template for Neoplasia Vol. 10, No. 11, 2008 TMA construction. All TMAs were assembled using the manual tissue arrayer (Beecher Instruments, Silver Spring, MD). At least three tissue cores were sampled from each donor block. Histologic diagnosis of the tissue cores was verified by standard hematoxylin and eosin staining of the initial TMA slide. This radical prostatectomy series is part of the University of Michigan Prostate Cancer Specialized Program of Research Excellence Tissue Core, with informed consent of the patients and prior institutional review board approval. Standard biotin-avidin complex immunohistochemistry was performed using a polyclonal anti-GOLM1 antibody. Digital images were acquired Figure 1. GOLM1 expression in prostate cancer. (A) GOLM1 transcript levels were collected from DNA microarray analysis of 101 prostate samples. Cy3/Cy5 ratios indicate the expression of GOLM1 in prostate tissue RNA compared to RNA from normal prostate pool. BPH indicates benign prostatic hyperlasia; MET, metastatic prostate cancer; NAP, normal adjacent prostate; PCA, prostate cancer. (B) GOLM1 expression in prostate cancer profiling studies from different publicly available cancer microarray data sets in Oncomine [3,18,26,40] and Yang et al. (unpublished data). Expression array analysis of multiple prostate cancer microarray data sets were collected and analyzed, and statistical significance was calculated. (For details visit: Class 1 represents the GOLM1 expression in normal tissues and class 2 represents GOLM1 expression in cancer. (C) Quantitative polymerase chain reaction confirmation of increased expression of GOLM1 transcripts in prostate cancer. Quantitative polymerase chain reaction was done using RNA from benign, prostate cancer, and metastatic prostate cancer tissue samples. Quantitative polymerase chain reaction of glyceraldehyde-3-phosphate dehydrogenase served as the internal control. Neoplasia Vol. 10, No. 11, 2008 GOLM1 Is a Tissue Biomarker of Prostate Cancer Varambally et al. 1289 Figure 2. Immunoblot analyses of the GOLM1 protein in prostate tissue lysates. (A) Immunoblot analysis of GOLM1 protein expression using benign and prostate cancer tissue lysates. Total prostate tissue lysates (10-μg total protein) were analyzed using anti–GOLM1 rabbit polyclonal antibody. Ten micrograms of total tissue lysate from benign prostate tissue, prostate cancer, and metastatic prostate cancer tissues were analyzed by immunoblot. The blot was reprobed with GAPDH antibody to confirm equal loading. (B) The prostate epithelial cells were isolated by LCM technique and were analyzed by ...
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I added more information in the literature review using the articles attached.



Expression of Gp73 or Golm1 in Hepatocellular and Prostate Carcinoma in the Saudi Population.
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Expression of Gp73 or Golm1 in Hepatocellular and Prostate Carcinoma in Saudi Arabia.
Cancer is increasingly becoming a common killer disease in the world. Prostate and
hepatocellular carcinomas, which are manifestations of the scourge, have affected a considerable
number of people. Almutairi et al. (2019) posit that in Saudi Arabia alone, the prevalence of
prostatic carcinoma is around 70 cases in a population of 100,000. This manifestation is in
comparison to 83 cases per 100,000 attributed to hepatocellular carcinoma (Alhaqbani et al. 2019).
However, with technological advancement, early detection, which is critical in cancer treatment,
is made possible through the identification of biomarkers. This development forms the basis of
this paper that seeks to investigate the expression of Gp73 or Golm1 in hepatocellular and prostate
carcinoma among the Saudi population.
In this study, the expression of the protein is a vital aspect in determining the identification
of, particularly, hepatocellular and prostatic carcinomas. The possibility of this incidence is due to
the fact the proto-oncogene may be detectable in serum or urine once manifestations of the
cancerous tissues of the two carcinomas occur in the specific sites. Therefore, the study will focus
on the expression of the proto-oncogenes in serum or urine in hepatocellular and prostatic
carcinomas patients in the Saudi population. Additionally, this study will seek to compare the
reviews in a bid to give a valid conclusion.
Literature review
Modern science possesses the capability to study specific genomes in any cell. With
advancements in technology, the gene profiling of the hepatocellular and prostatic tissues affected



by carcinomas is possible. Following the ability to profile the specific genes present in tissues
extracted from cancer patients, identification of Gp73 or Golm1 genes occurred. Arora (2016)
posits that Gp73 or Golm1 is the prevalent gene found to have a leading role in the metastasis of
carcinomas, especially hepatocellular carcinomas. The attribute of the gene makes a considerable
impact on the recurrence of the carcinomas leading to a poor prognosis.
The role of the gene in metastasis is made possible through the activation of the
EGFR/RTK pathway leading to the growth and proliferation of cancerous cells in other sites of
the human body. Ye et al. (2016) outline that, "the oncogene particularly interacts with epidermal
growth factor receptor (EGFR) and acts as a specific cargo adaptor to assist EGFR/RTK anchoring
on the trans-Golgi network (TGN) and recycling back to the plasma membrane, leading to
prolonged activation of the downstream kinases." This development is responsible for the
regulation of redistribution in a spatial manner, recycling as well as kinetics present in signaling
in the epidermal growth factor receptor /receptor tyrosine kinases (EGFR/RTK).
In a study conducted by Saraswati, et al. (2019) in Saudi Arabia, the researchers discovered
that phloretin inactivated the action of STAT-3. Additionally, it also defied sorafenib resistance
through the targeting of SHP-1–mediated inhibition of STAT3 and Akt/VEGFR2 pathways. More
startling, however, was the reduction of the levels of the GOLM1 proteins in serum and urine. This
incidence occurred since phloretin inhibited the action of epidermal growt...

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