Article Data

  • Views 1311
  • Dowloads 155

Original Research

Open Access

PROL1 is essential for xenograft tumor development in mice injected with the human prostate cancer cell-line, LNCaP, and modulates cell migration and invasion

  • Amarnath Mukherjee1,†
  • Augene Park1,†
  • Kelvin Paul Davies1,2,*,

1Department of Urology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

2Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA

DOI: 10.31083/jomh.2021.131 Vol.18,Issue 2,February 2022 pp.1-9

Submitted: 28 July 2021 Accepted: 23 September 2021

Published: 28 February 2022

*Corresponding Author(s): Kelvin Paul Davies E-mail:

† These authors contributed equally.


Background and objective: A growing body of literature suggests modulated expression of members of the opiorphin family of genes (PROL1, SMR3A and SMR3B) is associated with cancer. Recently, overexpression of PROL1 was shown to be associated with prostate cancer, with evidence of a role in overcoming the hypoxic barrier that develops as tumors grow. The primary goal of the present studies was to support and expand evidence for a role of PROL1 in the development and progression of prostate cancer.

Material and methods: We engineered knock-out of the opiorphin gene, PROL1, in LNCaP, an androgen-sensitive, human prostate cancer derived, cell-line. Using xenograft assays, we compared the ability of injected LNCaP PROL1 knock-out cell-lines to develop tumors in both castrated and intact male mice with the parental LNCaP and LNCaP PROL1 overexpressing cell-lines. We used RNAseq to compare global gene expression between the parental and LNCaP PROL1 knock-out cell-lines. Wound closure and 3D spheroid invasion assays were used to compare cell motility and migration between parental LNCaP cells and LNCaP cells overexpressing of PROL1.

Results: The present studies demonstrate that LNCaP cell-lines with consisitutive knock-out of PROL1 fail to develop tumors when injected into both castrated and intact male mice. Using RNAseq to compare global gene expression between the parental and LNCaP PROL1 knock-out cell-lines, we confirmed a role for PROL1 in regulating molecular pathways associated with angiogenesis and tumor blood supply, and also identified a potential role in pathways related to cell motility and migration. Through the use of wound closure and 3D spheroid invasion assays, we confirmed that overexpression of PROL1 in LNCaP cells leads to greater cell motility and migration compared to parental cells, suggesting that PROL1 overexpression results in a more invasive phenotype.

Conclusion: Overall, our studies add to the growing body of evidence that opiorphin-encoding genes play a role in cancer development and progression. PROL1 is essential for establishment and growth of tumors in mice injected with LNCaP cells, and we provide evidence that PROL1 has a possible role in progression towards a more invasive, metastatic and castration resistant prostate cancer (PrCa).


Cell motility; Cell invasiveness; Opiorphin; PROL1; Prostate cancer

Cite and Share

Amarnath Mukherjee,Augene Park,Kelvin Paul Davies. PROL1 is essential for xenograft tumor development in mice injected with the human prostate cancer cell-line, LNCaP, and modulates cell migration and invasion. Journal of Men's Health. 2022. 18(2);1-9.


[1] Global Burden of Disease Cancer C, Fitzmaurice C, Abate D, Abbasi N, Abbastabar H, Abd-Allah F, et al. Global, Regional, and National Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life-Years for 29 Cancer Groups, 1990 to 2017: A Systematic Analysis for the Global Burden of Disease Study. Lancet. 2018; 392: 1789–1858.

[2] Wang G, Zhao D, Spring DJ, DePinho RA. Genetics and biology of prostate cancer. Genes & Development. 2018; 32: 1105–1140.

[3] Lang Z, Wu Y, Pan X, Qu G, Zhang T. Study of differential gene expression between invasive multifocal/ multicentric and unifocal breast cancer. Journal of BUON. 2018; 23: 134–142.

[4] Pihur V, Datta S, Datta S. Finding common genes in multiple cancer types through meta–analysis of microarray experiments: a rank aggregation approach. Genomics. 2008; 92: 400–403.

[5] Lv X, He M, Zhao Y, Zhang L, Zhu W, Jiang L, et al. Identification of potential key genes and pathways predicting pathogenesis and prognosis for triple-negative breast cancer. Cancer Cell International. 2019; 19: 172.

[6] Wu Q, Cao R, Chen J, Xie X. Screening and identification of biomarkers associated with clinicopathological parameters and prog-nosis in oral squamous cell carcinoma. Experimental and Therapeutic Medicine. 2019; 18: 3579–3587.

[7] Koffler J, Holzinger D, Sanhueza GA, Flechtenmacher C, Zaoui K, Lahrmann B, et al. Submaxillary gland androgen-regulated protein 3a expression is an unfavorable risk factor for the survival of oropha-ryngeal squamous cell carcinoma patients after surgery. European Archives of Oto-Rhino-Laryngology. 2013; 270: 1493–1500.

[8] Grünow J, Rong C, Hischmann J, Zaoui K, Flechtenmacher C, Weber K, et al. Regulation of submaxillary gland androgen-regulated protein 3a via estrogen receptor 2 in radioresistant head and neck squamous cell carcinoma cells. Journal of Experimental & Clinical Cancer Research. 2017; 36: 25.

[9] Thierauf J, Veit JA, Grunow J, Doscher J, Weissinger S, Whiteside T, et al. Expression of Submaxillary Gland Androgen-regulated Protein 3A (SMR3A) in Adenoid Cystic Carcinoma of the Head and Neck. Anticancer Research. 2016; 36: 611–615.

[10] Wu J, Sun B, Ren N, Ye Q, Qin L. Genomic aberrations in hepatocellular carcinoma related to osteopontin expression detected by array-CGH. Journal of Cancer Research and Clinical Oncology. 2010; 136: 595–601.

[11] Mukherjee A, Park A, Wang L, Davies KP. Role of opiorphin genes in prostate cancer growth and progression. Future Oncology. 2021; 17: 2209–2223.

[12] Wisner A, Dufour E, Messaoudi M, Nejdi A, Marcel A, Ungeheuer M-, et al. Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways. Proceedings of the National Academy of Sciences. 2006; 103: 17979–17984.

[13] Fu S, Tar MT, Melman A, Davies KP. Opiorphin is a master regulator of the hypoxic response in corporal smooth muscle cells. The FASEB Journal. 2014; 28: 3633–3644.

[14] Fu S, Davies KP. Opiorphin-dependent upregulation of CD73 (a key enzyme in the adenosine signaling pathway) in corporal smooth muscle cells exposed to hypoxic conditions and in corporal tissue in pre-priapic sickle cell mice. International Journal of Impotence Research. 2015; 27: 140–145.

[15] Siemann DW, Horsman MR. Modulation of the tumor vasculature and oxygenation to improve therapy. Pharmacology & Therapeutics. 2015; 153: 107–124.

[16] Movsas B, Chapman JD, Horwitz EM, Pinover WH, Greenberg RE, Hanlon AL, et al. Hypoxic regions exist in human prostate carcinoma. Urology. 1999; 53: 11–18.

[17] Emami Nejad A, Najafgholian S, Rostami A, Sistani A, Shojaeifar S, Esparvarinha M, et al. The role of hypoxia in the tumor microenvi-ronment and development of cancer stem cell: a novel approach to developing treatment. Cancer Cell International. 2021; 21: 62.

[18] Folkman J. What is the Evidence that Tumors are Angiogenesis Dependent? Journal of the National Cancer Institute. 1990; 82: 4–7.

[19] Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Research. 1989; 49: 6449–6465.

[20] Bacac M, Stamenkovic I. Metastatic Cancer Cell. Annual Review of Pathology: Mechanisms of Disease. 2008; 3: 221–247.

[21] Duffy M, McGowan P, Gallagher W. Cancer invasion and metastasis: changing views. The Journal of Pathology. 2008; 214: 283–293.

[22] Rudolfsson SH, Bergh A. Hypoxia drives prostate tumour progression and impairs the effectiveness of therapy, but can also promote cell death and serve as a therapeutic target. Expert Opinion on Therapeutic Targets. 2009; 13: 219–225.

[23] Vaupel P, Multhoff G. Fatal Alliance of Hypoxia-/HIF-1alpha-Driven Microenvironmental Traits Promoting Cancer Progression. Advancesin Experimental Medicine and Biology. 2020; 1232: 169–176.

[24] O’Reilly D, Johnson P, Buchanan PJ. Hypoxia induced cancer stem cell enrichment promotes resistance to androgen deprivation therapy in prostate cancer. Steroids. 2019; 152: 108497.

[25] Martinotti S, Ranzato E. Scratch Wound Healing Assay. Methods in Molecular Biology. 2019; 26: 225–229.

[26] Berens EB, Holy JM, Riegel AT, Wellstein A. A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting. Journal of Visualized Experiments. 2015; 53409.

[27] Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols. 2009; 4: 44–57.

[28] Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Research. 2009; 37: 1–13.

[29] Lee SO, Dutt SS, Nadiminty N, Pinder E, Liao H, Gao AC. Develop-ment of an androgen-deprivation induced and androgen suppressed human prostate cancer cell line. The Prostate. 2007; 67: 1293–1300.

[30] Umekita Y, Hiipakka RA, Kokontis JM, Liao S. Human prostate tumor growth in athymic mice: inhibition by androgens and stimulation by finasteride. Proceedings of the National Academy of Sciences. 1996; 93: 11802–11807.

[31] Jaikumarr Ram A, Girija AS S, Jayaseelan VP, Arumugam P. Overexpression of BASP1 Indicates a Poor Prognosis in Head and Neck Squamous Cell Carcinoma. Asian Pacific Journal of Cancer Prevention. 2020; 21: 3435–3439.

[32] Tang H, Wang Y, Zhang B, Xiong S, Liu L, Chen W, et al. High brain acid soluble protein 1(BASP1) is a poor prognostic factor for cervical cancer and promotes tumor growth. Cancer Cell International. 2017; 17: 97.

[33] Wu S, Yuan W, Shen Y, Lu X, Li Y, Tian T, et al. The miR-608 rs4919510 polymorphism may modify cancer susceptibility based on type. Tumor Biology. 2017; 39: 1010428317703819.

[34] Sohn HM, Kim B, Park M, Ko YJ, Moon YH, Sun JM, et al. Effect of CD133 overexpression on bone metastasis in prostate cancer cell line LNCaP. Oncology Letters. 2019; 18: 1189–1198.

[35] Sun C, Zhang G, Cheng S, Qian H, Li D, Liu M. URG11 promotes proliferation and induced apoptosis of LNCaP cells. International Journal of Molecular Medicine. 2019; 43: 2075–2085.

[36] Nakonechnaya AO, Shewchuk BM. Growth hormone enhances LNCaP prostate cancer cell motility. Endocrine Research. 2015; 40: 97–105.

[37] Thomas S, Chiriva-Internati M, Shah GV. Calcitonin receptor-stimulated migration of prostate cancer cells is mediated by urokinase receptor-integrin signaling. Clinical & Experimental Metastasis. 2007; 24: 363–377.

[38] Chin YR, Yuan X, Balk SP, Toker A. PTEN-Deficient Tumors Depend on AKT2 for Maintenance and Survival. Cancer Discovery. 2014; 4: 942–955.

[39] Li Y, Donmez N, Sahinalp C, Xie N, Wang Y, Xue H, et al. SRRM4 Drives Neuroendocrine Transdifferentiation of Prostate Adenocar-cinoma under Androgen Receptor Pathway Inhibition. European Urology. 2017; 71: 68–78.

[40] Mizerska-Dudka M, Kandefer-Szerszeń M. Opioids, Neutral En-dopeptidase, its Inhibitors and Cancer: is there a Relationship among them? Archivum Immunologiae Et Therapiae Experimentalis. 2015; 63: 197–205.

[41] Namekawa T, Ikeda K, Horie-Inoue K, Inoue S. Application of Prostate Cancer Models for Preclinical Study: Advantages and Limitations of Cell Lines, Patient-Derived Xenografts, and Three-Dimensional Culture of Patient-Derived Cells. Cells. 2019; 8: 74.

[42] Okada S, Vaeteewoottacharn K, Kariya R. Application of Highly Immunocompromised Mice for the Establishment of Patient-Derived Xenograft (PDX) Models. Cells. 2019; 8: 889.

[43] Rea D, del Vecchio V, Palma G, Barbieri A, Falco M, Luciano A, et al. Mouse Models in Prostate Cancer Translational Research: from Xenograft to PDX. BioMed Research International. 2016; 2016: 9750795.

[44] Dockhorn RJ, Green AW, Green E. Assessing the efficacy and safety of q. d. theophylline therapy: a multicenter study. Annals of Allergy. 1985; 55: 658–664.

[45] van Weerden WM, Romijn JC. Use of nude mouse xenograft models in prostate cancer research. The Prostate. 2000; 43: 263–271.

[46] Calenda G, Tong Y, Kanika ND, Tar MT, Suadicani SO, Zhang X, et al. Reversal of diabetic vasculopathy in a rat model of type 1 diabetes by opiorphin-related peptides. American Journal of Physiology-Heart and Circulatory Physiology. 2011; 301: H1353–H1359.

[47] Tong Y, Tar M, Monrose V, DiSanto M, Melman A, Davies KP. HSMR3a as a Marker for Patients with Erectile Dysfunction. Journal of Urology. 2007; 178: 338–343.

[48] Kanika ND, Tar M, Tong Y, Kuppam DSR, Melman A, Davies KP. The mechanism of opiorphin-induced experimental priapism in rats involves activation of the polyamine synthetic pathway. American Journal of Physiology-Cell Physiology. 2009; 297: C916–C927.

[49] Thoma CR, Zimmermann M, Agarkova I, Kelm JM, Krek W. 3D cell culture systems modeling tumor growth determinants in cancer target discovery. Advanced Drug Delivery Reviews. 2014; 69-70: 29–41.

[50] Koussounadis A, Langdon SP, Um IH, Harrison DJ, Smith VA. Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system. Scientific Reports. 2015; 5: 10775.

[51] Shankavaram UT, Reinhold WC, Nishizuka S, Major S, Morita D, Chary KK, et al. Transcript and protein expression profiles of the NCI-60 cancer cell panel: an integromic microarray study. Molecular Cancer Therapeutics. 2007; 6: 820–832.

Abstracted / indexed in

Science Citation Index Expanded (SciSearch) Created as SCI in 1964, Science Citation Index Expanded now indexes over 9,200 of the world’s most impactful journals across 178 scientific disciplines. More than 53 million records and 1.18 billion cited references date back from 1900 to present.

Journal Citation Reports/Science Edition Journal Citation Reports/Science Edition aims to evaluate a journal’s value from multiple perspectives including the journal impact factor, descriptive data about a journal’s open access content as well as contributing authors, and provide readers a transparent and publisher-neutral data & statistics information about the journal.

Directory of Open Access Journals (DOAJ) DOAJ is a unique and extensive index of diverse open access journals from around the world, driven by a growing community, committed to ensuring quality content is freely available online for everyone.

SCImago The SCImago Journal & Country Rank is a publicly available portal that includes the journals and country scientific indicators developed from the information contained in the Scopus® database (Elsevier B.V.)

Publication Forum - JUFO (Federation of Finnish Learned Societies) Publication Forum is a classification of publication channels created by the Finnish scientific community to support the quality assessment of academic research.

Scopus: CiteScore 0.9 (2023) Scopus is Elsevier's abstract and citation database launched in 2004. Scopus covers nearly 36,377 titles (22,794 active titles and 13,583 Inactive titles) from approximately 11,678 publishers, of which 34,346 are peer-reviewed journals in top-level subject fields: life sciences, social sciences, physical sciences and health sciences.

Norwegian Register for Scientific Journals, Series and Publishers Search for publication channels (journals, series and publishers) in the Norwegian Register for Scientific Journals, Series and Publishers to see if they are considered as scientific. (

Submission Turnaround Time