Skip to main content

Advertisement

Springer Nature Link
Log in

Hypoxic Regulation of the KLK4 Gene in two Different Prostate Cancer Cells Treated with TGF- β

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

The human kallikrein-related peptidase (KLK) family which consists of 15 members is associated with prostate cancer and other cancers. It has been reported that overexpression of KLK4 in prostate cancer correlates with bone metastasis or advanced stage. Hypoxia occurs in the early stages of prostate cancer due to the accumulation of acidic metabolites or reactive oxygen species (ROS). In our study, KLK4 gene expression in hypoxic conditions in PC-3 and LNCaP cells which are treated with TGF-β was evaluated with mRNA, protein, and promoter activity levels. A chemical hypoxia model was created and confirmed at mRNA and protein level. No statistically significant cytotoxic effect of CoCl2 and TGF-β was observed in PC-3 and LNCaP cells with the MTT test. Four different truncated KLK4 gene promoter constructs were cloned in pmetLuc expression vector and basal activities of all promoter fragments were analyzed. The activities of P1 (−447/ + 657), P2 (−103/ + 657), and P3 (−267/ + 657) promoter fragments increased in hypoxic conditions except P4 (+555/ + 657), which does not contain the SMAD and HRE region. KLK4 mRNA levels in both PC-3 and LNCaP cells increased in the hypoxia and hypoxia/TGF groups compared to the non-treated groups. The stimulating effect of TGF-β is correlated with the increase in SMAD2/3 mRNA levels. KLK4 expression is up-regulated by TGF-β, especially under hypoxic conditions, and its interaction with the SMAD pathway is determined with different inhibitor experiments. HIF-1α and SMAD transcription factors bind to the KLK4 promoter showing the direct interaction of HIF-1α (−80/−52) and SMAD (+163/+194) regions with EMSA.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

Data Availability

The sequence used in KLK4 promoter cloning was obtained from NCBI NG_012154.2 RefSeqGene on Chromosome 10 and the clones were confirmed by sequence analysis. Sequence analysis results can be sent if desired. The KLK4 promoter sequence will also be registered with NCBI.

References

  1. Emami, N., & Diamandis, E. P. (2008). Utility of Kallikrein-Related Peptidases (KLKs) as cancer biomarkers. Clinical Chemistry, 54, 1600–1607. https://doi.org/10.1373/clinchem.2008.105189.

    Article  CAS  PubMed  Google Scholar 

  2. Kontos, C. K., & Scorilas, A. (2012). Kallikrein-related peptidases (KLKs): a gene family of novel cancer biomarkers. Clinical Chemistry and Laboratory Medicine, 50, 1877–1891. https://doi.org/10.1515/cclm-2012-0247.

    Article  CAS  PubMed  Google Scholar 

  3. Kryza, T., Silva, L. M., Bock, N., Fuhrman-Luck, R. A., Stephens, C. R., Gao, J., Samaratunga, H., Lawrence, M. G., Hooper, J. D., Dong, Y., Risbridger, G. P., & Clements, J. A. (2017). Kallikrein-related peptidase 4 induces cancer-associated fibroblast features in prostate-derived stromal cells. Molecular Oncology, 11, 1307–1329. https://doi.org/10.1002/1878-0261.12075.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Fuhrman-Luck, R. A., Stansfield, S. H., Stephens, C. R., Loessner, D., & Clements, J. A. (2016). Prostate Cancer-Associated Kallikrein-Related Peptidase 4 Activates Matrix Metalloproteinase-1 and Thrombospondin-1. Journal of Proteome Research, 15, 2466–2478. https://doi.org/10.1021/acs.jproteome.5b01148.

    Article  CAS  PubMed  Google Scholar 

  5. Okuyan, D., Turkoglu, S. A., & Kockar, F. (2020). Carbonic anhydrase III is a new target of HIF1α in prostate cancer model. Gene, 762, 145034 https://doi.org/10.1016/j.gene.2020.145034.

    Article  CAS  PubMed  Google Scholar 

  6. Selvendiran, K., Bratasz, A., Kuppusamy, M. L., Tazi, M. F., Rivera, B. K., & Kuppusamy, P. (2009). Hypoxia induces chemoresistance in ovarian cancer cells by activation of signal transducer and activator of transcription 3. International Journal of Cancer, 125, 2198–2204. https://doi.org/10.1002/ijc.24601.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Marignol, L., Lawler, M., Coffey, M., & Hollywood, D. (2005). Achieving hypoxiainducible gene expression in tumours. Cancer Biology &Therapy, 4, 365–370. https://doi.org/10.4161/cbt.4.4.1646.

    Article  Google Scholar 

  8. Türkoğlu, S. A., Dayi, G., & Köçkar, F. (2020). Upregulation of PSMD4 gene by hypoxia in prostate cancer cells. Turkish Journal of Biology, 44, 275–283. https://doi.org/10.3906/biy-2002-71.

    Article  CAS  Google Scholar 

  9. Turkoglu, S. A., Okuyan, D., & Kockar, F. (2019). TGF-β downregulates CAIII expression via MAPK and PI3K signaling pathways in colon carcinoma and osteosarcoma cells. Archives of Biological Sciences, 71, 393–401. https://doi.org/10.2298/ABS181008020A.

    Article  Google Scholar 

  10. Massague, J. (1998). TGF-β signal transduction. Annual Review of Biochemistry, 67, 753–791. https://doi.org/10.1146/annurev.biochem.67.1.753.

    Article  CAS  PubMed  Google Scholar 

  11. Thompson-Elliott, B., Johnson, R., & Khan, S. A. (2021). Alterations in TGFβ signaling during prostate cancer progression. American Journal of Clinical and Experimental Urology, 9, 318–328.

    PubMed  PubMed Central  Google Scholar 

  12. Massague, J., Seoane, J., & Wotton, D. (2005). Smad transcription factors. Genes & Development, 19, 2783–2810. https://doi.org/10.1101/gad.1350705.

    Article  CAS  Google Scholar 

  13. Korkmaz, S. K., Korkmaz, G. C., Pretlow, G. T., & Saatcioglu, F. (2001). Distinctly different gene structure of KLK4/KLK-L1/Prostase/ARM1 compared with other members of the Kallikrein family: intracellular localization, alternative cDNA forms, and regulation by multiple hormones. DNA and Cell Biology, 20, 435–445. https://doi.org/10.1089/104454901750361497.

    Article  CAS  PubMed  Google Scholar 

  14. Suzuki, M., Shin, M., Simmer, J. P., & Bartlett, J. D. (2014). Fluoride affects enamel protein content via TGF-β1-mediated KLK4 inhibition. Journal of Dental Research, 93, 1022–1027. https://doi.org/10.1177/0022034514545629.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Shahinian, H., Loessner, D., Biniossek, M. L., Kizhakkedathu, J. N., Clements, J. A., Magdolen, V., & Schilling, O. (2014). Secretome and degradome profiling shows that Kallikrein-related peptidases 4, 5, 6, and 7 induce TGFβ-1 signaling in ovarian cancer cells. Molecular Oncology, 8, 68–82. https://doi.org/10.1016/j.molonc.2013.09.003.

    Article  CAS  PubMed  Google Scholar 

  16. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods, 25, 402–408. https://doi.org/10.1006/meth.2001.1262.

    Article  CAS  PubMed  Google Scholar 

  17. Altuntaş, C., Alper, M., Keleş, Y., Sav, F. N., & Köçkar, F. (2023). Hypoxic regulation of ADAMTS-2 and-3 (a disintegrin and matrix metalloproteinase with thrombospondin motifs 2 and 3) procollagen N proteinases by HIF-1α in endothelial cells. Molecular and Cellular Biochemistry, 478, 1151–1160.

    Article  PubMed  Google Scholar 

  18. Poyrazlı, F., Türkoğlu, S. A., Babacan, D., & Köçkar, F. (2022). Farklı Hücre Hatlarında KLK4 Gen İfadesinin Belirlenmesi. Karaelmas Fen ve Mühendislik Dergisi, 12, 207–215. https://doi.org/10.7212/karaelmasfen.1080507.

    Article  Google Scholar 

  19. Xi, Z., Klokk, T. I., Korkmaz, K., Kurys, P., Elbi, C., Risberg, B., Danielsen, H., Loda, M., & Saatcioglu, F. (2004). Kallikrein 4 is a Predominantly Nuclear Protein and Is Overexpressed in Prostate Cancer. Cancer Research, 64, 2365–2370. https://doi.org/10.1158/0008-5472.CAN-03-2025.

    Article  CAS  PubMed  Google Scholar 

  20. Dong, Y., Kaushal, A., Bui, L., Chu, S., Fuller, P. J., Nicklin, J., Samaratunga, H., & Clements, J. A. (2001). Human kallikrein 4 (KLK4) is highly expressed in serous ovarian carcinomas. Clinical Cancer Research, 7, 2363–2371.

    CAS  PubMed  Google Scholar 

  21. Lai, J., Myers, S. A., Lawrence, M. G., Odorico, D. M., & Clements, J. A. (2009). Direct progesterone receptor and indirect androgen receptor interactions with the kallikrein-related peptidase 4 gene promoter in breast and prostate cancer. Molecular Cancer Research, 7, 129–141. https://doi.org/10.1158/1541-7786.MCR-08-0218.

    Article  CAS  PubMed  Google Scholar 

  22. Veveris-Lowe, T. L., Lawrence, M. G., Collard, R. L., Bui, L., Herington, A. C., Nicol, D. L., & Clements, J. A. (2005). Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells. Endocrine-related cancer, 12, 631–643. https://doi.org/10.1677/erc.1.00958.

    Article  CAS  PubMed  Google Scholar 

  23. Mukai, S., Yorit, K., Yamasaki, K., Nagai, T., Kamibeppu, T., Sugie, S., Kida, K., Onizuka, C., Tsukino, H., Kamimura, T., Kamoto, T., & Kataoka, H. (2015). Expression of human kallikrein 1-related peptidase 4 (KLK4) and MET phosphorylation in prostate cancer tissue: immunohistochemical analysis. Human Cell, 28, 133–142.

    Article  CAS  PubMed  Google Scholar 

  24. Obiezu, C. V., Scorilas, A., Katsaros, D., Massobrio, M., Yousef, G. M., Fracchioli, S., Rigault de la Longrais, I. A., Arisio, R., & Diamandis, E. P. (2001). Higher human kallikrein gene 4 (KLK4) expression indicates poor prognosis of ovarian cancer patients. Clinical Cancer Research, 7, 2380–2386.

    CAS  PubMed  Google Scholar 

  25. Avgeris, M., Mavridis, K., & Scorilas, A. (2012). Kallikrein-related peptidases in prostate, breast, and ovarian cancers: from pathobiology to clinical relevance. Biological Chemistry, 393, 301–317. https://doi.org/10.1515/hsz-2011-0260.

    Article  CAS  PubMed  Google Scholar 

  26. Yang, F., Chen, Y., Shen, T., Guo, D., Dakhova, O., Ittmann, M. M., Creighton, C. Z., Zhang, Y., Dang, T. D., & Rowley, D. R. (2014). Stromal TGF-β signaling induces AR activation in prostate cancer. Oncotarget, 5, 10854–10869. https://doi.org/10.18632/oncotarget.2536.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Yousef, G. M., Obiezu, C. V., Luo, L. Y., Black, M. H., & Diamandis, E. P. (1999). Prostase/KLK-L1 is a new member of the human kallikrein gene family, is expressed in prostate and breast tissues, and is hormonally regulated. Cancer Research, 59, 4252–4256.

    CAS  PubMed  Google Scholar 

  28. Nelson, P. S., Gan, L., Ferguson, C., Moss, P., Gelinas, R., Hood, L., & Wang, K. (1999). Molecular cloning and characterization of prostase, an androgen-regulated serine protease with prostate-restricted expression. Proceedings of Nationall Academy of Sciences, 96, 3114–3119. https://doi.org/10.1073/pnas.96.6.3114.

    Article  CAS  Google Scholar 

  29. Niu, Y., Yeh, S., Miyamoto, H., Li, G., Altuwaijri, S., Yuan, J., Han, R., Ma, T., Kuo, H. C., & Chang, C. (2008). Tissue prostate-specific antigen facilitates refractory prostate tumor progression via enhancing ARA70-regulated androgen receptor transactivation. Cancer Research, 68, 7110–7119. https://doi.org/10.1158/0008-5472.CAN-07-6507.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lai, J., An, J., Nelson, C. C., Lehman, M. L., Batra, J., & Clements, J. A. (2014). Analysis of androgen and anti-androgen regulation of KLK-related peptidase 2, 3, and 4 alternative transcripts in prostate cancer. Biological Chemistry, 395, 1127–1132. https://doi.org/10.1515/hsz-2014-0149.

    Article  CAS  PubMed  Google Scholar 

  31. Kryza, T., Silva, M. L., Loessner, D., Heuzé-Vourc’h, N., & Clements, J. A. (2016). The kallikrein-related peptidase family: Dysregulation and functions during cancer progression. Biochimie, 122, 283–299. https://doi.org/10.1016/j.biochi.2015.09.002.

    Article  CAS  PubMed  Google Scholar 

  32. Lawrence, M. G., Lai, J., & Clements, J. A. (2010). Kallikreins on steroids: Structure, function, and hormonal regulation of prostate-specific antigen and the extended kallikrein locus. Endocrine Review, 31, 407–446. https://doi.org/10.1210/er.2009-0034.

    Article  CAS  Google Scholar 

  33. Sanchez, W. Y., de Veer, S. J., Swedberg, J. E., Hong, E. J., Reid, J. C., Walsh, T. P., Hooper, J. D., Hammond, G. L., Clements, J. A., & Harris, J. M. (2012). Selective cleavage of human sex hormone-binding globulin by kallikrein-related peptidases and effects on androgen action in LNCaP prostate cancer cells. Endocrinology, 153, 3179–3189. https://doi.org/10.1210/en.2012-1011.

    Article  CAS  PubMed  Google Scholar 

  34. Costanza, B., Umelo, I. A., Bellier, J., Castronovo, V., & Turtoi, A. (2017). Stromal modulators of TGF-beta in cancer. Journal of Clinical Medicine, 6, 7 https://doi.org/10.3390/jcm6010007.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Dallas, S. L., Zhao, S., Cramer, S. D., Chen, Z., Peehl, D. M., & Bonewald, L. F. (2005). Preferential production of latent transforming growth factor beta-2 by primary prostatic epithelial cells and its activation by prostate-specific antigen. Journal of Cellular Physiology, 202, 361–370. https://doi.org/10.1002/jcp.20147.

    Article  CAS  PubMed  Google Scholar 

  36. Park, S. Y., Kim, Y. J., Gao, A. C., Mohler, J. L., Onate, S. A., Hidalgo, A. A., Ip, C., Park, E. M., Yoon, S. Y., & Park, Y. M. (2006). Hypoxia increases androgen receptor activity in prostate cancer cells. Cancer Research, 66, 5121–5129. https://doi.org/10.1158/0008-5472.CAN-05-1341.

    Article  CAS  PubMed  Google Scholar 

  37. Mitani, T., Yamaji, R., Higashimura, Y., Harada, N., Nakano, Y., & Inui, H. (2011). Hypoxia enhances transcriptional activity of androgen receptor through hypoxia-inducible factor-1α in a low androgen environment. The Journal of Steroid Biochemistry and Molecular Biology, 123, 58–64. https://doi.org/10.1016/j.jsbmb.2010.10.009.

    Article  CAS  PubMed  Google Scholar 

  38. Mohamed, O. A., Tesen, H. S., Hany, Sherif, A., Abdelwaha, M. M., & Elnaggar, M. H. (2023). The role of hypoxia on prostate cancer progression and metastasis. Molecular Biology Report, 50, 3873–3884.

    Article  CAS  Google Scholar 

  39. Cameron, S., Deblois, G., Hawley, J. R., Qamra, A., Zhou, S., Tonekaboni, S. A. M., Murison, A., Vliet, R. V., Liu, J., Locasale, J. W., & Lupien, M. (2023). Chronic hypoxia favours adoption to a castration-resistant cell state in prostate cancer. Oncogene, 42, 1693–1703.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ke, Q., & Costa, M. (2006). Hypoxia-inducible factor-1 (HIF-1). Molecular Pharmacology, 70, 1469–1480. https://doi.org/10.1124/mol.106.027029.

    Article  CAS  PubMed  Google Scholar 

  41. Lin, M. F., Meng, T. C., Rao, P. S., Chang, C., Schonthal, A. H., & Lin, F. F. (1998). Expression of human prostatic acid phosphatase correlates with androgen-stimulated cell proliferation in prostate cancer cell lines. Journal of Biological Chemistry, 273, 5939–5947. https://doi.org/10.1074/jbc.273.10.5939.

    Article  CAS  PubMed  Google Scholar 

  42. Chlenski, A., Nakashiro, K., Ketels, K. V., Korovaitseva, G. I., & Oyasu, R. (2001). Androgen receptor expression in androgen-independent pros-tate cancer cell lines. The Prostate, 47, 66–75. https://doi.org/10.1002/pros.1048.

    Article  CAS  PubMed  Google Scholar 

  43. Tilley, W. D., Bentel, J. M., Aspinall, J. O., Hall, R. E., & Horsfall, D. J. (1995). Evidence for a novel mechanism of androgen resistance in thehuman prostate cancer cell line, PC-3. Steroids, 60, 180–186. https://doi.org/10.1016/0039-128X(94)00031-7.

    Article  CAS  PubMed  Google Scholar 

  44. Yuan, S., Trachtenberg, J., Mills, G. B., Brown, T. J., Xu, F., & Keating, A. (1993). Androgen-induced inhibition of cell proliferation in an andro-gen-insensitive prostate cancer cell line (PC-3) transfected with ahuman androgen receptor complementary DNA. Cancer Research, 53, 1304–1311.

    CAS  PubMed  Google Scholar 

  45. Heisler, L. E., Evangelou, A., Lew, A. M., Trachtenberg, J., Elsholtz, H. P., & Brown, T. J. (1997). Androgen-dependent cell cycle arrest and apoptoticdeath in PC-3 prostatic cell cultures expressing a full-lengthhuman androgen receptor. Molecular and Cellular Endocrinology, 126, 59–73. https://doi.org/10.1016/S0303-7207(96)03970-6.

    Article  CAS  PubMed  Google Scholar 

  46. Marcelli, M., Haidacher, S. J., Plymate, S. R., & Birnbaum, R. S. (1995). Alteredgrowth and insulin-like growth factor-binding protein-3 pro-duction in PC3 prostate carcinoma cells stably transfected with aconstitutively active androgen receptor complementary deox-yribonucleic acid. Endocrinology, 136, 1040–1048. https://doi.org/10.1210/en.136.3.1040.

    Article  CAS  PubMed  Google Scholar 

  47. Turkoglu, S. A., & Kockar, F. (2016). SP1 and USF differentially regulate ADAMTS1 gene expression under normoxic and hypoxic conditions in hepatoma cells. Gene, 575, 48–57. https://doi.org/10.1016/j.gene.2015.08.035.

    Article  CAS  PubMed  Google Scholar 

  48. Smale, S. T., & Kadonaga, J. T. (2003). The RNA polymerase II core promoter. Annual Review of Biochemistry, 72, 449–479. https://doi.org/10.1146/annurev.biochem.72.121801.161520.

    Article  CAS  PubMed  Google Scholar 

  49. Thomas, M. C., & Chiang, C. M. (2006). The general transcription machinery and general cofactors. Critical Review in Biochemistry and Molecular Biology, 41, 105–178. https://doi.org/10.1080/10409230600648736.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Turkish Research Council (TUBITAK) project (TBAG 118Z369).

Author information

Authors and Affiliations

  1. University of Balikesir, Faculty of Science and Literature, Department of Molecular Biology and Genetics, Balikesir, Turkey

    Fatma Poyrazlı, Feray Köçkar & Sümeyye Aydoğan Türkoğlu

  2. University of Bandırma, Susurluk Vocational Training Schools, Laboratory and Veterinary Health Program, Balikesir, Turkey

    Derya Okuyan

Authors
  1. Fatma Poyrazlı
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Derya Okuyan
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Feray Köçkar
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Sümeyye Aydoğan Türkoğlu
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

Sümeyye Aydoğan Türkoğlu designed the research; Fatma Poyrazlı (FP) performed the experiments; Derya Okuyan (DO) performed the IFC experiment; Sümeyye Aydoğan Türkoğlu (SAT) and Feray Köçkar (FK) checked the results; FP, DO and SAT wrote the paper. FK checked the language of the paper.

Corresponding author

Correspondence to Sümeyye Aydoğan Türkoğlu.

Ethics declarations

Conflict of interest

The authors declar no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Poyrazlı, F., Okuyan, D., Köçkar, F. et al. Hypoxic Regulation of the KLK4 Gene in two Different Prostate Cancer Cells Treated with TGF- β. Cell Biochem Biophys 82, 2797–2812 (2024). https://doi.org/10.1007/s12013-024-01396-5

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-024-01396-5

Key words

Profiles

  1. Fatma Poyrazlı View author profile