Background:
Several years ago, we have identified a number of genes that are differentially regulated in response to androgens and had enriched expression in the prostate, a major target organ for androgens. Three of the genes that we identified in this work are Six TrAnsMembrane Protein of prostate 1 and 2 (STAMP1 and STAMP2) (Korkmaz et al., 2002, 2005; Wellen et al., 2007; Wang et al., 2010; Lindstad et al., 2010), and Kallikrein 4 (KLK4) (Xi et al., 2004; Klokk et al., 2007; Jin et al., 2013).
Confocal immunoflourescence microscopy and live cell imaging techniques have shown that STAMPs fused to Green Fluorescent Protein (GFP) are localized in the Golgi/ER and shuttle between these organelles and the plasma membrane. In addition, STAMPs are targeted to the endocytic pathway and may be receptors for a ligand. Furthermore, STAMP1 is highly enriched to prostate for expression and is expressed only in AR positive prostate cancer cell lines whereas STAMP2 is more widely expressed, but exquisitely androgen regulated. Consistent with a role in cancer development, both STAMP1 and STAMP2 expression is increased in prostate cancer compared with normal prostate. Overexpression of STAMPs in prostate cancer cells increases their proliferation whereas siRNA-mediated knockdown decreases cell growth. Furthermore, both STAMP1 and STAMP2 inhibit apoptosis in prostate cancer cells. Interestingly, STAMP2 knockout mice spontaneously develop all the hallmarks of the metabolic syndrome on a regular diet indicating that it has an important role in regulating metabolism (Wellen et al., 2007; Lindstad et al., 2010). Consistently, STAMP2 also affects metabolic pathways in prostate cancer cells. Futhermore, STAMP2 is involved in regulating macrophage inflammatory responses and atherosclerosis in mouse models (ten Freyhaus et al., 2012). These findings warrant more detailed analysis of STAMP function in normal cells, as well as under pathological conditions.
We found that KLK4 is the first member of the normally secreted KLK family that is intracellular (Xi et al., 2004). Similar to STAMPs, KLK4 expression is significantly increased in prostate cancer compared with normal prostate. Overexpression of KLK4 in prostate cancer cells increases their proliferation as well as anchorage independent growth, whereas siRNA-mediated knockdown decreases cell growth and sensitizes cells to apoptosis inducing agents. Remarkably, KLK4 knockdown by systemic nanoliposomal siRNA delivery results in profound tumor regression in two independent preclinical models of prostate cancer (Figure 1) (Jin et al., 2013).
Projects:
Various lines of investigation are under way regarding the STAMPs and KLK4 which are related with each other using similar approaches. For each project, the incoming MSc student will work with a postdoctoral fellow and/or a Ph.D. candidate. The two different areas are summarized below:
Project 1. Characterization of the interacting partners of STAMPs/KLK4 and the respective signaling pathways. The knowledge of the interacting partners of any protein is important to delineate their function in the cell. We have thus used the yeast two-hybrid assay and identified several potential STAMP and KLK4 interactors. For example, for KLK4 we found that it interacts with the Promyelocytic Leukemia Zinc Finger (PLZF) which act as a transcriptional repressor and a tumor supressor. Based on KLK4-PLZF interactions, we have charted a new network of interactions that integrate two of the most significant signaling pathways in the cell, androgen and PI3K/mTOR signaling (Jin et al., 2013; Figure 2). The validation of the interactors for STAMPs and their functional consequence(s), if any, is in progress, and has already been established for some of the interactors. The different lines of investigation in this project is to carry these forward, validate them in prostate cancer cells, and determine if they have functional consequences. To that end, several approaches are being used. First, coimmunoprecipitation assays are utilized to study the interactions in vivo. Second, GST-pulldown assays are used to complement the coimmunoprecipitations and study interactions in more detail. Third, possible colocalization in cells is determined by immunoflourescence microscopy. Foruth, functional interactions are assessed based by using a variety of assays based on the interacting protein involved.
Project 2. Functional analysis of STAMPs and KLK4 in prostate cancer cells. As noted above, we have strong evidence for the involvement of STAMPs and KLK4 in the biology prostate cancer cells. Building on our current findings, we are investigating the possible mechanisms through which these proteins increase proliferation, inhibit apoptosis, and affect metabolic pathways in prostate cancer cells and adipocytes. As exemplified by KLK4, we have evidence which indicates that these proteins are involved in regulating the activity of some of the central intracellular signaling pathways (Figure 2). The goal of the studies in this project is to use both cell lines in vitro, as well as xenografts in vivo, to establish if these pathways are indeed affected, and if so, determine the exact molecular mechanisms that are involved. We have generated Stamp1 and Stamp2 knockout mice which are also available for use in these studies. We are also interested in manipulating expression of these three proteins in different permutations to see if we can have synergistic effects on prostate cancer viability in vitro and in vivo.
In both of these projects the student will gain in depth experience with mammalian cell culture, working with DNA, RNA, and protein, and becoming proficient in basic and some advanced molecular and cell biology methodology. If interested, there are possibilities of learning and using mouse models to complement in vitro data which is encouraged.
For questions and more information, please contact Fahri Saatcioglu at 22854569, fahris@ibv.uio.no, Rm. 3611, or come talk to us during the poster presentations of the different groups in September. We are especially interested in those students who are already considering a career in science and would like to work in an internationally competitive laboratory. For more detailed information on the projects and the lab, please see our website
References:
Jin, Y., Qu, S., Wang, L., Kristian, A., Maelandsmo, G.M., Jeronimo, C., Teixeira, M.R., Tekedereli, I., Sood, A., Lopez-Berestein, G., Ozpolat, B., Danielsen, H., and Saatcioglu, F. (2013). Kallikrein 4 integrates androgen and mTOR signalling in prostate cancer. Proc. Nat. Acad. Sci. USA 110(28):E2572-81.
Jin, Y., Wang, L., Qu, S., Kristian, A., Mælandsmo, G.M., Yuca, E., Tekedereli, I., Gorgulu, K., Alpay, N., Sood, A., Lopez-Berestein, G., Fazli, L., Rennie, P., Risberg, B., Wæhre, H., Danielsen, H.D., Ozpolat, B.,and Fahri Saatcioglu (2015). STAMP2 increases oxidative stress and is critical for prostate cancer. EMBO Mol Med, 7(3):315-31
Klokk, T.I., Kilander, A., Xi, Z., Wæhre, H., Risberg, B., Danielsen, H.E., and Saatcioglu, F. (2007). Kallikrein 4 is a proliferative factor that is overexpressed in prostate cancer. Cancer Res. 67, 5221-5230.
Korkmaz, K., Elbi, C., Korkmaz, C., Kurys, P., Hager, G., Loda, M., and Saatcioglu, F. (2002). Cloning and characterization of STAMP1--A highly prostate restricted six transmembrane protein overexpressed in prostate cancer. J. Biol. Chem. 277, 36689-96696.
Korkmaz, C., Korkmaz, K., Kurys, P., Elbi, C., Klokk, T.I., Hammarstrom, C. Svindland, A., Hager, G., and Saatcioglu, F. (2005). Molecular cloning and characterization of STAMP2, an androgen regulated six-trans-membrane protein that is overexpressed in a subset of prostate cancers. Oncogene 24, 4934-4945.
Lindstad, T., Jin, Y., Wang, L., Qu, S., Saatcioglu, F. (2010). STAMPs at the crossroads of cancer and nutrition. Nutr Cancer 62:891-895.
Sikkeland, J., and Saatcioglu, F. (2013). Differential regulation and function of STAMP family proteins during adipogenesis. PloS One 8(7):e68249.
Sheng, X., Arnoldussen, Y.W., Storm, M., Tesikova, M., Jin, Y., Nenseth, H.Z., Sen Zhao, S., Mills, I.G., Fazli, L., Rennie, P., Risberg, B., Wæhre, H., Danielsen, H.E. Hotamisligil, G.S., and Saatcioglu, F. (2015). Divergent androgen regulation of unfolded protein response pathways drives prostate cancer. EMBO Mol Med, e201404509.
Wellen, K.E., Fucho, R., Gregor, M.F., Furuhashi, M., Morgan, C., Lindstad, T., Gorgun, C., Vaillencourt, C., Saatcioglu, F., and Hotamisligil, G.S. (2007). Coordinated regulation of nutrient and inflammatory responses by STAMP2 is essential for metabolic homeostasis. Cell 129, 537-548.
Figure 1. KLK4 depletion by nanoliposomal siRNA delivery induces tumor regression in vivo. Two preclinical models of human PCa (A: LNCaP and B: VCaP) were established in the right flank of male nude mice. After tumors have grown to 5 mm in size, mice were given either empty liposomes, DOPC-nanoliposome encapsulated non-silencing control siRNA, or KLK4 siRNA from the tail vein, twice a week. At the indicated weeks after the first injection, tumor sizes were measured. n=6 for each group of mice. (Jin et al., 2013).
Figure 2. Model depicting interactions between AR, KLK4, and PLZF, and the functional consequences for PCa. Overexpression of KLK4 or dysregulation of PLZF will result in sustained activation of the AR and mTOR signaling pathways which leads to increased proliferation and survival of PCa cells. Conversely, knockdown of KLK4 will increase PLZF and REDD1 levels and thus inhibit both the AR and mTOR pathways. (Jin et al., 2013)
Figure 3. Regulation of UPR pathways by androgens in PCa cells. Androgen receptor (AR) directly binds to the regulatory elements in the IRE1, XBP1, and XBP1 target genes and activate their expression. Simultaneously, AR inhibits JNK activation induced by IRE1 as well as inactivating the PERK pathway. As a result, apoptosis is inhibited and proliferation/survival is increased (Sheng et al., 2015)