Molecular mechanisms of stress signaling in cancer ¨C Identification of biomarkers and therapeutic targets

The overall focus of our laboratory is to understand the molecular mechanisms of hormone action, especially that of androgens (testosterone) and estrogens, as well as stress signalling as they relate to prostate and breast cancer. To achieve this, we use molecular, biochemical, cellular, and genetic approaches. Part of this work is translational in nature from basic science to the clinic. We thus have collaborations with clinical colleagues in Norway and internationally and have identified a number of potential biomarkers and therapeutic targets for cancer. This MSc project focuses on characterization of stress signaling pathways in prostate cancer that may have implications for other cancer types as well.

Background

Prostate cancer (PCa) is the most common non-cutaneous cancer in men and remains a key challenge for men¡¯s health and a significant economic burden on society (Siegel et al., 2019). Majority of primary prostate tumors are treated successfully by radical prostatectomy or external beam radiotherapy, but often tumors progress to invasive and disseminated disease, and in many cases it is already metastatic at diagnosis (Attard and Antonarakis, 2016). Patients with metastatic PCa are treated with androgen deprivation therapy (ADT); whereas the initial response is very high, complete remissions are rare and disease progression resumes after a median of 2-3 years. The emergent castration-resistant PCa (CRPC) generally responds to recently developed androgen receptor (AR) inhibitory drugs, but their effect is transient and resistance develops. AR-independent disease, such as neuroendocrine PCa emerges and at this point no effective therapy exists, with prognosis very unfavorable and a median survival of 18 months. The goal of our laboratory is to provide a detailed mechanistic understanding of stress signaling in PCa cells, which we recently found to be critical for PCa, and to develop biomarkers for disease progression as well as identifying potential therapeutic targets and testing of new therapies.

In this context, we have recently discovered that androgen signaling significantly regulates the endoplasmic reticulum (ER) stress pathways and the cellular response to establish homeostasis, the unfolded protein response (UPR) ((Sheng et al., 2015) (Sheng et al., 2019) (Unal et al., unpublished data). We found that androgen receptor (AR) directly activated one of the canonical arms of the UPR, inositol-requiring enzyme 1 (IRE1) pathway. Consistent with the importance of IRE1 signaling in PCa, its genetic or pharmacological inhibition dramatically inhibited PCa growth in vitro and in vivo (Sheng et al., 2015) (Sheng et al., 2019). In keeping with these observations, IRE1 expression and its gene signature, are deregulated in human PCa specimens. Further molecular characterization of IRE1 signaling in PCa showed that IRE1 pathway is involved in regulating oncogene signaling that drives PCa progression (Sheng et al., 2019). In addition, we have shown that IRE1 signaling may be involved in CRPC progression and resistance to therapy (Sheng et al., 2019). 

We have also recently found that one of  the other three canonical UPR pathways, mediated by PKR-like ER Kinase (PERK), eukaryotic Translation Initiation Factor 2 alpha (eIF2a), and Activating Transcription Factor 4 (ATF4) signaling, is also required for PCa growth and survival (Pallmann et al., 2019). In addition, AR signaling also affects the PERK-peIF2a-ATF4 pathway that was previously implicated in a number of cancer types (Sheng et al., 2015). We found that ATF4 expression is increased in human PCa compared with benign prostate; consistently, siRNA-mediated ATF4 knockdown inhibited PCa growth in vitro and in vivo in multiple PCa models (Pallmann et al., 2019). Parallel global transcriptomic and proteomic analysis of ATF4 knockdown confirmed previously reported target genes, but also identified novel ones; all gene classes were confirmed in rescue experiments with ATF4 re-expression. We have started to characterize the function of ATF4 target genes in PCa at least one of which provide a feedback loop on PERK pathway regulation.

Project

Functional analysis of ER stress pathways in PCa cells. As noted above, we have strong evidence for the involvement of UPR signaling on PCa cellular phenotype and disesase progression (e.g. Sheng et al., 2019; Pallmann et al., 2019). Building on our recent findings, we are investigating the possible mechanisms through which IRE1 and PERK-ATF4 signaling arms of UPR increase proliferation and inhibit cell death in PCa. We use both cell lines in vitro, as well as xenografts in vivo, to determine the exact molecular mechanisms that are involved. We have established both genetic manipulation as well as pharmacological targeting of these pathways to evaluate their potential as therapeutic targets/biomarkers in PCa. Molecular details and potential feedback mechanisms within each arm of the UPR are being investigated to discover the molecular details of their regulation. In addition, we are exploring the possibility that these two closely related pathways may crosstalk with each other, which may also be provide nodes of control that can be targeted for improved therapeutic applications. To that end, we are using novel reporter cell lines that we have generated that enable examination of UPR signalling simultaneously as well as genome wide CRISPR screen approaches.

In this project, you will gain expertise in the following experimental techniques: mammalian cell culture, siRNA-mediated gene silencing in vitro and in vivo, biochemical assays, qPCR and Western analysis, advanced microscopy methods, and experiments in mice, as well as some bioinformatics tools. Since we are a molecular and cell biology laboratory with a keen interest in translation into the clinic, we use a variety of methods, which is one of the strengths of our research environment for an MSc project. In addition, during the weekly lab meetings, everyone take turns (one person per week) to either present her/his work or an article from the field, which will improve your scientific presentation skills.

You will work closely with a PhD student and/or a postdoctoral fellow. We are especially interested in those students who are already considering a career in science and would like to get experience in an internationally competitive laboratory. For more detailed information on the project and our laboratory, please see our website at https://saatcioglulab.org. For questions and more information, please do not hesitate to contact Fahri Saatcioglu at fahris@ibv.uio.no and request a meeting.

Figure 1. In PCa cells, the androgen receptor (AR) directly activates IRE1¦Á expression, which splices XBP1 mRNA and allows for XBP1s generation. XBP1s transcriptionally induces c-Myc expression, a potent oncoprotein that contributes to tumor progression (left panel). MKC8866 or siXBP1 inhibit XBP1s production, resulting in a significantly decreased c-Myc levels leading to tumor regression (right panel) (Sheng et al., 2019).

References

1. Attard, G., and Antonarakis, E.S. (2016). Prostate cancer: AR aberrations and resistance to abiraterone or enzalutamide. Nature Rev Urol. 13(12): 697-698.
2. 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., W?hre, H., Danielsen, H.E. Hotamisligil, G.S., and Saatcioglu, F. (2015). Divergent androgen regulation of unfolded protein response pathways drives PCa. EMBO Mol Med, e201404509.
3. Sheng, X., Nenseth, H.Z., Qu, S., Fazli, L., Frahnow, T., Simon, L., Rennie, P., Danielsen, H., Theis, F.J., Patterson, J.B., Mills, I.G., Jin, Y., Saatcioglu, F. (2019). IRE1¦Á-XBP1s pathway promotes prostate cancer by activating oncogenic signaling pathways. Nature Comm., Jan 24;10(1):323. doi: 10.1038/s41467-018-08152-3.
4. Siegel, R. L., Miller, K. D., and Jemal, A. (2019). Cancer Statistics 2019. CA: a cancer journal for clinicians 69(1):7-34.
5. Pallmann, N., Livgard, M., Nenseth, H. Z., Xi, Y., Koc, D., Kahraman, N., Mokhlis, H.M., Ozpolat, B., Fazli, L., Rennie, P., Danielsen, H., Jin, Y., Saatcioglu, F. (2019). Regulation of the Integrated Stress Response through ATF4 and FAM129A is Critical for Prostate Cancer. Oncogene, in press.