Therapeutic response in human cancers : the dual role of STAT signalling

STAT proteins were discovered as the mediators of interferon (IFN) signalling in response to viral infections. Later, it has become evident that STATs are activated by many stimuli and that they exert multifaceted effects by regulating gene transcription. Of seven members of the STAT family, my thesis dealt with STAT1, STAT2, and STAT3 and the genes regulated by transcriptional complexes containing them. STAT1 and STAT3 were believed to regulate the opposing functions, the former acting as a tumour suppressor and the latter being an oncogene. Type I IFNs have been used for the treatment of infectious diseases and some types of cancer. In Paper I, we investigated pathways involved in the IFN-induced apoptosis in a myeloma cell line, and what role the STAT1 phosphorylation plays in this model. We used chemical inhibitors of pSTAT1, Akt, mTOR, and cells with a dominant-negative mutant form of STAT1 to evaluate which pathways are essential for the pro-apoptotic effect of IFNα. We have found that pSTAT1 is important, but cooperation with other signalling pathways is necessary to maximize the proapoptotic effect of IFNα. In paper II, we used multicellular spheroids (3D culture) as a model to study a gene signature associated with drug resistance. We have found that STAT1, STAT2, and IRF9, as well as IFNstimulated genes (ISGs), have increased expression in this drug resistance model. Moreover, a similar gene signature is induced in cells cultured for a prolonged time with no trace of IFN detected. The expression of ISGs was not STAT1 dependent but was controlled by STAT2 and IRF9. Overexpression of IRF9 alone was sufficient to drive the transcription of the ISGs and to induce drug resistance in the cells. Therefore, IRF9-induced gene signature can be explored as a marker for therapy response in cancer. In Paper III, we studied the role of the constitutively activated STAT3 in the sensitivity of multiple myeloma cells to the Hsp90 inhibitors treatment. We used a panel of cell lines categorised by different basal levels of the pTyr705STAT3 and of CD45 and found that the sensitivity of myeloma cells to an Hsp90 inhibitor correlated with the presence of pSTAT3. Using samples from multiple myeloma patients, we have demonstrated that it is the pSTAT3+CD45+ cell population that undergoes apoptosis in response to the Hsp90 inhibitor treatment. Thus, pSTAT3/CD45 can be used as a stratification marker for the use of these drugs. In Paper IV, we attempted to develop new inhibitors targeting STAT3. After the screening campaign, we have chosen several inhibitors that preferentially affect the viability in STAT3dependent cell lines. The inhibitors have different effects on the phosphorylation of STAT3 and STAT1, but regardless of that, all the compounds interfere with the STAT3-driven gene transcription. One of the compounds, KI16, preferentially inhibits the phosphorylation of STAT3 over STAT1. It also docks well to the SH2-domain of STAT3 and has a potential to be developed as a STAT3-targeting drug. Other compounds act through a different mechanism(s), but are also plausible for chemical modifications and development, both as drugs and as molecular probes to identify novel targets important for the full oncogenic function of STAT3. Taken together, our findings demonstrate that the roles of STAT1 and STAT3 in cancer are not strictly determined, but are highly context-dependent. It appears that the IFN/STAT1 signalling can be both pro-apoptotic and pro-survival, whereas the oncogenic JAK/STAT3 axis can be targeted to induce cancer cell death. List of Scientific Papers I. Arulampalam, V. , Kolosenko, I. , Hjortsberg, L. , Bjorklund, A.C. , Grander, D. and Tamm, K.P. (2011) Activation of STAT1 is required for interferon-alpha-mediated cell death. Experimental cell research, 317, 9-19. II. Kolosenko, I. , Fryknas, M. , Forsberg, S. , Johnsson, P., Cheon, H. , Holvey-Bates, E.G., Edsbacker, E. , Pellegrini, P., Rassoolzadeh, H. , Brnjic, S. , Larsson, R. , Stark, GR. , Grander, D. , Linder, S. , Tamm, K.P.* and De Milito, A*. (2015) Cell crowding induces interferon regulatory factor 9, which confers resistance to chemotherapeutic drugs. International journal of cancer. 136, E51-61. III. Lin, H*. , Kolosenko, I*. , Bjorklund, A.C. , Protsyuk, D. , Osterborg, A. , Grander, D. and Tamm, K.P. (2013) An activated JAK/STAT3 pathway and CD45 expression are associated with sensitivity to Hsp90 inhibitors in multiple myeloma. Experimental cell research, 319, 600-611. IV. Kolosenko, I. , Yu, Y. ,* Busker, S. ,* Page, B. , Liu, J., Reese, A. , Volk, N. , Haraldsson, M. , Lundbäck, T. , Tamm K.P. and Grander D. (2016) Development and characterization of novel STAT inhibitors. Manuscript in preparation. *equal contribution Publications not included in the thesis: Kolosenko, I. , Grander, D. and Tamm, K.P. (2014) IL-6 activated JAK/STAT3 pathway and sensitivity to Hsp90 inhibitors in multiple myeloma. Current medicinal chemistry, 21, 3042-3047. (Review) Akcakaya, P., Ekelund, S. , Kolosenko, I. , Caramuta, S. , Ozata, D.M. , Xie, H. , Lindforss, U. , Olivecrona, H. and Lui, W.O. (2011) miR-185 and miR-133b deregulation is associated with overall survival and metastasis in colorectal cancer. International journal of oncology, 39, 311-318.