Tumors are not merely masses of neoplastic cells but complex tissues composed of cellular and noncellular elements. This review provides recent data on the main components of a dynamic system, such as carcinoma associated fibroblasts that change the extracellular matrix (ECM) topology, induce stemness and promote metastasis-initiating cells. Altered production and characteristics of collagen, hyaluronan and other ECM proteins induce increased matrix stiffness. Stiffness along with tumor growth-induced solid stress and increased interstitial fluid pressure contribute to tumor progression and therapy resistance. Second, the role of immune cells, cytokines and chemokines is outlined. We discuss other noncellular characteristics of the tumor microenvironment such as hypoxia and extracellular pH in relation to neoangiogenesis. Overall, full understanding of the events driving the interactions between tumor cells and their environment is of crucial importance in overcoming treatment resistance and improving patient outcome.

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Asporin has been reported as a tumor suppressor in breast cancer, while asporin-activated invasion has been described in gastric cancer. According to our in silico search, high asporin expresion associates with significantly better relapse free survival (RFS) in patients with low-grade tumors but RFS is significantly worse in patients with grade 3 tumors. In line with other studies, we have confirmed asporin expression by RNA scope in situ hybridization in cancer associated fibroblasts. We have also found asporin expression in the Hs578T breast cancer cell line which we confirmed by quantitative RT-PCR and western blotting. From multiple testing, we found that asporin can be downregulated by bone morphogenetic protein 4 while upregulation may be facilited by serum-free cultivation or by three dimensional growth in stiff Alvetex scaffold. Downregulation by shRNA inhibited invasion of Hs578T as well as of CAFs and T47D cells. Invasion of asporin-negative MDA-MB-231 and BT549 breast cancer cells through collagen type I was enhanced by recombinant asporin. Besides other investigations, large scale analysis of aspartic acid repeat polymorphism will be needed for clarification of the asporin dual role in progression of breast cancer.

Background. The Wnt signaling pathway is crucial for cell fate decisions, stem cell renewal, regulation of cell proliferation and differentiation. Deregulated Wnt signaling is also implicated in a number of hereditary and degenerative diseases and cancer.Methods and results. This review highlights the role of the Wnt pathway in the regulation of stem/progenitor cell renewal and prostate gland development and how this signaling is altered in prostate cancer. Recent evidence suggests that Wnt signaling regulates androgen activity in prostate cancer cells, enhances androgen receptor expression and promotes the growth of prostate cancer even after androgen ablation therapy. There is also strong evidence that Wnt signaling is enhanced in androgen-ablation resistant tumors and bone metastases.Conclusions. Further study of the modulators of this pathway will be of therapeutic relevance as inhibition of Wnt signaling may have the potential to reduce the self-renewal and aggressive behaviour of prostate cancer while Wnt signaling activation might enhance stem cell activity when tissue regeneration is required.

Background. Small leucine rich proteoglycans (SLRPs), major non-collagen components of the extracellular matrix (ECM), have multiple biological roles with diverse effects. Asporin, a member of the SLRPs class I, competes with other molecules in binding to collagen and affects its mineralization. Its role in cancer is only now being elucidated. Methods. The PubMed online database was used to search relevant reviews and original articles. Furthermore, altered asporin expression was analysed in publicly available genome-wide expression data at the Gene Expression Omnibus database. Results. Polymorphisms in the N-terminal polyaspartate domain, which binds calcium, are associated with osteoarthritis and prostate cancer. Asporin also promotes the progression of scirrhous gastric cancer where it is required for coordinated invasion by cancer associated fibroblasts and cancer cells. Besides the enhanced expression of asporin observed in multiple cancer types, such as breast, prostate, gastric, pancreas and colon cancer, tumour suppressive effects of asporin were described in triple-negative breast cancer. We also discuss a number of factors modulating asporin expression in different cell types relevant for alterations toing the tumour microenvironment. Conclusion. The apparent contradicting tumour promoting and suppressive effects of asporin require further investigation. Deciphering the role of asporin and other SLRPs in tumour-stroma interactions is needed for a better understanding of cancer progression and potentially also for novel tumour microenvironment based therapies.

Background Castration‐resistant prostate cancer (PCa) represents a serious health challenge. Based on mechanistically‐supported rationale we explored new therapeutic options based on clinically available drugs with anticancer effects, including inhibitors of PARP1 enzyme (PARPi), and histone deacetylases (vorinostat), respectively, and disulfiram (DSF, known as alcohol‐abuse drug Antabuse) and its copper‐chelating metabolite CuET that inhibit protein turnover. Methods Drugs and their combination with ionizing radiation (IR) were tested in various cytotoxicity assays in three human PCa cell lines including radio‐resistant stem‐cell like derived cells. Mechanistically, DNA damage repair, heat shock and unfolded protein response (UPR) pathways were assessed by immunofluorescence and immunoblotting. Results We observed enhanced sensitivity to PARPi/IR in PC3 cells consistent with lower homologous recombination (HR) repair. Vorinostat sensitized DU145 cells to PARPi/IR and decreased mutant p53. Vorinostat also impaired HR‐mediated DNA repair, as determined by Rad51 foci formation and downregulation of TOPBP1 protein, and overcame radio‐resistance of stem‐cell like DU145‐derived cells. All PCa models responded well to CuET or DSF combined with copper. We demonstrated that DSF interacts with copper in the culture media and forms adequate levels of CuET indicating that DSF/copper and CuET may be considered as comparable treatments. Both DSF/copper and CuET evoked hallmarks of UPR in PCa cells, documented by upregulation of ATF4, CHOP and phospho‐eIF2α, with ensuing heat shock response encompassing activation of HSF1 and HSP70. Further enhancing the cytotoxicity of CuET, combination with an inhibitor of the anti‐apoptotic protein survivin (YM155, currently undergoing clinical trials) promoted the UPR‐induced toxicity, yielding synergistic effects of CuET and YM155. Conclusions We propose that targeting genotoxic and proteotoxic stress responses by combinations of available drugs could inspire innovative strategies to treat castration‐resistant PCa.

Iron chelation therapy is commonly used in patients with myelodysplastic syndrome (MDS), to prevent majorcomplications of iron overload. Besides effects on maintaining control of iron stores and preventing iron-induced cardiac disease, the impact of chelation therapy on overall survival and leukemia-free survival in MDS has been documented, but not well understood. Since MDS bone marrow cells are known to activate DNA damage response (DDR) signaling and iron chelators target cancer cells through multiple stress-response mechanisms (endoplasmic reticulum (ER) stress, autophagy), we hypothesized that iron chelation could reinforce DDR signaling and could thus support tumor-suppressing role of DDR. Also nucleotide deficiency was shown to contribute to DDR, and iron chelation is known to inhibit ribonucleotide reductase (RR), an iron-dependent enzyme, which supplies cells with deoxyribonucleotides (dNTPs). Here, we tested the effects of lysosomotropic iron chelator deferoxamine mesylate (DFO) in a preleukemia mouse model, wherein epigenetic oncogene-induced leukemogenesis is preceded with a long-lasting preleukemia stage (Takacova S, et al. Cancer Cell. 2012;21(4):517-31.). Preleukemic, aberrantly proliferating myeloid cells in this model activate a replication checkpoint and ATR-Chk1-mediated DDR (consistent with oncogene-induced replication stress) and attain hallmarks of senescence (with a long latency), resulting in the inhibition of leukemia progression. A group of 10 preleukemia mice and a group of 10 control mice aged 7 month were treated twice daily with DFO doses adjusted to 88,8 mg/kg (i.p. injection) in order to mimic serum concentrations of the drug achieved in patients. After 6 weeks of chelator administration, the treatment lead to the activation of Chk1(S345) in the bone marrow (BM) of control mice, but did not result in accumulation of γH2AX, a marker of DNA damage, in BM of these mice. In contrast, in preleukemia mice, with already activated threshold of ATR-Chk1 signaling (marker of ongoing oncogene-induced replication stress), Chk1(S345) remained unchanged after DFO treatment. However, we observed significant accumulation of γH2AX foci in oncogene-positive BM cells. These data suggested that iron removal may induce Chk1 activation in vivo, and, in addition, may reinforce activation of DDR in preleukemia cells perhaps due to synthetic effect of iron chelation with oncogene activation resulting in increased levels in DDR signaling (assessment of oxidative DNA damage (8-oxoguanine staining) is ongoing). Next, we analyzed whether iron chelation in both groups of mice influences DNA replication, in which the limiting step is the availability of dNTPs. The RR activity was significantly decreased in the BM of both groups of DFO-treated mice, however, with no impact on the concentration of BM dNTPs; in fact, dNTPs have accumulated in BM of these mice. We revealed that this was a consequence of the activation of S-phase checkpoint in control mice, and of a decrease of actively replicating myeloid cells and activation of G2/M checkpoint in preleukemia mice. Cellular iron depletion was shown to activate p38MAPK pathway (Yu Y, Richardson DR. J Biol Chem. 2011;286(17):15413-27.). p38MAPK pathway, and its component MK2, establishes intra-S-phase cell cycle checkpoint and activates G2/M checkpoint (as a part of DDR, in parallel to Chk1 activation (Reinhardt HC, et al. Curr Opin Cell Biol. 2009;21:245-55.)). Indeed, our preliminary result revealed phosphorylated MK2 specifically in preleukemia mouse BM treated with DFO. Since we did not detect increased apoptosis in BM of DFO treated mice, and because p38MAPK pathway is involved in the activation of ER stress and autophagy, we tested whether markers of ER stress and autophagy are detectable in the mice upon DFO treatment. MyD116 (marker of recovery from ER stress) and LC3-II (marker of autophagy), were specifically induced in preleukemia cells upon DFO treatment. Collectively, these data demonstrate that preleukemia cells exposed to DFO activate distinct but functionally overlapping signaling pathways, resulting in reinforced DDR. Whether this mechanism could increase a barrier against leukemia transformation of chelated MDS patients remains to be investigated. Authorship: LRK and ZS: equal credit as first authors. Acknowledgment: Supported by the Czech Science Foundation (P301/12/1503) and by IGA_LF_2015_015. Disclosures No relevant conflicts of interest to declare.

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