Moreover as shown in Fig f while the knockdown of

Moreover, as shown in Fig. 5f, while the knockdown of SMURF2 in wild-type AZ505 has no significant effect on HSP27, in CK2α/α′(−/−) cells it leads to an increase of HSP27 level. These results suggest that SMURF2 affects HSP27 protein stability only when overexpressed, at least in our cell model. The downregulation of HSP27 protein in response to CK2 inhibition prompted us to hypothesize that these cells could be more sensitive to heat shock treatment, being HSP27 generally considered an important player in cell thermotolerance [29]. The contribution of HSP27 to thermotolerance has been verified in one of our cell models. The knockout of HSP27 in HepG2 cells increases cell sensitivity to the heat shock treatment respect to the wild type cells (Fig. S3). This aspect is of particular relevance for its potential applications in the field of cancer treatment. Thermotherapy is indeed considered the fifth treatment of cancer along with surgery, chemotherapy, radiotherapy and immunotherapy [30]. A selective in vivo tumour killing is observed following hyperthermia treatment (40–44 °C) based on the different physiology between normal and tumour cells. To our knowledge, the efficacy of a therapy combining heath shock with CK2 inhibition has never been considered to date. Therefore, we tested the combined effects of CK2 inhibition and heat shock stress on the proliferation of the tumoral cell lines HepG2 and HeLa, by comparing dose-response curves of CX-4945 treatment alone and with sequential heat shock treatment at 43 °C for 45 min. A modest thermosensitivity of both cell lines was observed, with only a 10% reduction in cell proliferation after 24 h. However, CX-4945, applied 16 h prior to heat shock treatment, shows significant thermo-enhancement effects as observed comparing the EC50 of CX-4945 with or without thermal shock (Fig. 6).
Discussion HSP27 plays a major role in proteostasis; in particular, it prevents protein aggregation under different stress conditions, such as temperature, oxidative stress etc., and promotes proteins refolding or their proteolytic degradation. Notably, HSP27 is an anti-apoptotic protein [31], and accordingly to its pro-survival function, it is tightly implicated in cancer where its expression is correlated with cell proliferation and increased survival [7,8]. Indeed, overexpression of HSP27 has been detected in several human cancers (such as that affecting breast, colorectal, liver, tongue, ovarian, and pancreas) and it is associated with increased tumorigenicity, metastatic potential and resistance to chemotherapy, resulting in poor prognosis [32]. Several studies agree that HSP27-targeting represents an interesting strategy to treat cancer. For example, suppression of HSP27 increases colon cancer sensitivity to 5-fluorouracil (5-FU) in a mouse model [33]. A group of patients with esophageal adenocarcinoma presenting low level of HSP27, p-HSP27, and HSP90 expression, demonstrated a better response to cisplatin/oxaliplatin- or 5-FU-based chemotherapy [34]. In addition, downregulation of HSP27 enhanced doxorubicin sensitivity and induced apoptosis in human colon carcinoma cells [35]. Unfortunately, none of the HSP inhibitors has been approved by the FDA for the treatment of patients with cancer, yet [32]. Nevertheless, a second generation phosphorothiodate antisense oligonucleotide targeting HSP27 has been developed (Apatorsen, OGX-427) and it is now in clinical trials [36]. Our results show that genetic or pharmacological inhibition of protein kinase CK2, a kinase overexpressed in many different type of cancers [16,17], leads to the downregulation of HSP27 expression in all the four different cell lines tested so far. Therefore, treatment with CK2 inhibitors can prove a valuable strategy in all the conditions where a reduction of HSP27 is desirable. Pertinent to this may be the observation that quercetin, a flavonoid able to inhibit protein kinase CK2 with IC50 in the sub-μM range [37], also downregulates HSP27 expression [38].