The Hsp70 chaperone binds and inhibits proteins implicated in apoptotic signaling including Caspase-3. Induction of apoptosis is an important mechanism of anti-cancer drugs, therefore Hsp70 can act as a protective system in tumor cells against therapeutic agents. In this study we present an assessment of candidate compounds that are able to dissociate the complex of Hsp70 with Caspase-3, and thus sensitize cells to drug-induced apoptosis. Using the PASS program for prediction of biological activity we selected a derivative of benzodioxol (BT44) that is known to affect molecular chaperones and caspases. Drug affinity responsive target stability and microscale thermophoresis assays indicated that BT44 bound to Hsp70 and reduced the chaperone activity. When etoposide was administered, heat shock accompanied with an accumulation of Hsp70 led to an inhibition of etoposide-induced apoptosis. The number of apoptotic cells increased following BT44 administration, and forced Caspase-3 processing. Competitive protein–protein interaction and immunoprecipitation assays showed that BT44 caused dissociation of the Hsp70–Caspase-3 complex, thus augmenting the anti-tumor activity of etoposide and highlighting the potential role of molecular separators in cancer therapy.

Cancer cells are known to contain high levels of the heat shock protein 70 kDa (Hsp70), which mediates increased cell proliferation, escape from programmed cell death, enhanced invasion, and metastasis. A part of Hsp70 molecules may release from cancer cells and affect the behavior of adjacent stromal cells. To explore the effects of Hsp70 on the status of monocytes/macrophages in the tumor locale, we incubated human carcinoma cells of three distinct lines with normal and reduced content of Hsp70 with THP1 monocytes. Using two methods, we showed that the cells with knock-down of Hsp70 released a lower amount of protein in the extracellular medium. Three cycles of the co-cultivation of cancer and monocytic cells led to the secretion of several cytokines typical of the tumor microenvironment (TME) and to pro-cancer activation of the monocytes/macrophages as established by elevation of F4/80 and arginase-1 markers. Unexpectedly, the efficacy of epithelial–mesenchymal transition and resistance of carcinoma cells to anticancer drugs after incubation with monocytic cells were more pronounced in cells with lower Hsp70, e.g., releasing less Hsp70 into the extracellular milieu. These data suggest that Hsp70 released from tumor cells into the TME is able, together with the development of an anti-cancer immune response, to limit the conversion of a considerable part of monocytic cells to the pro-tumor phenotype.

Alpha-actinin 4 (ACTN4) is an actin-binding protein of the spectrin superfamily. ACTN4 is found both in the cytoplasm and nucleus of eukaryotic cells. The main function of cytoplasmic ACTN4 is stabilization of actin filaments and their binding to focal contacts. Nuclear ACTN4 takes part in the regulation of gene expression following by activation of certain transcription factors, but the mechanisms of regulation are not completely understood. Our previous studies have demonstrated the interaction of ACTN4 with the RelA/p65 subunit of NF-kappaB factor and the effect on its transcriptional activity in A431 and HEK293T cells. In the present work, we investigated changes in the composition of nuclear ACTN4-interacting proteins in non-small cell lung cancer cells H1299 upon stable RELA overexpression. We showed that ACTN4 was present in the nuclei of H1299 cells, regardless of the RELA expression level. The presence of ectopic RelA/p65 in H1299 cells increased the number of proteins interacting with nuclear ACTN4. Stable expression of RELA in these cells suppressed cell proliferation, which was further affected by simultaneous ACTN4 overexpression. We detected no significant effect on cell cycle but the apoptosis rate was increased in cells with a double RELA/ACTN4 overexpression. Interestingly, when expressed individually ACTN4 promoted proliferation of lung cancer cells. Furthermore, the bioinformatics analysis of gene expression in lung cancer patients suggested that overexpression of ACTN4 correlated with poor survival prognosis. We hypothesize that the effect of RELA on proliferation and apoptosis of H1299 cells can be mediated via affecting the interactome of ACTN4.

Hyperglycemia may contribute to the progression of carcinomas by triggering epithelial-to-mesenchymal transition (EMT). Some proteostasis systems are involved in metastasis; in this paper, we sought to explore the mechanism of Hsp70 chaperone in EMT. We showed that knockdown of Hsp70 reduced cell migration capacity concomitantly with levels of mRNA of the Slug, Snail, and Twist markers of EMT, in colon cancer cells incubated in high glucose medium. Conversely, treatment of cells with Hsp70 inducer U-133 were found to elevate cell motility, along with the other EMT markers. To prove that inhibiting Hsp70 may reduce EMT efficiency, we treated cells with a CL-43 inhibitor of the HSF1 transcription factor, which lowered Hsp70 and HSF1 content in the control and induced EMT in carcinoma cells. Importantly, CL-43 reduced migration capacity, EMT-linked transcription factors, and increased content of epithelial marker E-cadherin in colon cancer cells of three lines, including one derived from a clinical sample. To prove that Hsp70 chaperone should be targeted when inhibiting the EMT pathway, we treated cancer cells with 2-phenylethynesulfonamide (PES) and demonstrated that the compound inhibited substrate-binding capacity of Hsp70. Furthermore, PES suppressed EMT features, cell motility, and expression of specific transcription factors. In conclusion, the Hsp70 chaperone machine efficiently protects mechanisms of the EMT, and the safe inhibitors of the chaperone are needed to hamper metastasis at its initial stage.

Despite significant progress in the diagnosis and treatment of colorectal cancer, drug resistance continues to be a major limitation of therapy. In this regard, studies aimed at creating combination therapy are gaining popularity. One of the most promising adjuvants are inhibitors of the proteostasis system, chaperone machinery, and autophagy. The main HSP regulator, HSF1, is overactivated in cancer cells and autophagy sustains the survival of malignant cells. In this work, we focused on the selection of combination therapy for the treatment of rectal cancer cells obtained from patients after tumor biopsy without prior treatment. We characterized the migration, proliferation, and chaperone status in the resulting lines and also found them to be resistant to a number of drugs widely used in the clinic. However, these cells were sensitive to the autophagy inhibitor, chloroquine. For combination therapy, we used an HSF1 activity inhibitor discovered earlier in our laboratory, the cardenolide CL-43, which has already been proven as an auxiliary component of combined therapy in established cell lines. CL-43 effectively suppressed HSF1 activity and Hsp70 expression in all investigated cells. We tested the autophagy inhibitor, chloroquine, in combination with CL-43. Our results indicate that the use of an inhibitor of HSF1 activity in combination with an autophagy inhibitor results in effective cancer cell death, therefore, this therapeutic approach may be a promising treatment regimen for certain patients.

Heat shock factor 1 (HSF1) plays a central role in orchestrating the heat shock response (HSR), leading to the activation of multiple heat shock proteins (HSPs) genes and approximately thousands of other genes involved in various cellular functions. In cancer cells, HSPs play a particular role in coping with the accumulation of damaged proteins resulting from dysregulated translation and post-translational processes. This proteotoxic stress is a hallmark of cancer cells and causes constitutive activation of HSR. Beyond its role in the HSR, HSF1 regulates diverse processes critical for tumor cells, including proliferation, cell death, and drug resistance. Emerging evidence also highlights HSF1’s involvement in remodeling the tumor immune microenvironment as well as in the maintenance of cancer stem cells. Consequently, HSF1 has emerged as an attractive therapeutic target, prompting the development of specific HSF1 inhibitors that have progressed to clinical trials. Importantly, HSF1 possesses a broad interactome, forming protein–protein interactions (PPIs) with components of signaling pathways, transcription factors, and chromatin regulators. Many of these interactors modulate HSF1’s activity and HSF1-dependent gene expression and are well-recognized targets for cancer therapy. This review summarizes the current knowledge on HSF1 interactions with molecular chaperones, protein kinases, and other regulatory proteins. Understanding the key HSF1 interactions promoting cancer progression, along with identifying factors that disrupt these protein complexes, may offer valuable insights for developing innovative therapeutic strategies against cancer.