Mouse models for cancer research - current state and the perspective.

Cancer is one of the leading causes of death worldwide. We still do not understand all the details of carcinogenesis, and effective treatment is lacking for many oncological diseases. Animal models provide an irreplaceable tool to observe the growth and spreading of neoplastic cells in an environment of living organisms, to test the efficacy of cancer treatment, side effects, and toxicity, and to study the tumor microenvironment. Mice are the most often used model organisms because of their easy handling, short reproductive period, multiple strains, and complete DNA sequencing. An ideal model should accurately recapitulate each step of tumor development. Recent techniques have established models that enable the study of different aspects of cancer, but choosing a particular model depends on the application of output data. This article aimed to review induced, transplantable, and engineered mice and highlight their significance for recent and future cancer research.

Overexpression of TNFα induces senescence, autophagy and mitochondrial dysfunctions in melanoma cells.

BACKGROUND: Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with both anti-tumorigenic and pro-tumorigenic activity, affecting tumor cell biology, the balance between cell survival and death. The final effect of TNFα is dependent on the type of malignant cells, with the potential to arrest cancer progression. METHODS: In order to explain the diverse cellular response to TNFα, we engineered melanoma and colorectal carcinoma cell lines stably overexpressing this cytokine. RESULTS: Under the TNFα overexpression, significant upregulation of two genes was observed: proinflammatory cytokine IL6 gene in melanoma cells A375 and gene for pro-apoptotic ligand TRAIL in colorectal carcinoma cells HT29, both mediated by TNFα/TNFR1 signaling. Malignant melanoma line A375 displayed also increased autophagy on day 3, followed by premature senescence on day 6. Both processes seem to be interconnected, following earlier apoptosis induction and deregulation of mitochondrial functions. We documented altered mitochondrial status, lowered ATP production, lowered mitochondrial mass, and changes in mitochondrial morphology (shortened and condensed mitochondria) both in melanoma and colorectal carcinoma cells. Overexpression of TNFα was not linked with significant affection of the subpopulation of cancer stem-like cells in vitro. However, we could demonstrate a decrease in aldehyde dehydrogenase (ALDH) activity up to 50%, which is associated with to the stemness phenotype. CONCLUSIONS: Our in vitro study of direct TNFα influence demonstrates two distinct outcomes in tumor cells of different origin, in non-epithelial malignant melanoma cells of neural crest origin, and in colorectal carcinoma cells derived from the epithelium.

Inhibition of Glycolysis Suppresses Cell Proliferation and Tumor Progression In Vivo: Perspectives for Chronotherapy.

A high rate of glycolysis is considered a hallmark of tumor progression and is caused by overexpression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Therefore, we analyzed the possibility of inhibiting tumor and endothelial cell metabolism through the inhibition of PFKFB3 by a small molecule, (E)-1-(pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one (PFK15), as a promising therapy. The effects of PFK15 on cell proliferation and apoptosis were analyzed on human umbilical vein endothelial cells (HUVEC) and the human colorectal adenocarcinoma cell line DLD1 through cytotoxicity and proliferation assays, flow cytometry, and western blotting. The results showed that PFK15 inhibited the proliferation of both cell types and induced apoptosis with decreasing the Bcl-2/Bax ratio. On the basis of the results obtained from in vitro experiments, we performed a study on immunodeficient mice implanted with DLD1 cells. We found a reduced tumor mass after morning PFK15 treatment but not after evening treatment, suggesting circadian control of underlying processes. The reduction in tumor size was related to decreased expression of Ki-67, a marker of cell proliferation. We conclude that inhibition of glycolysis can represent a promising therapeutic strategy for cancer treatment and its efficiency is circadian dependent.

Targeting of Deregulated Wnt/ß-Catenin Signaling by PRI-724 and LGK974 Inhibitors in Germ Cell Tumor Cell Lines.

The majority of patients with testicular germ cell tumors (GCTs) can be cured with cisplatin-based chemotherapy. However, for a subset of patients present with cisplatin-refractory disease, which confers a poor prognosis, the treatment options are limited. Novel therapies are therefore urgently needed to improve outcomes in this challenging patient population. It has previously been shown that Wnt/ß-catenin signaling is active in GCTs suggesting that its inhibitors LGK974 and PRI-724 may show promise in the management of cisplatin-refractory GCTs. We herein investigated whether LGK-974 and PRI-724 provide a treatment effect in cisplatin-resistant GCT cell lines. Taking a genoproteomic approach and utilizing xenograft models we found the increased level of ß-catenin in 2 of 4 cisplatin-resistant (CisR) cell lines (TCam-2 CisR and NCCIT CisR) and the decreased level of ß-catenin and cyclin D1 in cisplatin-resistant NTERA-2 CisR cell line. While the effect of treatment with LGK974 was limited or none, the NTERA-2 CisR exhibited the increased sensitivity to PRI-724 in comparison with parental cell line. Furthermore, the pro-apoptotic effect of PRI-724 was documented in all cell lines. Our data strongly suggests that a Wnt/ß-catenin signaling is altered in cisplatin-resistant GCT cell lines and the inhibition with PRI-724 is effective in NTERA-2 CisR cells. Further evaluation of Wnt/ß-catenin pathway inhibition in GCTs is therefore warranted.

Recent advances in understanding tumor stroma-mediated chemoresistance in breast cancer.

Although solid tumors comprise malignant cells, they also contain many different non-malignant cell types in their micro-environment. The cellular components of the tumor stroma consist of immune and endothelial cells combined with a heterogeneous population of stromal cells which include cancer-associated fibroblasts. The bi-directional interactions between tumor and stromal cells therefore substantially affect tumor cell biology.Herein, we discuss current available information on these interactions in breast cancer chemo-resistance. It is acknowledged that stromal cells extrinsically alter tumor cell drug responses with profound consequences for therapy efficiency, and it is therefore essential to understand the molecular mechanisms which contribute to these substantial alterations because they provide potential targets for improved cancer therapy. Although breast cancer patient survival has improved over the last decades, chemo-resistance still remains a significant obstacle to successful treatment.Appreciating the important experimental evidence of mesenchymal stromal cells and cancer-associated fibroblast involvement in breast cancer clinical practice can therefore have important therapeutic implications.

Sodium/calcium exchanger is involved in apoptosis induced by H2S in tumor cells through decreased levels of intracellular pH.

We explored possibility that sodium/calcium exchanger 1 (NCX1) is involved in pH modulation and apoptosis induction in GYY4137 treated cells. We have shown that although 10 days treatment with GYY4137 did not significantly decreased volume of tumors induced by colorectal cancer DLD1 cells in nude mice, it already induced apoptosis in these tumors. Treatment of DLD1 and ovarian cancer A2780 cells with GYY4137 resulted in intracellular acidification in a concentration-dependent manner. We observed increased mRNA and protein expression of both, NCX1 and sodium/hydrogen exchanger 1 (NHE1) in DLD1-induced tumors from GYY4137-treated mice. NCX1 was coupled with NHE1 in A2780 and DLD1 cells and this complex partially disintegrated after GYY4137 treatment. We proposed that intracellular acidification is due to uncoupling of NCX1/NHE1 complex rather than blocking of the reverse mode of NCX1, probably due to internalization of NHE1. Results might contribute to understanding molecular mechanism of H2S-induced apoptosis in tumor cells.

ALDH1A3 upregulation and spontaneous metastasis formation is associated with acquired chemoresistance in colorectal cancer cells.

BACKGROUND: Efficiency of colorectal carcinoma treatment by chemotherapy is diminished as the resistance develops over time in patients. The same holds true for 5-fluorouracil, the drug used in first line chemotherapy of colorectal carcinoma. METHODS: Chemoresistant derivative of HT-29 cells was prepared by long-term culturing in increasing concentration of 5-fluorouracil. Cells were characterized by viability assays, flow cytometry, gene expression arrays and kinetic imaging. Immunomagnetic separation was used for isolation of subpopulations positive for cancer stem cells-related surface markers. Aldehyde dehydrogenase expression was attenuated by siRNA. In vivo studies were performed on SCID/bg mice. RESULTS: The prepared chemoresistant cell line labeled as HT-29/EGFP/FUR is assigned with different morphology, decreased proliferation rate and 135-fold increased IC50 value for 5-fluorouracil in comparison to parental counterparts HT-29/EGFP. The capability of chemoresistant cells to form tumor xenografts, when injected subcutaneously into SCID/bg mice, was strongly compromised, however, they formed distant metastases in mouse lungs spontaneously. Derived cells preserved their resistance in vitro and in vivo even without the 5-fluorouracil selection pressure. More importantly, they were resistant to cisplatin, oxaliplatin and cyclophosphamide exhibiting high cross-resistance along with alterations in expression of cancer-stem cell markers such as CD133, CD166, CD24, CD26, CXCR4, CD271 and CD274. We also detected increased aldehyde dehydrogenase (ALDH) activity associated with overexpression of specific ALDH isoform 1A3. Its inhibition by siRNA approach partially sensitized cells to various agents, thus linking for the first time the ALDH1A3 and chemoresistance in colorectal cancer. CONCLUSION: Our study demonstrated that acquired chemoresistance goes along with metastatic and migratory phenotype and can be accompanied with increased activity of aldehyde dehydrogenase. We describe here the valuable model to study molecular link between resistance to chemotherapy and metastatic dissemination.

Tyrosine kinase inhibitor SU11274 increased tumorigenicity and enriched for melanoma-initiating cells by bioenergetic modulation.

BACKGROUND: Small molecule inhibitor of tyrosine kinase activity, compound SU11274, was reported to have antitumorigenic and antimetastatic effect in melanoma. In this study, we evaluated, whether similar effect could be achieved also in other melanoma cells including highly tumorigenic and hypermetastatic variant. METHODS: The effect of SU11274 was evaluated in adherent and non-adherent melanosphere cultures of human melanoma cells M14, M4Beu, A375 and EGFP-A375/Rel3. Tumorigenicity of SU11274-treated cells was tested by limiting dilution assay in xenograft model in vivo. RESULTS: Here we show that SU11274 enriched for melanoma-initiating cells in vivo. SU11274 substantially decreased number of cells in adherent and spheroid cultures, but increased their tumorigenic potential as determined by higher frequency of tumor-initiating cells in vivo. SU11274 treatment was not associated with any significant alteration in the expression of stem cell markers, but the inhibitor stimulated higher level of pluripotent markers. SU11274-treated melanoma cells exhibited higher ATP content and lactate release indicative of increased glycolysis. Our data suggest that the SU11274 altered bioenergetic state of the cells. Indeed, pharmacological intervention with a glycolytic inhibitor dichloroacetate significantly reduced SU11274-promoted increase in melanoma-initiating cells and decreased their tumorigenicity. CONCLUSIONS: Our data suggest critical role of glycolysis regulation in melanoma-initiating cells. Moreover, these data unravel substantial plasticity of melanoma cells and their adoptive mechanisms, which result in ambivalent response to therapeutic targeting.

Cisplatin-induced mesenchymal stromal cells-mediated mechanism contributing to decreased antitumor effect in breast cancer cells.

BACKGROUND: Cells of the tumor microenvironment are recognized as important determinants of the tumor biology. The adjacent non-malignant cells can regulate drug responses of the cancer cells by secreted paracrine factors and direct interactions with tumor cells. RESULTS: Human mesenchymal stromal cells (MSC) actively contribute to tumor microenvironment. Here we focused on their response to chemotherapy as during the treatment these cells become affected. We have shown that the secretory phenotype and behavior of mesenchymal stromal cells influenced by cisplatin differs from the naïve MSC. MSC were more resistant to the concentrations of cisplatin, which was cytotoxic for tumor cells. They did not undergo apoptosis, but a part of MSC population underwent senescence. However, MSC pretreatment with cisplatin led to changes in phosphorylation profiles of many kinases and also increased secretion of IL-6 and IL-8 cytokines. These changes in cytokine and phosphorylation profile of MSC led to increased chemoresistance and stemness of breast cancer cells. CONCLUSION: Taken together here we suggest that the exposure of the chemoresistant cells in the tumor microenvironment leads to substantial alterations and might lead to promotion of acquired microenvironment-mediated chemoresistance and stemness.

Genetically engineered mesenchymal stromal cells producing TNFα have tumour suppressing effect on human melanoma xenograft.

BACKGROUND: Mesenchymal stromal cells (MSC) are a promising tool for targeted cancer therapy due to their tumour-homing ability. Intrinsic resistance enables the MSC to longer tolerate therapeutic factors, such as prodrug converting enzymes, cytokines and pro-apoptotic proteins. Tumour necrosis factor alpha (TNFα) is known to be cytotoxic to a variety of cancer cells and exert a tumour-destructive capacity. METHODS: MSC were retrovirally transduced to stable express an exogenous gene encoding the desired therapeutic agent hTNFα. The effect of a TNFα-producing adipose tissue-derived MSC (AT-MSC/hTNFα) was tested on the tumour cell lines of different origins: melanoma (A375), breast carcinoma (SKBR3, MDA-MB-231), colon carcinoma (HT29), ovarian carcinoma (SKOV3) and glioblastoma (U87-MG) cells. The tumour suppressing effect of AT-MSC/hTNFα on A375 melanoma xenografts was monitored in an immunodeficient mouse model in vivo. RESULTS: Engineered AT-MSC are able to constitutively secrete human TNFα protein, induce apoptosis of tumour cell lines via caspase 3/7 activation and inhibit the tumour cell proliferation in vitro. Melanoma A375 and breast carcinoma SKBR3 cells were the most sensitive, and their proliferation in vitro was reduced by conditioned media produced by AT-MSC/hTNFα to 60% and 40%, respectively. The previously reported tumour supportive effect of AT-MSC on subcutaneous A375 melanoma xenograft growth was neutralised and suppressed by engineered AT-MSC stably producing hTNFα. When AT-MSC/hTNFα were coinjected with A375 melanoma cells, the tumour mass inhibition was up to 97.5%. CONCLUSIONS: The results of the present study demonstrate that tumour cells respond to hTNFα-based treatment mediated by genetically engineered AT-MSC/hTNFα both in vitro and in vivo.

Therapeutic dendritic cell-based cancer vaccines: the state of the art.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells, capable of initiating proper adaptive immune responses. Although tumor-infiltrating DCs are able to recognize cancer cells and uptake tumor antigens, they often have impaired functions because of the immunosuppressive tumor milieu. Therefore, DCs are targeted by therapeutic means either in vivo or ex vivo to facilitate tumor antigen presentation to T cells and induce or promote efficient antitumor immune responses in cancer patients. This immunotherapeutical approach is defined as specific active tumor immunotherapy or therapeutic cancer vaccination. In this review we briefly discuss general aspects of DC biology, followed by a thorough description of the current knowledge and optimization trends of DC vaccine production ex vivo, including various approaches for the induction of proper DC maturation and efficient loading with tumor antigens. We also discuss critical clinical aspects of DC vaccine application in cancer patients, including protocols of administration (routes and regimens), individualization of tumor immunotherapy, prediction and proper evaluation of immune and clinical responses to immunotherapy, and the critical role of combining tumor immunotherapy with other cancer treatment strategies to achieve maximal therapeutic effects.

Altered features and increased chemosensitivity of human breast cancer cells mediated by adipose tissue-derived mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) represent heterogeneous cell population suitable for cell therapies in regenerative medicine. MSCs can also substantially affect tumor biology due to their ability to be recruited to the tumor stroma and interact with malignant cells via direct contacts and paracrine signaling. The aim of our study was to characterize molecular changes dictated by adipose tissue-derived mesenchymal stromal cells (AT-MSCs) and the effects on drug responses in human breast cancer cells SKBR3. METHODS: The tumor cells were either directly cocultured with AT-MSCs or exposed to MSCs-conditioned medium (MSC-CM). Changes in cell biology were evaluated by kinetic live cell imaging, fluorescent microscopy, scratch wound assay, expression analysis, cytokine secretion profiling, ATP-based viability and apoptosis assays. The efficiency of cytotoxic treatment in the presence of AT-MSCs or MSCs-CM was analyzed. RESULTS: The AT-MSCs altered tumor cell morphology, induced epithelial-to-mesenchymal transition, increased mammosphere formation, cell confluence and migration of SKBR3. These features were attributed to molecular changes induced by MSCs-secreted cytokines and chemokines in breast cancer cells. AT-MSCs significantly inhibited the proliferation of SKBR3 cells in direct cocultures which was shown to be dependent on the SDF-1α/CXCR4 signaling axis. MSC-CM-exposed SKBR3 or SKBR3 in direct coculture with AT-MSCs exhibited increased chemosensitivity and induction of apoptosis in response to doxorubicin and 5-fluorouracil. CONCLUSIONS: Our work further highlights the multi-level nature of tumor-stromal cell interplay and demonstrates the capability of AT-MSCs and MSC-secreted factors to alter the anti-tumor drug responses.

Cystathionine ß-synthase affects organization of cytoskeleton and modulates carcinogenesis in colorectal carcinoma cells.

Background: Cystathionine ß-synthase (CBS), one of three enzymes that endogenously produce hydrogen sulfide, is extensively studied for its relevance in the cells of various tumors. In our previous work, we observed that the immunofluorescence pattern of CBS is very similar to that of tubulin and actin. Therefore, we focused on the potential interaction of CBS with cytoskeletal proteins ß-actin and ß-tubulin and the functional relevance of the potential interaction of these proteins in colorectal carcinoma cell lines. Methods: To study the potential interaction of CBS with cytoskeletal proteins and its functional consequences, a CBS-knockout DLD1 (DLDx) cell line was established by using the CRISPR/Cas9 gene editing method. The interaction of the selected cytoskeletal protein with CBS was studied by immunoprecipitation, Western blot analysis, immunofluorescence, and proximity ligation assay. The functional consequences were studied by proliferation and migration assays and by generation of xenografts in SCID/bg mice. Results: We have found that CBS, an enzyme that endogenously produces H2S, binds to cytoskeletal ß-tubulin and, to a lesser extent, also to ß-actin in colorectal carcinoma-derived cells. When CBS was knocked out by the CRISPR/Cas9 technique (DLDx), we observed a de-arranged cytoskeleton compared to the unmodified DLD1 cell line. Treatment of these cells with a slow sulfide donor GYY4137 resulted in normal organization of the cytoskeleton, thus pointing to the role of CBS in microtubule dynamics. To evaluate the physiological importance of this observation, both DLD1 and DLDx cells were injected into SCID/bg mice, and the size and mass of the developed xenografts were evaluated. Significantly larger tumors developed from DLDx compared to the DLD1 cells, which correlated with the increased proliferation of these cells. Conclusions: Taken together, in colorectal cancer DLD1 cells, CBS binds to the cytoskeleton, modulates microtubule dynamics, and thus affects the proliferation and migration in the colorectal carcinoma stable cell line.

Intrinsic properties of tumour cells have a key impact on the bystander effect mediated by genetically engineered mesenchymal stromal cells.

BACKGROUND: Engineered mesenchymal stromal cells (MSC) have been used in many preclinical studies of gene directed enzyme/prodrug therapy. We aimed to compare the efficacy of two most frequently used systems, as well as evaluate the extent of a bystander effect mediated by therapeutic MSC towards cell lines derived from different tumours. METHODS: Two approaches were compared: (i) herpes simplex virus thymidine kinase (TK)/ganciclovir (GCV) and (ii) yeast cytosine deaminase fused with uracil phosphoribosyltransferase (CD::UPRT)/5-fluorocytosine (5-FC). The cytotoxic effect mediated by therapeutic MSC was evaluated in direct co-culture by a fluorimetric assay. The expression profile of tumour cells was analyzed by a quantitative polymerase chain reaction, and the ability of gap-junctional intercellular communication (GJIC) was evaluated by a dye transfer assay. RESULTS: Both systems were effective only on glioblastoma cells (8-MG-BA). The CD::UPRT-MSC/5-FC system showed efficiency on melanoma A375 cells. We decreased the sensitivity of 8-MG-BA cells and A375 cells to the CD::UPRT-MSC/5-FC system by pharmacological inhibition of thymidylate synthase, and we achieved a similar result in A375 cells by inhibition of thymidine phosphorylase. Although we demonstrated functional GJIC in A375 cells, TK-MSC were ineffective in mediating the bystander effect similarly to HeLa cells, which were also relatively resistant to CD::UPRT-MSC/5-FC treatment. TK-MSC/GCV treatment had a strong cytotoxic effect on MDA-MB-231 cells (breast carcinoma), whereas CD::UPRT-MSC/5-FC treatment failed as a result of overexpression of the gene for ABCC11. Transfection of the MDA-MB-231 cell line with small interference RNA specific to ABCC11 led to a significantly increased sensitivity to the CD::UPRT-MSC/5-FC approach. CONCLUSIONS: GJIC, expression of enzymes involved in drug metabolism and ABC transporters correlate with the response of tumour cells to treatment by MSC-expressing prodrug-converting genes.

The evaluation of the efficacy and potential genotoxic hazard of combined SAHA and 5-FU treatment in the chemoresistant colorectal cancer cell lines.

5-Fluorouracil (5-FU) is an essential chemotherapeutic drug for colorectal cancer (CRC) treatment. However, the frequent development of drug resistance has dramatically affected its clinical use. Therefore, novel treatment strategies are critical to improving patient outcomes. Herein, we investigated the ability of the epigenetic drug SAHA to increase the sensitivity of chemoresistant CRC cells to 5-FU. In addition, we evaluated the potential genotoxic risk of SAHA+5-FU combination treatment. As a model system, we used three CRC cell lines, HT-29, SW480, and HT-29/EGFP/FUR, differing in their resistance to 5-FU. CRC cell lines were exposed to sub-toxic SAHA concentrations for 24 h, followed by a 48 h treatment with 5-FU. The cytotoxicity of SAHA, 5-FU, and SAHA+5-FU was measured by the MTT test, the genotoxicity by the comet assay, and the micronucleus test. The apoptotic/necrotic activity was assessed using morphological criteria. We found a synergic decrease in the viability of HT-29 and SW480 cells, but not the most resistant HT-29/EGFP/FUR cells after combined SAHA+5-FU exposure compared to 5-FU. Remarkably, SAHA most efficiently induced apoptosis in HT-29/EGFP/FUR cells compared to HT-29 and SW480 cells. Combined SAHA+5-FU treatment resulted in a synergistic increase in apoptotic/necrotic cells in HT-29 cell line, while rather additive/sub-additive effect was determined in the SW480 and HT-29/EGFP/FUR cells. At the same time, however, a synergistic rise in micronuclei was found in CRC cell lines (at least at some concentrations). We have shown that SAHA can sensitize CRC cells to 5-FU; therefore, epigenetic and convential drug combinations could be beneficial for the patients. However, the increase in micronucleus formation after combined SAHA+5-FU treatment indicates a potential health hazard. The clastogenic activity could contribute to cancer heterogeneity, favoring progeny of such aberrant cells to clonal expansion. Therefore, developing new specific epigenetic drugs or nanocarriers for targeted drug delivery might reduce the potential genotoxic risk.

In vitro antiproliferative and cytotoxic activities of novel triphenyltin isoselenocyanate in human breast carcinoma cell lines MCF 7 and MDA-MB-231.

Intensive investigation for novel antiproliferative and cytotoxic effective chemical compounds is currently concentrated on structurally modified agents of natural or synthetic source. The selenium derivative of triorganotin compound, triphenyltin isoselenocyanate (TPT-NCSe) caused higher cytotoxicity in hormone sensitive MCF 7 (IC 50-250 nM) in comparison with triple-negative MDA-MB-231 breast carcinoma cell line (IC 50-450 nM) as determined by MTT assay. Measurement of DNA damage showed presence of crosslinks in both cell lines treated by increasing TPT-NCSe concentrations. This compound decreased mitochondrial membrane potential shown by JC-1 staining in a concentration-dependent manner in both cell lines. Activation of caspases-3/7 was observed in MDA-MB-231 cells and was significant only by concentrations causing significant level of crosslinks. On the other hand, migration assay revealed inhibitory effect of viability keeping 100 nM concentration of TPT-NCSe on migration of MDA-MB-231 cells. Our data has shown that this selenium containing triorganotin molecule exerts DNA damage-linked antineoplastic activity in breast carcinoma cell lines studied.

Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC's effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs' changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.

Decitabine potentiates efficacy of doxorubicin in a preclinical trastuzumab-resistant HER2-positive breast cancer models.

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.

Adipose tissue-derived mesenchymal stem cells expressing prodrug-converting enzyme inhibit human prostate tumor growth.

The ability of human adipose tissue-derived mesenchymal stem cells (AT-MSCs), engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT), to convert the relatively nontoxic 5-fluorocytosine (5-FC) into the highly toxic antitumor 5-fluorouracil (5-FU) together with their ability to track and engraft into tumors and micrometastases makes these cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we tested the feasibility and efficacy of these therapeutic cells to function as cellular vehicles of prodrug-activating enzymes in prostate cancer (PC) therapy. In in vitro migration experiments we have shown that therapeutic AT-MSCs migrated to all the prostate cell lines tested. In a pilot preclinical study, we observed that coinjections of human bone metastatic PC cells along with the transduced AT-MSCs into nude mice treated with 5-FC induced a complete tumor regression in a dose dependent manner or did not even allow the establishment of the tumor. More importantly, we also demonstrated that the therapeutic cells were effective in significantly inhibiting PC tumor growth after intravenous administration that is a key requisite for any clinical application of gene-directed enzyme prodrug therapies.