Pre-clinical Evaluation of Karanjin Against DMBA-Induced Breast Cancer in Female Sprague-Dawley Rats Through Modulation of SMAR1 and CDP/CUx genes.

PURPOSE: To investigate the chemoprotective potential of karanjin against 7,12-dimethylbenz(α)anthracene (DMBA)-induced breast cancer. METHODOLOGY: Thirty-six female rats were utilized for the study. Breast cancer was induced through a subcutaneous injection of 35 mg/kg DMBA. The animals were allocated to six groups. Three groups were allocated for karanjin (50 mg/kg, 100 mg/kg, and 200 mg/kg), and received daily treatment for 20 weeks (including 2 weeks as pre-treatment). Doxorubicin (4 mg/kg) was administered to the standard control group twice a week for 20 weeks. The disease control (DC) and normal control (NC) groups received daily treatment with saline. After the treatment, oxidative stress parameters, biochemical parameters, and inflammatory parameters were estimated. CCAAT-displacement protein/cut homeobox (CUP/Cux) and scaffold/matrix attachment region binding protein 1 (SMAR1) expression levels were measured through gene expression analysis. Immunohistochemical (IHC) analysis was performed to estimate the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). RESULTS: Tumor growth reduced significantly (P-value < 0.01) in karanjin-treated animals compared to the DC group. Karanjin significantly (P-value < 0.01) regulated the levels of oxidative stress parameters, biochemical parameters, and inflammatory parameters compared to the DC group. Karanjin treatment significantly (P-value < 0.001) regulated the expression levels of SMAR1 and CDP/Cux. A notable reduction in the IHC scores was observed for ER, PR, and HER2 expression in karanjin groups. CONCLUSION: Karanjin demonstrated chemoprotective activity against DMBA-induced breast cancer in animals potentially through modulation of SMAR1 and CDP/Cux gene expression and reduction of ER, PR and HER2 expression levels.

A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors.

Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for a sufficient time. iPSCs are typically generated using viruses or virus-based methods, which have drawbacks such as vector persistence, risk of insertional mutagenesis, and oncogenesis. The application of less harmful nonviral vectors is limited as conventional plasmids cannot deliver the levels or duration of the factors necessary from a single transfection. Hence, plasmids that are most often used for reprogramming employ the potentially oncogenic Epstein-Barr nuclear antigen 1 (EBNA-1) system to ensure adequate levels and persistence of expression. In this study, we explored the use of nonviral SMAR DNA vectors to reprogram human fibroblasts into iPSCs. We show for the first time that iPSCs can be generated using nonviral plasmids without the use of EBNA-1 and that these DNA vectors can provide sufficient expression to induce pluripotency. We describe an optimised reprogramming protocol using these vectors that can produce high-quality iPSCs with comparable pluripotency and cellular function to those generated with viruses or EBNA-1 vectors.

A novel perspective on eugenol as a natural anti-quorum sensing molecule against Serratia sp.

Serratia marcescens is commonly noted to be an opportunistic pathogen and is often associated with nosocomial infections. In addition to its high antibiotic resistance, it exhibits a wide range of virulence factors that confer pathogenicity. Targeting quorum sensing (QS) presents a potential therapeutic strategy for treating bacterial infections caused by S. marcescens, as it regulates the expression of various virulence factors. Inhibiting QS can effectively neutralize S. marcescens' bacterial virulence without exerting stress on bacterial growth, facilitating bacterial eradication by the immune system. In this study, the antibacterial and anti-virulence properties of eugenol against Serratia sp. were investigated. Eugenol exhibited inhibitory effects on the growth of Serratia, with a minimal inhibitory concentration (MIC) value of 16.15 mM. At sub-inhibitory concentrations, eugenol also demonstrated antiadhesive and eradication activities by inhibiting biofilm formation. Furthermore, it reduced prodigiosin production and completely inhibited protease production. Additionally, eugenol effectively decreased swimming and swarming motilities in Serratia sp. This study demonstrated through molecular modeling, docking and molecular dynamic that eugenol inhibited biofilm formation and virulence factor production in Serratia by binding to the SmaR receptor and blocking the formation of the HSL-SmaR complex. The binding of eugenol to SmaR modulates biofilm formation and virulence factor production by Serratia sp. These findings highlight the potential of eugenol as a promising agent to combat S. marcescens infections by targeting its virulence factors through quorum sensing inhibition.

Secreted cyclophilin A is non-genotoxic but acts as a tumor promoter.

Chronic inflammation is associated with malignant transformation and creates the microenvironment for tumor progression. Cyclophilin A (CypA) is one of the major pro-inflammatory mediators that accumulates and persists in the site of inflammation in high doses over time. According to multiomics analyses of transformed cells, CypA is widely recognized as a pro-oncogenic factor. Vast experimental data define the functions of intracellular CypA in carcinogenesis, but findings on the role of its secreted form in tumor formation and progression are scarce. In the studies here, we exploit short-term in vitro and in vivo tests to directly evaluate the mutagenic, recombinogenic, and blastomogenic effects, as well as the promoter activity of recombinant human CypA (rhCypA), an analogue of secreted CypA. Our findings showed that rhCypA had no genotoxicity and, thus, was neither involved in nor influenced the initiation stage of carcinogenesis. At high doses, rhCypA could disrupt gap junctions in rat liver epithelial IAR-2 cells in vitro by decreasing the expression of connexins 26 and 43 in these cells and inhibit A549 cell adhesion. These data suggested that rhCypA could contribute to epithelial-mesenchymal transition in malignant cells. The research presented here elucidated the role of secreted CypA in carcinogenesis, revealing that it is not a tumor initiator but can act as a tumor promoter at high concentrations.

Gene Augmentation of CHM Using Non-Viral Episomal Vectors in Models of Choroideremia.

Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the CHM gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human CHM coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) CHM patient-derived fibroblasts and (2) chmru848 zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in CHM patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected chmru848 zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.

Non-Viral Episomal Vector Mediates Efficient Gene Transfer of the ß-Globin Gene into K562 and Human Haematopoietic Progenitor Cells.

ß-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPß-globin for the physiological ß-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the ß-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the ß-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce ß-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPß-globin provides the basis for the development of therapeutic EV for gene therapy of ß-thalassemias.

SMAR1 inhibits proliferation, EMT and Warburg effect of bladder cancer cells by suppressing the activity of the Wnt/ß-catenin signaling pathway.

This study aimed to investigate the effects of scaffold matrix attachment region binding protein 1 (SMAR1) on the development of bladder cancer (BCa). SMAR1 expression in paired tumor and corresponding adjacent normal tissues from 55 BCa patients was detected by quantitative reverse transcription-polymerase chain reaction. BCa cells were transfected to regulate SMAR1 expression. BCa cells were treated with XAV-939, LiCl and 2-deoxyglucose. The effect of SMAR1 on the viability, proliferation, migration, invasion and Warburg effect of BCa cells was researched by counting kit-8, colony formation assay, Transwell and aerobic glycolysis assays. Western blot was performed to detect protein expression. BCa cell growth in vivo was recorded in nude mice. Immunohistochemical staining was performed for clinical and xenografted tumor tissue specimens. SMAR1 expression was down-regulated in BCa patients, associating with worse prognoses. SMAR1 knockdown enhanced the viability, proliferation, migration, invasion, EMT and Warburg effect of BCa cells. The opposite effect was found in the SMAR1 overexpression BCa cells. XAV-939 treatment reversed the elevation of ß-catenin, c-Myc and Cyclin D1 proteins expression and Warburg effect in Bca cells post-SMAR1 knockdown. LiCl treatment abrogated the inhibition of ß-catenin, c-Myc and Cyclin D1 proteins expression and Warburg effect proteins due to SMAR1 overexpression in BCa cells. SMAR1 overexpression inhibited the growth of BCa cells in vivo. SMAR1 might suppress the Wnt/ß-catenin signaling pathway activity to inhibit the progression of BCa. It might be an effective treatment target for BCa.

Extending the novel |ρ|-based phasing algorithm to the solution of anomalous scattering substructures from SAD data of protein crystals.

Owing to the importance of the single-wavelength anomalous diffraction (SAD) technique, the recently developed |ρ|-based phasing algorithm (SM,|ρ|) incorporating the inner-pixel preservation (ipp) procedure [Rius & Torrelles (2021). Acta Cryst A77, 339-347] has been adapted to the determination of anomalous scattering substructures and its applicability tested on a series of 12 representative experimental data sets, mostly retrieved from the Protein Data Bank. To give an idea of the suitability of the data sets, the main indicators measuring their quality are also given. The dominant anomalous scatterers are either SeMet or S atoms, or metals/clusters incorporated by soaking. The resulting SAD-adapted algorithm solves the substructures of the test protein crystals quite efficiently.

Episomes and Transposases-Utilities to Maintain Transgene Expression from Nonviral Vectors.

The efficient delivery and stable transgene expression are critical for applications in gene therapy. While carefully selected and engineered viral vectors allowed for remarkable clinical successes, they still bear significant safety risks. Thus, nonviral vectors are a sound alternative and avoid genotoxicity and adverse immunological reactions. Nonviral vector systems have been extensively studied and refined during the last decades. Emerging knowledge of the epigenetic regulation of replication and spatial chromatin organisation, as well as new technologies, such as Crispr/Cas, were employed to enhance the performance of different nonviral vector systems. Thus, nonviral vectors are in focus and hold some promising perspectives for future applications in gene therapy. This review addresses three prominent nonviral vector systems: the Sleeping Beauty transposase, S/MAR-based episomes, and viral plasmid replicon-based EBV vectors. Exemplarily, we review different utilities, modifications, and new concepts that were pursued to overcome limitations regarding stable transgene expression and mitotic stability. New insights into the nuclear localisation of nonviral vector molecules and the potential consequences thereof are highlighted. Finally, we discuss the remaining limitations and provide an outlook on possible future developments in nonviral vector technology.

Harnessing artificial intelligence in cardiac rehabilitation, a systematic review.

Aim: This systematic review aims to evaluate the current body of research surrounding the efficacy of artificial intelligence (AI) in cardiac rehabilitation. Presently, AI can be incorporated into personal devices such as smart watches and smartphones, in diagnostic and home monitoring devices, as well as in certain inpatient care settings. Materials & methods: The PRISMA guidelines were followed in this review. Inclusion and exclusion criteria were set using the Population, Intervention, Comparison and Outcomes (PICO) tool. Results: Eight studies meeting the inclusion criteria were found. Conclusion: Incorporation of AI into healthcare, cardiac rehabilitation delivery, and monitoring holds great potential for early detection of cardiac events, allowing for home-based monitoring, and improved clinician decision making.

Lay abstract Artificial intelligence (AI) involves the use of technologies capable of making decisions based on data provided. AI can be used in healthcare to provide actionable data for a clinician by analyzing patterns in patient data to predict outcomes and guide treatment. Cardiovascular disease is the leading cause of death worldwide. Cardiac rehabilitation is a therapy proven to reduce mortality and morbidity from cardiovascular disease. This study outlines three cases of AI based healthcare tools in cardiac rehabilitation. This includes the provision of personalized, home-based cardiac rehabilitation, the early detection of cardiac events through smart watch monitoring and by providing clinician decision making support in cardiac failure rehabilitation.

RING finger protein TOPORS modulates the expression of tumor suppressor SMAR1 in colorectal cancer via the TLR4-TRIF pathway.

TOP1-binding arginine/serine-rich protein (TOPORS), a really interesting new gene finger protein, has the ability to bind to a palindromic consensus DNA sequence that enables it to function as a potential transcriptional regulator. However, its role in regulating the transcription of cancer-associated genes is yet to be explored. As Toll-like receptor 4 (TLR4) agonists are known to regress solid tumors, we observed that lipopolysaccharide (LPS) induces TOPORS via a TLR4-TIR domain-containing adapter-inducing interferon-ß-dependent pathway, which in turn modulates the transcription of tumor suppressor scaffold/matrix attachment region-binding protein 1 (SMAR1, also known as BANP). ChIP analysis showed that TOPORS binds on the SMAR1 promoter and its occupancy increases upon LPS treatment. A previous study from our laboratory revealed that SMAR1 acts as a repressor of signal transducer and activator of transcription 3 (STAT3) transcription. Tumor growth, as well as tumor-associated macrophage polarization, depends on the status of the STAT1:STAT3 ratio. LPS-induced SMAR1 expression decreases STAT3 expression and also skews the macrophage polarization toward M1 phenotype. In contrast, LPS failed to polarize tumor-associated macrophages to M1 phenotype in a SMAR1-silenced condition, which shows the involvement of SMAR1 in dictating the fate of colorectal cancer progression. Identification of the molecular mechanism behind LPS-mediated tumor regression would be crucial for designing cancer treatment strategies involving bacterial components.

An episomal DNA vector platform for the persistent genetic modification of pluripotent stem cells and their differentiated progeny.

The genetic modification of stem cells (SCs) is typically achieved using integrating vectors, whose potential integrative genotoxicity and propensity for epigenetic silencing during differentiation limit their application. The genetic modification of cells should provide sustainable levels of transgene expression, without compromising the viability of a cell or its progeny. We developed nonviral, nonintegrating, and autonomously replicating minimally sized DNA nanovectors to persistently genetically modify SCs and their differentiated progeny without causing any molecular or genetic damage. These DNA vectors are capable of efficiently modifying murine and human pluripotent SCs with minimal impact and without differentiation-mediated transgene silencing or vector loss. We demonstrate that these vectors remain episomal and provide robust and sustained transgene expression during self-renewal and targeted differentiation of SCs both in vitro and in vivo through embryogenesis and differentiation into adult tissues, without damaging their phenotypic characteristics.

SMAR1 suppresses the cancer stem cell population via hTERT repression in colorectal cancer cells.

One of the hallmarks of a cancer cell is the ability for indefinite proliferation leading to the immortalization of the cell. Activation of several signaling pathways leads to the immortalization of cancer cells via the reactivation of enzyme telomerase (hTERT). hTERT is active in germ cells, stem cells and also cancer cells. An earlier report from our lab suggests that SMAR1, a tumor suppressor protein, is significantly downregulated in the higher grades of colorectal cancers. Our study identifies SMAR1 as a transcriptional repressor of hTERT. We find that SMAR1 interacts with HDAC1/mSin3a co-repressor complex at the hTERT promoter and brings about HDAC1-mediated transcriptional repression of the promoter. Most solid tumors including colorectal cancer reactivate hTERT expression as it confers several advantages to the cancer cells like increased proliferation and angiogenesis. One of these non-canonical functions of hTERT is inducing the pool of cancer stem cell population. We find that in the CD133HighCD44High cancer stem cells population, SMAR1 expression is highly diminished leading to elevated hTERT expression. We also find that knockdown of SMAR1 promotes total CD133+CD44+ population and impart enhanced sphere-forming ability to the colorectal cancer cells. SMAR1 also inhibits invasion and metastasis in colorectal cancer cell lines via repression of hTERT. Our study provides evidence that downregulation of SMAR1 causes activation of hTERT leading to an increase in the cancer stem cell phenotype in colorectal cancer cells.

Chromatin remodeling protein SMAR1 regulates adipogenesis by modulating the expression of PPARγ.

Adipogenesis is described as the process of conversion of pre-adipocytes into differentiated lipid-laden adipocytes. Adipogenesis is known to be regulated by a myriad of transcription factors and co-regulators. However, there is a dearth of information regarding the mechanisms that regulate these transcription factors and hence control adipogenesis. PPARγ is the master transcriptional regulator of adipogenesis and its expression is essential for adipocyte differentiation. Herein, we identified that scaffold/matrix attachment region-binding protein 1 (SMAR1) negatively regulates adipogenesis. We observed that SMAR1 gets downregulated during adipocyte differentiation and knockdown of SMAR1 promotes lipid accumulation and adipocyte differentiation. Mechanistically, we have shown that SMAR1 suppresses PPARγ through recruitment of the HDAC1/mSin3a repressor complex to the PPARγ promoter. We further identified cell division cycle 20 (cdc20) mediated proteasomal degradation of SMAR1 during adipogenesis. Moreover, knockdown of cdc20 resulted in stabilization of SMAR1 and a reduction in adipocyte differentiation. Taken together, our observations suggest that SMAR1 functions as a negative regulator of adipogenesis by inhibiting PPARγ expression in differentiating adipocytes.

Advances in the Development and the Applications of Nonviral, Episomal Vectors for Gene Therapy.

Nonviral and nonintegrating episomal vectors are reemerging as a valid, alternative technology to integrating viral vectors for gene therapy, due to their more favorable safety profile, significantly lower risk for insertional mutagenesis, and a lesser potential for innate immune reactions, in addition to their low production cost. Over the past few years, attempts have been made to generate highly functional nonviral vectors that display long-term maintenance within cells and promote more sustained gene expression relative to conventional plasmids. Extensive research into the parameters that stabilize the episomal DNA within dividing and nondividing cells has shed light into the genetic and epigenetic mechanisms that govern replication and transcription of episomal DNA within a mammalian nucleus in long-term cell culture. Episomal vectors based on scaffold/matrix attachment regions (S/MARs) do not integrate into the genomic DNA and address the serious problem of plasmid loss during mitosis by providing mitotic stability to established plasmids, which results in long-term transfection and transgene expression. The inclusion, in such vectors, of an origin of replication-initiation region-from the human genome has greatly enhanced their performance in primary cell culture. A number of vectors that function as episomes have arisen, which are either devoid or depleted of harmful CpG sequences and bacterial genes, and their effectiveness, as well as that of nonintegrating viral episomes, is enhanced when combined with S/MAR elements. As a result of these advances, an "S/MAR technology" has emerged for the production of efficient episomal vectors. Significant research continues in this field and innovations, in combination with promising systems based on nanoparticles and potentially combined with physical delivery methods, will enable the generation of optimized systems with scale-up and clinical application suitability utilizing episomal vectors.

Enhanced Estimation of Root Zone Soil Moisture at 1 km Resolution Using SMAR Model and MODIS-Based Downscaled AMSR2 Soil Moisture Data.

Root zone soil moisture (RZSM) is an essential variable for weather and hydrological prediction models. Satellite-based microwave observations have been frequently utilized for the estimation of surface soil moisture (SSM) at various spatio-temporal resolutions. Moreover, previous studies have shown that satellite-based SSM products, coupled with the soil moisture analytical relationship (SMAR) can estimate RZSM variations. However, satellite-based SSM products are of low-resolution, rendering the application of the above-mentioned approach for local and pointwise applications problematic. This study initially attempted to estimate SSM at a finer resolution (1 km) using a downscaling technique based on a linear equation between AMSR2 SM data (25 km) with three MODIS parameters (NDVI, LST, and Albedo); then used the downscaled SSM in the SMAR model to monitor the RZSM for Rafsanjan Plain (RP), Iran. The performance of the proposed method was evaluated by measuring the soil moisture profile at ten stations in RP. The results of this study revealed that the downscaled AMSR2 SM data had a higher accuracy in relation to the ground-based SSM data in terms of MAE (↓0.021), RMSE (↓0.02), and R (↑0.199) metrics. Moreover, the SMAR model was run using three different SSM input data with different spatial resolution: (a) ground-based SSM, (b) conventional AMSR2, and (c) downscaled AMSR2 products. The results showed that while the SMAR model itself was capable of estimating RZSM from the variation of ground-based SSM data, its performance increased when using downscaled SSM data suggesting the potential benefits of proposed method in different hydrological applications.

K-mer Content Changes with Node Degree in Promoter-Enhancer Network of Mouse ES Cells.

Maps of Hi-C contacts between promoters and enhancers can be analyzed as networks, with cis-regulatory regions as nodes and their interactions as edges. We checked if in the published promoter-enhancer network of mouse embryonic stem (ES) cells the differences in the node type (promoter or enhancer) and the node degree (number of regions interacting with a given promoter or enhancer) are reflected by sequence composition or sequence similarity of the interacting nodes. We used counts of all k-mers (k = 4) to analyze the sequence composition and the Euclidean distance between the k-mer count vectors (k-mer distance) as the measure of sequence (dis)similarity. The results we obtained with 4-mers are interpretable in terms of dinucleotides. Promoters are GC-rich as compared to enhancers, which is known. Enhancers are enriched in scaffold/matrix attachment regions (S/MARs) patterns and depleted of CpGs. Furthermore, we show that promoters are more similar to their interacting enhancers than vice-versa. Most notably, in both promoters and enhancers, the GC content and the CpG count increase with the node degree. As a consequence, enhancers of higher node degree become more similar to promoters, whereas higher degree promoters become less similar to enhancers. We confirmed the key results also for human keratinocytes.

Novel vectors and approaches for gene therapy in liver diseases.

Gene therapy is becoming an increasingly valuable tool to treat many genetic diseases with no or limited treatment options. This is the case for hundreds of monogenic metabolic disorders of hepatic origin, for which liver transplantation remains the only cure. Furthermore, the liver contains 10-15% of the body's total blood volume, making it ideal for use as a factory to secrete proteins into the circulation. In recent decades, an expanding toolbox has become available for liver-directed gene delivery. Although viral vectors have long been the preferred approach to target hepatocytes, an increasing number of non-viral vectors are emerging as highly efficient vehicles for the delivery of genetic material. Herein, we review advances in gene delivery vectors targeting the liver and more specifically hepatocytes, covering strategies based on gene addition and gene editing, as well as the exciting results obtained with the use of RNA as a therapeutic molecule. Moreover, we will briefly summarise some of the limitations of current liver-directed gene therapy approaches and potential ways of overcoming them.

Tumor suppressor SMAR1 regulates PKM alternative splicing by HDAC6-mediated deacetylation of PTBP1.

BACKGROUND: Highly proliferating cancer cells exhibit the Warburg effect by regulation of PKM alternative splicing and promoting the expression of PKM2. Majority of the alternative splicing events are known to occur in the nuclear matrix where various MARBPs actively participate in the alternative splicing events. SMAR1, being a MARBP and an important tumor suppressor, is known to regulate the splicing of various cancer-associated genes. This study focuses on the regulation of PKM alternative splicing and inhibition of the Warburg effect by SMAR1. METHODS: Immunohistochemistry was performed in breast cancer patient samples to establish the correlation between SMAR1 and PKM isoform expression. Further, expression of PKM isoforms upon modulation in SMAR1 expression in breast cancer cell lines was quantified by qRT-PCR and western blot. The acetylation status of PTBP1 was estimated by immunoprecipitation along with its enrichment on PKM pre-mRNA by CLIP in SMAR1 knockdown conditions. The role of SMAR1 in tumor metabolism and tumorigenesis was explored by in vitro enzymatic assays and functional assays upon SMAR1 knockdown. Besides, in vivo tumor formation by injecting adeno-SMAR1-transduced MDA-MB-231 cells in NOD/SCID mice was performed. RESULTS: The expression profile of SMAR1 and PKM isoforms in breast cancer patients revealed that SMAR1 has an inverse correlation with PKM2 and a positive correlation with PKM1. Further quantitative PKM isoform expression upon modulation in SMAR1 expression also reflects that SMAR1 promotes the expression of PKM1 over tumorigenic isoform PKM2. SMAR1 deacetylates PTBP1 via recruitment of HDAC6 resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. SMAR1 inhibits the Warburg effect, tumorigenic potential of cancer cells, and in vivo tumor generation in a PKM2-dependent manner. CONCLUSIONS: SMAR1 regulates PKM alternative splicing by causing HDAC6-dependent deacetylation of PTBP1, resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. Additionally, SMAR1 suppresses glucose utilization and lactate production via repression of PKM2 expression. This suggests that tumor suppressor SMAR1 inhibits tumor cell metabolism and tumorigenic properties of cancer cells via regulation of PKM alternative splicing.

SMAR1 attenuates the stemness of osteosarcoma cells via through suppressing ABCG2 transcriptional activity.

The promoting roles of the transcriptional regulator SMAR1 have been revealed in several tumors, such as colorectal and breast cancer, however, its roles in osteosarcoma (OS) progression are still confusing. Here, we find that SMAR1 expression is positively correlated with the overall survival of OS patients and negatively correlated with the expression of stemness markers by analyzing the online datasets. Through analyzing different Gene Expression Omnibus (GEO) datasets, SMAR1 is found to be lowly expressed in OS tissues relative to that in adjacent tissues. Functional experiments indicate that SMAR1 overexpression attenuates the stemness of OS cells, characterized as the decrease of stemness marker expression, sphere-formation ability and ALDH activity. Mechanistically, it is shown that SMAR1 increases the deacetylation level of the drug efflux pump ABCG2 via recruiting HDAC2 to the promoter of the gene coding ABCG2, and thus decreases ABCG2 transcriptional activity. Additionally, overexpression of ABCG2 rescues the inhibition of SMAR1 overexpression on the stemness of OS cells. Moreover, this SMAR1/ABCG2 axis positively regulates the chemotherapeutic sensitivity of OS cells. This work indicates that SMAR1 is a critical suppressor for OS progression through transcriptionally regulating ABCG2 expression.