The role of SWI/SNF chromatin remodelers in the repair of DNA double strand breaks and cancer therapy.

Switch/Sucrose non-fermenting (SWI/SNF) chromatin remodelers hydrolyze ATP to push and slide nucleosomes along the DNA thus modulating access to various genomic loci. These complexes are the most frequently mutated epigenetic regulators in human cancers. SWI/SNF complexes are well known for their function in transcription regulation, but more recent work has uncovered a role for these complexes in the repair of DNA double strand breaks (DSBs). As radiotherapy and most chemotherapeutic agents kill cancer cells by inducing double strand breaks, by identifying a role for these complexes in double strand break repair we are also identifying a DNA repair vulnerability that can be exploited therapeutically in the treatment of SWI/SNF-mutated cancers. In this review we summarize work describing the function of various SWI/SNF subunits in the repair of double strand breaks with a focus on homologous recombination repair and discuss the implication for the treatment of cancers with SWI/SNF mutations.

The SWI/SNF ATPase BRG1 stimulates DNA end resection and homologous recombination by reducing nucleosome density at DNA double strand breaks and by promoting the recruitment of the CtIP nuclease.

DNA double strand breaks (DSBs) are among the most toxic DNA lesions and can be repaired accurately through homologous recombination (HR). HR requires processing of the DNA ends by nucleases (DNA end resection) in order to generate the required single-stranded DNA (ssDNA) regions. The SWI/SNF chromatin remodelers are 10-15 subunit complexes that contain one ATPase (BRG1 or BRM). Multiple subunits of these complexes have recently been identified as a novel family of tumor suppressors. These complexes are capable of remodeling chromatin by pushing nucleosomes along the DNA. More recent studies have identified these chromatin remodelers as important factors in DNA repair. Using the DR-U2OS reporter system, we show that the down regulation of BRG1 significantly reduces HR efficiency, while BRM has a minor effect. Inactivation of BRG1 impairs DSB repair and results in a defect in DNA end resection, as measured by the amount of BrdU-containing ssDNA generated after DNA damage. Inactivation of BRG1 also impairs the activation of the ATR kinase, reduces the levels of chromatin-bound RPA, and reduces the number of RPA and RAD51 foci after DNA damage. This defect in DNA end resection is explained by the defective recruitment of GFP-CtIP to laser-induced DSBs in the absence of BRG1. Importantly, we show that BRG1 reduces nucleosome density at DSBs. Finally, inactivation of BRG1 renders cells sensitive to anti-cancer drugs that induce DSBs. This study identifies BRG1 as an important factor for HR, which suggests that BRG1-mutated cancers have a DNA repair vulnerability that can be exploited therapeutically.

Cell-mediated immune resistance in cancer.

The genetic and epigenetic aberrations that underlie immune resistance lead to tumors that are refractory to clinically established and experimental immunotherapies, including monoclonal antibodies and T cell-based therapies. From various forms of cytotoxic T cells to small molecule inhibitors that revamp the tumor microenvironment, these therapies have demonstrated notable responses in cancer models and a resistant subset of cancer patients, used both alone and in combination. However, even current approaches, such as those targeting checkpoint molecules, tumor ligands, and involving gene-related therapies, present a challenge in non-responding patients. In this perspective, we discuss the most common mechanisms of immune resistance, including tumor heterogeneity, tumor ligand and major histocompatibility complex modulation, anti-apoptotic pathways, checkpoint inhibitory ligands, immunosuppressive cells and factors in the tumor microenvironment, and activation-induced cell death. In addition, we discuss the strategies designed to circumvent these resistance pathways to showcase the potential of emerging technologies in battling the rise of resistance.

Nitric Oxide-Mediated Enhancement and Reversal of Resistance of Anticancer Therapies.

In the last decade, immune therapies against human cancers have emerged as a very effective therapeutic strategy in the treatment of various cancers, some of which are resistant to current therapies. Although the clinical responses achieved with many therapeutic strategies were significant in a subset of patients, another subset remained unresponsive initially, or became resistant to further therapies. Hence, there is a need to develop novel approaches to treat those unresponsive patients. Several investigations have been reported to explain the underlying mechanisms of immune resistance, including the anti-proliferative and anti-apoptotic pathways and, in addition, the increased expression of the transcription factor Yin-Yang 1 (YY1) and the programmed death ligand 1 (PD-L1). We have reported that YY1 leads to immune resistance through increasing HIF-1α accumulation and PD-L1 expression. These mechanisms inhibit the ability of the cytotoxic T-lymphocytes to mediate their cytotoxic functions via the inhibitory signal delivered by the PD-L1 on tumor cells to the PD-1 receptor on cytotoxic T-cells. Thus, means to override these resistance mechanisms are needed to sensitize the tumor cells to both cell killing and inhibition of tumor progression. Treatment with nitric oxide (NO) donors has been shown to sensitize many types of tumors to chemotherapy, immunotherapy, and radiotherapy. Treatment of cancer cell lines with NO donors has resulted in the inhibition of cancer cell activities via, in part, the inhibition of YY1 and PD-L1. The NO-mediated inhibition of YY1 was the result of both the inhibition of the upstream NF-κB pathway as well as the S-nitrosylation of YY1, leading to both the downregulation of YY1 expression as well as the inhibition of YY1-DNA binding activity, respectively. Also, treatment with NO donors induced the inhibition of YY1 and resulted in the inhibition of PD-L1 expression. Based on the above findings, we propose that treatment of tumor cells with the combination of NO donors, at optimal noncytotoxic doses, and anti-tumor cytotoxic effector cells or other conventional therapies will result in a synergistic anticancer activity and tumor regression.

YY1 regulates cancer cell immune resistance by modulating PD-L1 expression.

Recent advances in the treatment of various cancers have resulted in the adaptation of several novel immunotherapeutic strategies. Notably, the recent intervention through immune checkpoint inhibitors has resulted in significant clinical responses and prolongation of survival in patients with several therapy-resistant cancers (melanoma, lung, bladder, etc.). This intervention was mediated by various antibodies directed against inhibitory receptors expressed on cytotoxic T-cells or against corresponding ligands expressed on tumor cells and other cells in the tumor microenvironment (TME). However, the clinical responses were only observed in a subset of the treated patients; it was not clear why the remaining patients did not respond to checkpoint inhibitor therapies. One hypothesis stated that the levels of PD-L1 expression correlated with poor clinical responses to cell-mediated anti-tumor immunotherapy. Hence, exploring the underlying mechanisms that regulate PD-L1 expression on tumor cells is one approach to target such mechanisms to reduce PD-L1 expression and, therefore, sensitize the resistant tumor cells to respond to PD-1/PD-L1 antibody treatments. Various investigations revealed that the overexpression of the transcription factor Yin Yang 1 (YY1) in most cancers is involved in the regulation of tumor cells' resistance to cell-mediated immunotherapies. We, therefore, hypothesized that the role of YY1 in cancer immune resistance may be correlated with PD-L1 overexpression on cancer cells. This hypothesis was investigated and analysis of the reported literature revealed that several signaling crosstalk pathways exist between the regulations of both YY1 and PD-L1 expressions. Such pathways include p53, miR34a, STAT3, NF-kB, PI3K/AKT/mTOR, c-Myc, and COX-2. Noteworthy, many clinical and pre-clinical drugs have been utilized to target these above pathways in various cancers independent of their roles in the regulation of PD-L1 expression. Therefore, the direct inhibition of YY1 and/or the use of the above targeted drugs in combination with checkpoint inhibitors should result in enhancing the cell-mediated anti-tumor cell response and also reverse the resistance observed with the use of checkpoint inhibitors alone.

Reevaluating Biochemistry and Nutrition Education Through Opinions of Clinicians and Educators.

Biochemistry is a core component of medical education as it contributes to the fundamental basis and understanding of molecular mechanisms in pathophysiological processes. The convergence of nutritional factors also gives insight to many chronic diseases. Topics of nutrition are often incorporated into biochemistry coursework and must be integrated in a way that makes sense within the overall curriculum. An important issue raised by this structure is determining which topics are most important to a student's understanding and what topics are most relevant to future clinical practice. Previous surveys show medical undergraduates feel that much of current medical biochemistry coursework lacks clinical relevance and pays too much attention to small details. Here we report the results of a survey that aims to determine the biochemical and nutritional topics that physicians and educators feel are most important to teach in medical school. This information is important for medical schools to better prepare their students for what they will see and apply in their future clinical practice. Physicians and medical educators were surveyed, asked demographic questions, and then requested to provide a prioritized list of the top 10 biochemistry and nutrition topics that they believed should be focused on in undergraduate medical education. Topics suggested by participants were normalized for spelling, acronyms, and abbreviations and given a weight from 10 to 1. A prioritized list was then created based on the suggested topics. This list provides insight into the topics that medical educators and physicians consider important to cover in undergraduate medical education.

"Pharming out" support: a promising approach to integrating clinical pharmacists into established primary care medical home practices.

Objective Embedding clinical pharmacists into ambulatory care settings needs to be assessed in the context of established medical home models. Methods A retrospective, observational study examined the effectiveness of the Intermountain Healthcare Collaborative Pharmacist Support Services (CPSS) program from 2012-2015 among adult patients diagnosed with diabetes mellitus (DM) and/or high blood pressure (HBP). Patients who attended this program were considered the intervention (CPSS) cohort. These patients were matched using propensity scores with a reference group (no-CPSS cohort) to determine the effect of achieving disease management goals and time to achievement. Results A total of 17,684 patients had an in-person office visit with their provider and 359 received CPSS (the matched no-CPSS cohort included 999 patients). CPSS patients were 93% more likely to achieve a blood pressure goal < 140/90 mmHg, 57% more likely to achieve HbA1c values < 8%, and 87% more likely to achieve both disease management goals compared with the reference group. Time to goal achievement demonstrated increasing separation between the study cohorts across the entire study period ( P < .001), and specifically, at 180 days post-intervention (HBP: 48% vs 27% P < .001 and DM: 39% vs 30%, P < .05). Conclusions CPSS participation is associated with significant improvement in achievement of disease management goals, time to achievement, and increased ambulatory encounters compared with the matched no-CPSS cohort.

Strength or Motor Control: What Matters in High-Functioning Stroke?

Background: The two primary motor impairments that hinder function after stroke are declines in strength and motor control. The impact of motor impairments on functional capacity may vary with the severity of stroke motor impairments. In this study, we focus on high-functioning stroke individuals who experience mild to moderate motor impairments and often resume prior activities or return to work. These tasks require the ability to move independently, placing high demands on their functional mobility. Therefore, the purpose of this study was to quantify impairments in strength and motor control and their contribution to functional mobility in high-functioning stroke. Methods:Twenty-one high-functioning stroke individuals (Fugl Meyer Lower Extremity Score = 28.67 ± 4.85; Functional Activity Index = 28.47 ± 7.04) and 21 age-matched healthy controls participated in this study. To examine motor impairments in strength and motor control, participants performed the following tasks with the paretic ankle (1) maximum voluntary contractions (MVC) and (2) visuomotor tracking of a sinusoidal trajectory. Strength was quantified as the maximum force produced during ankle plantarflexion and dorsiflexion. Motor control was quantified as (a) the accuracy and (b) variability of ankle movement during the visuomotor tracking task. For functional mobility, participants performed (1) overground walking for 7 meters and (2) simulated driving task. Functional mobility was determined by walking speed, stride length variability, and braking reaction time. Results: Compared with the controls, the stroke group showed decreased plantarflexion strength, decreased accuracy, and increased variability of ankle movement. In addition, the stroke group demonstrated decreased walking speed, increased stride length variability, and increased braking reaction time. The multiple-linear regression model revealed that motor accuracy was a significant predictor of the walking speed and braking reaction time. Further, motor variability was a significant predictor of stride length variability. Finally, the dorsiflexion or plantarflexion strength did not predict walking speed, stride length variability or braking reaction time. Conclusions: The impairments in motor control but not strength predict functional deficits in walking and driving in high-functioning stroke individuals. Therefore, rehabilitation interventions assessing and improving motor control will potentially enhance functional outcomes in high-functioning stroke survivors.

Gene expression profiles resulting from stable loss of p53 mirrors its role in tissue differentiation.

The tumor suppressor gene p53 is involved in a variety of cellular activities such as cellular stress responses, cell cycle regulation and differentiation. In our previous studies we have shown p53's transcription activating role to be important in osteoblast differentiation. There is still a debate in the literature as to whether p53 inhibits or promotes differentiation. We have found p53 heterozygous mice to show a p53 dependency on some bone marker gene expression that is absent in knockout mice. Mice heterozygous for p53 also show a higher incidence of osteosarcomas than p53 knockout mice. This suggests that p53 is able to modify the environment within osteoblasts. In this study we compare changes in gene expression resulting after either a transient or stable reduction in p53. Accordingly we reduced p53 levels transiently and stably in C2C12 cells, which are capable of both myoblast and osteoblast differentiation, and compared the changes in gene expression of candidate genes regulated by the p53 pathway. Using a PCR array to assay for p53 target genes, we have found different expression profiles when comparing stable versus transient knockdown of p53. As expected, several genes with profound changes after transient p53 loss were related to apoptosis and cell cycle regulation. In contrast, stable p53 loss produced a greater change in MyoD and other transcription factors with tissue specific roles, suggesting that long term loss of p53 affects tissue homeostasis to a greater degree than changes resulting from acute loss of p53. These differences in gene expression were validated by measuring promoter activity of different pathway specific genes involved in differentiation. These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression affecting normal tissue physiology more than acute loss of p53.

Vitamin D directly regulates Mdm2 gene expression in osteoblasts.

While Mdm2 is an important negative regulator of the p53 tumor suppressor, it also possesses p53-independent functions in cellular differentiation processes. Mdm2 expression is alternatively regulated by two P1 and P2 promoters. In this study we show that the P2-intiated transcription of Mdm2 gene is activated by 1,25-dihydroxy vitamin D3 in MC3T3 cells. By using P1 and P2-specific reporters, we demonstrate that only the P2-promoter responds to vitamin D treatment. We have further identified a potential vitamin D receptor responsive element proximal to the two p53 response elements within the Mdm2 P2 promoter. Using cell lines that are p53-temperature sensitive and p53-null, we show requirement of p53 for VDR-mediated up regulation of Mdm2 expression. Our results indicate that 1,25-dihydroxy vitamin D3 and its receptor have a role in the regulation of P2-initiated Mdm2 gene expression in a p53-dependent way.

p53 and MDM2 are involved in the regulation of osteocalcin gene expression.

Osteocalcin (OC) is a major noncollagenous bone matrix protein and an osteoblast marker whose expression is limited to mature osteoblasts during the late differentiation stage. In previous studies we have shown osteosarcomas to lose p53 function with a corresponding loss of osteocalcin gene expression. Introduction of wild type p53 resulted in re expression of the osteocalcin gene. Using gel shift and chromatin immunoprecipitation assays, we have identified a putative p53 binding site within the rat OC promoter region and observed an increase in OC promoter activity when p53 accumulates using a CAT assay. The p53 inducible gene Mdm2 is a well-known downstream regulator of p53 levels. Our results showed a synergistic increase in the OC promoter activity when both p53 and MDM2 were transiently overexpressed. We further demonstrate that p53 is not degraded during overexpression of MDM2 protein. Increased OC expression was observed with concomitantly increased p53, VDR, and MDM2 levels in ROS17/2.8 cells during treatment with differentiation promoting (DP) media, but was significantly decreased when co-treated with DP media and the small molecule inhibitor of MDM2-p53 interaction, Nutlin-3. We have also observed a dramatic increase of the OC promoter activity in the presence of p53 and Mdm2 with inclusion of Cbfa-1 and p300 factors. Our results suggest that under some physiological conditions the oncoprotein MDM2 may cooperate with p53 to regulate the osteocalcin gene during osteoblastic differentiation.

Osteocalcin gene expression is regulated by wild-type p53.

The tumor-suppressor p53 is a transcription factor that regulates a number of genes in the process of cell-cycle inhibition, apoptosis, and DNA damage. Recent studies have revealed a crucial role for p53 in bone remodeling. In our previous studies we have shown that p53 is an important regulator of osteoblast differentiation. In this study we investigated the role of p53 in the regulation of human osteocalcin gene expression. We observed that osteocalcin promoter activity could be upregulated by both exogenous and endogenous p53 and downregulated by p53-specific small interfering RNA. DNA affinity immunoblotting assay showed that p53 can bind to the human osteocalcin promoter in vitro. We further identified a p53 response element within the osteocalcin promoter region using a chromatin immunoprecipitation assay. Furthermore, we observed an additive effect of p53 and VDR on the regulation of osteocalcin promoter activity. Our findings suggest that p53 may directly target the human osteocalcin gene and positively affect osteocalcin gene expression.

Beta-catenin is not activated by downregulation of PTEN in osteoblasts.

Selective knockdown of phosphatase and tensin homolog (PTEN) has been recently shown to increase life long accumulation of bone and its ability to increase osteoblast lifespan. In order to determine how loss of PTEN function affects osteoblast differentiation, we created cell lines with stable knockdown of PTEN expression using short hairpin RNA vectors and characterized several clones. The effect of deregulated PTEN in osteoblasts was studied in relationship to cell proliferation and differentiation. Downregulation of PTEN initially affected the cell's attachment and spreading on plastic but cells recovered after a brief period of time. When cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, we noticed a small but significant increase in growth rates with PTEN reduction. The size of individual cells appeared larger when compared to control cells. Differentiation properties of these osteoblasts were increased as evidenced by higher expression of several of the bone markers tested (alkaline phosphatase, osteocalcin, osterix, bone morphogenetic protein 2, Cbfa1, osteoprotegerin, and receptor activator of NF-kappaB ligand) and their mineralization capacity in culture. As stabilization of beta-catenin is known to be responsible for growth deregulation with PTEN loss in other cell types, we investigated the activation of the canonical Wnt pathway in our cell lines. Immunofluorescence staining, protein expression in subcellular fractions for beta-catenin, and assays for activation of the canonical Wnt/beta-catenin signaling were studied in the PTEN downregulated cells. There was an overall decrease in beta-catenin expression in cells with PTEN knockdown. The distribution of beta-catenin was more diffuse within the cell in the PTEN-reduced clones when compared to controls where they were mostly present in cell borders. Signaling through the canonical pathway was also reduced in the PTEN knockdown cells when compared to control. The results of this study suggest that while decreased PTEN augments cell proliferation and positively affects differentiation, there is a decrease in beta-catenin levels and activity in osteoblasts. Therefore, at least in osteoblasts, beta-catenin is not responsible for mediating the activation of osteoblast differentiation with reduction in PTEN function.