Novel Dormancy Mechanism of Castration Resistance in Bone Metastatic Prostate Cancer Organoids.

Advanced prostate cancer (PCa) patients with bone metastases are treated with androgen pathway directed therapy (APDT). However, this treatment invariably fails and the cancer becomes castration resistant. To elucidate resistance mechanisms and to provide a more predictive pre-clinical research platform reflecting tumor heterogeneity, we established organoids from a patient-derived xenograft (PDX) model of bone metastatic prostate cancer, PCSD1. APDT-resistant PDX-derived organoids (PDOs) emerged when cultured without androgen or with the anti-androgen, enzalutamide. Transcriptomics revealed up-regulation of neurogenic and steroidogenic genes and down-regulation of DNA repair, cell cycle, circadian pathways and the severe acute respiratory syndrome (SARS)-CoV-2 host viral entry factors, ACE2 and TMPRSS2. Time course analysis of the cell cycle in live cells revealed that enzalutamide induced a gradual transition into a reversible dormant state as shown here for the first time at the single cell level in the context of multi-cellular, 3D living organoids using the Fucci2BL fluorescent live cell cycle tracker system. We show here a new mechanism of castration resistance in which enzalutamide induced dormancy and novel basal-luminal-like cells in bone metastatic prostate cancer organoids. These PDX organoids can be used to develop therapies targeting dormant APDT-resistant cells and host factors required for SARS-CoV-2 viral entry.

AID-induced genotoxic stress promotes B cell differentiation in the germinal center via ATM and LKB1 signaling.

During an immune response, B cells undergo rapid proliferation and activation-induced cytidine deaminase (AID)-dependent remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, the genotoxic stress associated with the GC reaction also promotes most B cell malignancies. Here, we report that exogenous and intrinsic AID-induced DNA strand breaks activate ATM, which signals through an LKB1 intermediate to inactivate CRTC2, a transcriptional coactivator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation while opposing lymphomagenesis. Inhibition of this pathway results in increased GC B cell proliferation, reduced antibody secretion, and impaired terminal differentiation. Multiple distinct pathway disruptions were also identified in human GC B cell lymphoma patient samples. Combined, our data show that CRTC2 inactivation, via physiologic DNA damage response signaling, promotes B cell differentiation in response to genotoxic stress.

PCSD1, a new patient-derived model of bone metastatic prostate cancer, is castrate-resistant in the bone-niche.

INTRODUCTION: Prostate cancer bone metastasis occurs in 50-90% of men with advanced disease for which there is no cure. Bone metastasis leads to debilitating fractures and severe bone pain. It is associated with therapy resistance and rapid decline. Androgen deprivation therapy (ADT) is standard of care for advanced prostate cancer, however, bone metastatic prostate cancer (PCa) often becomes resistant to ADT. There are few pre-clinical models to understand the interaction between the bone microenvironment and prostate cancer. Here we report the castrate resistant growth in the bone niche of PCSD1, a patient-derived intra-femoral xenograft model of prostate bone metastatic cancer treated with the anti-androgen, bicalutamide. METHODS: PCSD1 bone-niche model was derived from a human prostate cancer femoral metastasis resected during hemiarthroplasty and serially transplanted into Rag2(-/-); γ c(-/-) mice intra-femorally (IF) or sub-cutaneously (SC). At 5 weeks post-transplantation mice received bicalutamide or vehicle control for 18 days. Tumor growth of PCSD1 was measured with calipers. PSA expression in PCSD1 xenograft tumors was determined using quantitative RT-PCR and immunohistochemistry. Expression of AR and PSMA, were also determined with qPCR. RESULTS: PCSD1 xenograft tumor growth capacity was 24 fold greater in the bone (intra-femoral, IF) than in the soft tissue (sub-cutaneous, SC) microenvironment. Treatment with the anti-androgen, bicalutamide, inhibited tumor growth in the sub-cutaneous transplantation site. However, bicalutamide was ineffective in suppressing PCSD1 tumor growth in the bone-niche. Nevertheless, bicalutamide treatment of intra-femoral tumors significantly reduced PSA expression (p < = 0.008) and increased AR (p < = 0.032) relative to control. CONCLUSIONS: PCSD1 tumors were castrate resistant when growing in the bone-niche compared to soft tissue. Bicalutamide had little effect on reducing tumor burden in the bone yet still decreased tumor PSA expression and increased AR expression, thus, this model closely recapitulated castrate-resistant, human prostate cancer bone metastatic disease. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study bone metastatic disease and for pre-clinical drug development of novel therapies for inhibiting therapy resistant prostate cancer growth in the bone-niche.

A novel patient-derived intra-femoral xenograft model of bone metastatic prostate cancer that recapitulates mixed osteolytic and osteoblastic lesions.

UNLABELLED: Prostate cancer metastasizes to bone in the majority of patients with advanced disease leading to painfully debilitating fractures, spinal compression and rapid decline. In addition, prostate cancer bone metastases often become resistant to standard therapies including androgen deprivation, radiation and chemotherapy. There are currently few models to elucidate mechanisms of interaction between the bone microenvironment and prostate cancer. It is, thus, essential to develop new patient-derived, orthotopic models. Here we report the development and characterization of PCSD1 (Prostate Cancer San Diego 1), a novel patient-derived intra-femoral xenograft model of prostate bone metastatic cancer that recapitulates mixed osteolytic and osteoblastic lesions. METHODS: A femoral bone metastasis of prostate cancer was removed during hemiarthroplasty and transplanted into Rag2(-/-);γc(-/-) mice either intra-femorally or sub-cutaneously. Xenograft tumors that developed were analyzed for prostate cancer biomarker expression using RT-PCR and immunohistochemistry. Osteoblastic, osteolytic and mixed lesion formation was measured using micro-computed tomography (microCT). RESULTS: PCSD1 cells isolated directly from the patient formed tumors in all mice that were transplanted intra-femorally or sub-cutaneously into Rag2(-/-);γc(-/-) mice. Xenograft tumors expressed human prostate specific antigen (PSA) in RT-PCR and immunohistochemical analyses. PCSD1 tumors also expressed AR, NKX3.1, Keratins 8 and 18, and AMACR. Histologic and microCT analyses revealed that intra-femoral PCSD1 xenograft tumors formed mixed osteolytic and osteoblastic lesions. PCSD1 tumors have been serially passaged in mice as xenografts intra-femorally or sub-cutaneously as well as grown in culture. CONCLUSIONS: PCSD1 xenografts tumors were characterized as advanced, luminal epithelial prostate cancer from a bone metastasis using RT-PCR and immunohistochemical biomarker analyses. PCSD1 intra-femoral xenografts formed mixed osteoblastic/osteolytic lesions that closely resembled the bone lesions in the patient. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study metastatic disease in the bone and to develop novel therapies for inhibiting prostate cancer growth in the bone-niche.

Differential effect of ionizing radiation exposure on multipotent and differentiation-restricted bone marrow mesenchymal stem cells.

Debilitating effects of bone marrow from ionizing radiation exposure has been well established for hematopoietic stem cells; however, radiation toxicity of mesenchymal stem cells (MSCs) has been controversial. The present study addressed if ionizing radiation exposure differently affected bone marrow MSCs with various differentiation commitments. Mouse bone-marrow-derived MSCs, D1 cells of early passages (≤ 5 passages; p5) maintained the complete characteristics of multipotent MSCs, whereas, after ≥ 45 passages (p45) the differentiation capability of D1 cells became partially restricted. Both p5 and p45 D1 cells were subjected to single dose irradiation by radioactive isotope (137)Cs. Radiation treatment impaired cell renewal and differentiation activities of p5 D1 cells; however, p45 D1 cells were less affected. Radiation treatment upregulated both pro- and anti-apoptotic genes of p5 D1 cells in a dose-dependent manner, potentially resulting in the various apoptosis thresholds. It was found that constitutive as well as radiation-induced phosphorylation levels of histone H2AX was significantly higher in p45 D1 cells than in p5 D1 cells. The increased repair activity of DNA double-strand breakage may play a role for p45 D1 cells to exhibit the relative radioresistance. In conclusion, the radiation toxicity predominantly affecting multipotent MSCs may occur at unexpectedly low doses, which may, in part, contribute to the catabolic pathology of bone tissue.

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient's bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Epidermal growth factor receptor vIII expression in U87 glioblastoma cells alters their proteasome composition, function, and response to irradiation.

Little is known about the factors that influence the proteasome structures in cells and their activity, although this could be highly relevant to cancer therapy. We have previously shown that, within minutes, irradiation inhibits substrate degradation by the 26S proteasome in most cell types. Here, we report an exception in U87 glioblastoma cells transduced to express the epidermal growth factor receptor vIII (EGFRvIII) mutant (U87EGFRvIII), which does not respond to irradiation with 26S proteasome inhibition. This was assessed using either a fluorogenic substrate or a reporter gene, the ornithine decarboxylase degron fused to ZsGreen (cODCZsGreen), which targets the protein to the 26S proteasome. To elucidate whether this was due to alterations in proteasome composition, we used quantitative reverse transcription-PCR to quantify the constitutive (X, Y, Z) and inducible 20S subunits (Lmp7, Lmp2, Mecl1), and 11S (PA28alpha and beta) and 19S components (PSMC1 and PSMD4). U87 and U87EGFRvIII significantly differed in expression of proteasome subunits, and in particular immunosubunits. Interestingly, 2 Gy irradiation of U87 increased subunit expression levels by 16% to 324% at 6 hours, with a coincident 30% decrease in levels of the proteasome substrate c-myc, whereas they changed little in U87EGFRvIII. Responses similar to 2 Gy were seen in U87 treated with a proteasome inhibitor, NPI0052, suggesting that proteasome inhibition induced replacement of subunits independent of the means of inhibition. Our data clearly indicate that the composition and function of the 26S proteasome can be changed by expression of the EGFRvIII. How this relates to the increased radioresistance associated with this cell line remains to be established.

Exosomes: Versatile Nano Mediators of Immune Regulation.

One of many types of extracellular vesicles (EVs), exosomes are nanovesicle structures that are released by almost all living cells that can perform a wide range of critical biological functions. Exosomes play important roles in both normal and pathological conditions by regulating cell-cell communication in cancer, angiogenesis, cellular differentiation, osteogenesis, and inflammation. Exosomes are stable in vivo and they can regulate biological processes by transferring lipids, proteins, nucleic acids, and even entire signaling pathways through the circulation to cells at distal sites. Recent advances in the identification, production, and purification of exosomes have created opportunities to exploit these structures as novel drug delivery systems, modulators of cell signaling, mediators of antigen presentation, as well as biological targeting agents and diagnostic tools in cancer therapy. This review will examine the functions of immunocyte-derived exosomes and their roles in the immune response under physiological and pathological conditions. The use of immunocyte exosomes in immunotherapy and vaccine development is discussed.

Regulation of IGFBP6 gene and protein is mediated by the inverse expression and function of c-jun N-terminal kinase (JNK) and NFkappaB in a model of oral tumor cells.

The aim of this study is to identify potential gene and protein targets when nuclear factor kappa B (NFkappaB) and c-jun N-terminal kinase (JNK) were inversely expressed in oral tumors. To determine which genes were regulated synergistically by the inverse expression of NFkappaB and JNK, a pathway specific microarray analysis was performed. While either inhibition of NFkappaB or activation of JNK alone was unable to affect the IGFBP6 gene expression in microarray analysis, concomitant increase in JNK activation in the presence of NFkappaB inhibition increased the expression of this gene significantly. Synergistic increase in IGFBP6 gene expression was also confirmed by RT-PCR and Northern blot analysis of transfected cells. Accordingly, the levels of IGFBP6 protein secretion rose synergistically when JNK was over-expressed in NFkappaB knock down cells. In addition, increased expression of JNK in the absence of NFkappaB resulted in a significant induction of cell death in oral tumors when either left untreated or treated with TNF-alpha and TPA. Moreover, when JNK was inhibited by dominant negative JNK (APF), a significant decrease in cell death could be observed in TNF-alpha and TPA treated NFkappaB knock down oral tumors. Therefore, increased induction of IGFBP6 gene or protein expression in oral tumors could be regarded as a potential predictive marker of tumor sensitivity and could be used for prognostic purposes, since a significant correlation could be observed between increased induction of apoptotic cell death and elevated levels of IGFBP6 in these tumors.

Reciprocal regulation of SOCS 1 and SOCS3 enhances resistance to ionizing radiation in glioblastoma multiforme.

PURPOSE: The expression of suppressors of cytokine signaling 1 (SOCS1) and SOCS3 genes is dysregulated in several solid tumors, causing aberrant activation of cell growth and survival signaling pathways. In this study, we analyzed SOCS1 and SOCS3 gene expression in glioblastoma multiforme (GBM) and studied the role of each protein in GBM cell signaling and radiation resistance. EXPERIMENTAL DESIGN: SOCS1 and SOCS3 gene expression was analyzed in 10 GBM cell lines by reverse transcription-PCR and Western blotting. SOCS3 expression was also studied in 12 primary GBM tissues by immunohistochemistry. The methylation status of the SOCS1 and SOCS3 loci was determined by methylation-specific PCR. Extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) activation in GBM cell lines overexpressing SOCS1 or lacking SOCS3 was determined by phosphorylated-specific Western blotting. Radiation responses in SOCS1-positive and SOCS3-deficient GBM cell lines and fibroblasts from wild-type and SOCS1 or SOCS3 knockout mice were studied in a clonogenic survival assay. RESULTS: All GBM cell lines tested lacked SOCS1 expression, whereas GBM cell lines and primary GBM tumor samples constitutively expressed SOCS3. SOCS1 gene repression was linked to hypermethylation of the SOCS1 genetic locus in GBM cells. Reintroduction of SOCS1 or blocking SOCS3 expression sensitized cells to radiation and decreased the levels of activated ERK MAPKs in GBM cells. CONCLUSIONS: SOCS1 and SOCS3 are aberrantly expressed in GBM cell lines and primary tissues. Altered SOCS gene expression leads to increased cell signaling through the ERK-MAPK pathway and may play a role in disease pathogenesis by enhancing GBM radioresistance.

HDJ-2 as a target for radiosensitization of glioblastoma multiforme cells by the farnesyltransferase inhibitor R115777 and the role of the p53/p21 pathway.

Resistance of glioblastoma multiforme to radiotherapy poses a major clinical challenge. Farnesyltransferase inhibitors (FTI), such as R115777, have potential to increase radiotherapeutic benefit in this disease, although their mechanism of action is unclear. In our study with eight glioblastoma multiforme cell lines, the most sensitive ones underwent cell cycle arrest in response to FTI treatment. Radiosensitization by FTIs, however, seemed to involve other pathways. If R115777 treatment was initiated < 6 hours before irradiation, all eight glioblastoma multiforme lines were radiosensitized. However, if the time between drug and radiation was extended to 24 hours, cells harboring wild type but not mutated p53 were able to counteract drug-induced radiosensitization. The involvement of the p53/p21 pathway in the development of resistance was confirmed by showing that U87 cells transfected with human papillomavirus E6 to block p53 or interfering RNA to inhibit p21 stayed radiosensitive for 24 hours after drug treatment. The time dependency of R115777-induced radiosensitization suggested that the initial FTI target for early radiosensitization was short-lived, and that a p21-directed pathway restored resistance. Consideration of prenylated molecules that could potentially be involved led us to consider HDJ-2, a co-chaperone of heat shock protein 70. This hypothesis was strengthened by finding that cellular radiosensitivity was increased by genetic inhibition of HDJ-2, whereas overexpression conferred radioresistance. Importantly, irradiation of cells caused HDJ-2 to migrate from the cytoplasm to the nucleus, and this migration was inhibited by prior FTI treatment. These results have clinical relevance in that they help explain the variability in responses to FTIs that occurs following radiotherapy and elucidate some of the reasons for the complexity underlying FTI-induced radiosensitization.

Complete androgen insensitivity syndrome caused by c.1769-1G > C mutation and activation of a cryptic splice acceptor site in the androgen receptor gene.

Androgen insensitivity syndrome (AIS) is the most common cause of 46,XY disorders of sex development (46,XY DSD). This syndrome is an X-linked recessive genetic disease characterized by resistance to the actions of androgens in an individual with a male karyotype and it is caused by mutations in the androgen receptor (AR) gene. We evaluated two siblings with primary amenorrhea, normal secondary sex characteristics, absence of uterus and ovaries, intra-abdominal testis, and elevated testosterone levels. Sequence analysis of the AR gene revealed a splice acceptor site mutation in intron 2 (c.1769-1G > C). The analysis of mRNA showed that this mutation resulted in the activation of a cryptic splice acceptor site located in intron 2 and in the synthesis of an aberrant mRNA transcript with 69 nucleotides insertion between exon 2 and exon 3, leading to an insertion of 23 amino acids in the AR protein instead of generating a premature termination codon. The additional 23 amino acids insertion affects AR intracellular trafficking by impairing its translocation from the cytoplasm to the nucleus after hormone stimulation. The c.1769-1G > C mutation provides new insights into the molecular mechanism involved in splicing defects and expands the spectrum of mutations associated with the androgen insensitivity syndrome.

N-acetylcysteine protects dental pulp stromal cells from HEMA-induced apoptosis by inducing differentiation of the cells.

Resin-based materials are now widely used in dental restorations. Although the use of these materials is aesthetically appealing to patients, it carries the risk of local and systemic adverse effects. The potential risks are direct damage to the cells and induction of immune-based hypersensitivity reactions. Dental pulp stromal cells (DPSCs) and oral keratinocytes are the major cell types which may come in contact with dental resins such as 2-hydroxyethyl methacrylate (HEMA) after dental restorations. Here we show that N-acetylcysteine (NAC) inhibits HEMA-induced apoptotic cell death and restores the function of DPSCs and oral epithelial cells. NAC inhibits HEMA-mediated toxicity through induction of differentiation in DPSCs, because the genes for dentin sialoprotein, osteopontin (OPN), osteocalcin, and alkaline phosphatase, which are induced during differentiation, are also induced by NAC. Unlike NAC, vitamins E and C, which are known antioxidant compounds, failed to prevent either HEMA-mediated cell death or the decrease in VEGF secretion by human DPSCs. More importantly, when added either alone or in combination with HEMA, vitamin E and vitamin C did not increase the gene expression for OPN, and in addition vitamin E inhibited the protective effect of NAC on DPSCs. NAC inhibited the HEMA-mediated decrease in NF-kappaB activity, thus providing a survival mechanism for the cells. Overall, the studies reported in this paper indicate that undifferentiated DPSCs have exquisite sensitivity to HEMA-induced cell death, and their differentiation in response to NAC resulted in an increased NF-kappaB activity, which might have provided the basis for their increased protection from HEMA-mediated functional loss and cell death.

Coengagement of CD16 and CD94 receptors mediates secretion of chemokines and induces apoptotic death of naive natural killer cells.

Down-modulation of CD16 (FcgammaRIII) receptors and loss of natural killer (NK) cell function have been observed in oral cancer patients. However, neither the mechanisms nor the significance of the decrease in CD16 receptors have been fully understood. The cytotoxic activity and survival of NK cells are negatively regulated by antibodies directed against CD16 surface receptor. The addition of anti-CD94 antibody in combination with either F(ab')(2) fragment or intact anti-CD16 antibody to NK cells resulted in significant inhibition of NK cell cytotoxic function and induction of apoptosis in resting human peripheral blood NK cells. Addition of interleukin-2 to anti-CD16 and/or anti-CD94 antibody-treated NK cells significantly inhibited apoptosis and increased the function of NK cells. There was a significant increase in tumor necrosis factor-alpha (TNF-alpha) but not IFN-gamma secretion in NK cells treated either with anti-CD16 antibody alone or in combination with anti-CD94 antibodies. Consequently, the addition of anti-TNF-alpha antibody partially inhibited apoptosis of NK cells mediated by the combination of anti-CD94 and anti-CD16 antibodies. Increase in apoptotic death of NK cells also correlated with an increase in type 2 inflammatory cytokines and in the induction of chemokines. Thus, we conclude that binding of antibodies to CD16 and CD94 NK cell receptors induces death of the NK cells and signals for the release of chemokines.

Regulation of Natural Killer Cell Function by STAT3.

Natural killer (NK) cells, key members of a distinct hematopoietic lineage, innate lymphoid cells, are not only critical effectors that mediate cytotoxicity toward tumor and virally infected cells but also regulate inflammation, antigen presentation, and the adaptive immune response. It has been shown that NK cells can regulate the development and activation of many other components of the immune response, such as dendritic cells, which in turn, modulate the function of NK cells in multiple synergistic feed back loops driven by cell-cell contact, and the secretion of cytokines and chemokines that control effector function and migration of cells to sites of immune activation. The signal transducer and activator of transcription (STAT)-3 is involved in driving almost all of the pathways that control NK cytolytic activity as well as the reciprocal regulatory interactions between NK cells and other components of the immune system. In the context of tumor immunology, NK cells are a first line of defense that eliminates pre-cancerous and transformed cells early in the process of carcinogenesis, through a mechanism of "immune surveillance." Even after tumors become established, NK cells are critical components of anticancer immunity: dysfunctional NK cells are often found in the peripheral blood of cancer patients, and the lack of NK cells in the tumor microenvironment often correlates to poor prognosis. The pathways and soluble factors activated in tumor-associated NK cells, cancer cells, and regulatory myeloid cells, which determine the outcome of cancer immunity, are all critically regulated by STAT3. Using the tumor microenvironment as a paradigm, we present here an overview of the research that has revealed fundamental mechanisms through which STAT3 regulates all aspects of NK cell biology, including NK development, activation, target cell killing, and fine tuning of the innate and adaptive immune responses.

Specific bone region localization of osteolytic versus osteoblastic lesions in a patient-derived xenograft model of bone metastatic prostate cancer.

OBJECTIVE: Bone metastasis occurs in up to 90% of men with advanced prostate cancer and leads to fractures, severe pain and therapy-resistance. Bone metastases induce a spectrum of types of bone lesions which can respond differently to therapy even within individual prostate cancer patients. Thus, the special environment of the bone makes the disease more complicated and incurable. A model in which bone lesions are reproducibly induced that mirrors the complexity seen in patients would be invaluable for pre-clinical testing of novel treatments. The microstructural changes in the femurs of mice implanted with PCSD1, a new patient-derived xenograft from a surgical prostate cancer bone metastasis specimen, were determined. METHODS: Quantitative micro-computed tomography (micro-CT) and histological analyses were performed to evaluate the effects of direct injection of PCSD1 cells or media alone (Control) into the right femurs of Rag2-/-γc-/- male mice. RESULTS: Bone lesions formed only in femurs of mice injected with PCSD1 cells. Bone volume (BV) was significantly decreased at the proximal and distal ends of the femurs (p < 0.01) whereas BV (p < 0.05) and bone shaft diameter (p < 0.01) were significantly increased along the femur shaft. CONCLUSION: PCSD1 cells reproducibly induced bone loss leading to osteolytic lesions at the ends of the femur, and, in contrast, induced aberrant bone formation leading to osteoblastic lesions along the femur shaft. Therefore, the interaction of PCSD1 cells with different bone region-specific microenvironments specified the type of bone lesion. Our approach can be used to determine if different bone regions support more therapy resistant tumor growth, thus, requiring novel treatments.

Suppressor of cytokine signaling (SOCS) genes are silenced by DNA hypermethylation and histone deacetylation and regulate response to radiotherapy in cervical cancer cells.

Suppressor of cytokine signaling (SOCS) family is an important negative regulator of cytokine signaling and deregulation of SOCS has been involved in many types of cancer. All cervical cancer cell lines tested showed lower expression of SOCS1, SOCS3, and SOCS5 than normal tissue or cell lines. The immunohistochemistry result for SOCS proteins in human cervical tissue also confirmed that normal tissue expressed higher level of SOCS proteins than neighboring tumor. Similar to the regulation of SOCS in other types of cancer, DNA methylation contributed to SOCS1 downregulation in CaSki, ME-180, and HeLa cells. However, the expression of SOCS3 or SOCS5 was not recovered by the inhibition of DNA methylation. Histone deacetylation may be another regulatory mechanism involved in SOCS1 and SOCS3 expression, however, SOCS5 expression was neither affected by DNA methylation nor histone deacetylation. Ectopic expression of SOCS1 or SOCS3 conferred radioresistance to HeLa cells, which implied SOCS signaling regulates the response to radiation in cervical cancer. In this study, we have shown that SOCS expression repressed by, in part, epigenetically and altered SOCS1 and SOCS3 expression could contribute to the radiosensitive phenotype in cervical cancer.

Natural killer cells preferentially target cancer stem cells; role of monocytes in protection against NK cell mediated lysis of cancer stem cells.

Mounting effective anti-tumor immune responses by cytotoxic effectors is important for the clearance of tumors. However, accumulated evidence suggests that the cytotoxic function of immune effectors is largely suppressed in the tumor microenvironment by a number of distinct effectors and their secreted factors. The aims of this review are to provide a rationale and potential mechanism for immunosuppression in cancer, and to demonstrate the significance of such immunosuppression in cellular differentiation and tissue regeneration in pathological conditions, and progression of cancer. We have recently shown that increased NK cell function was seen when they were cultured with primary oral squamous carcinoma stem cells (OSCSCs) as compared to their more differentiated oral squamous carcinoma cells (OSCCs). In addition, human embryonic stem cells (hESCs), Mesenchymal Stem Cells (hMSCs), dental pulp stem cells (hDPSCs) and induced pluripotent stem cells (hiPSCs) were significantly more susceptible to NK cell mediated cytotoxicity than their differentiated counterparts or parental cells from which they were derived. We have also reported that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or targeted knock down of COX2 augmented NK cell function significantly. Total population of monocytes and those depleted of CD16(+) subsets were able to substantially prevent NK cell mediated lysis of OSCSCs, MSCs and DPSCs. Taken together, our results suggest that stem cells are significant targets of the NK cell cytotoxicity. The concept of split anergy in NK cells and its contribution to tissue repair and regeneration and in tumor resistance and progression will be discussed in this review. Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation at the site of the tumor for specific elimination of cancer stem cells.

Strategies to rescue mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) from NK cell mediated cytotoxicity.

BACKGROUND: The aim of this paper is to study the function of allogeneic and autologous NK cells against Dental Pulp Stem Cells (DPSCs) and Mesenchymal Stem Cells (MSCs) and to determine the function of NK cells in a three way interaction with monocytes and stem cells. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that freshly isolated untreated or IL-2 treated NK cells are potent inducers of cell death in DPSCs and MSCs, and that anti-CD16 antibody which induces functional split anergy and apoptosis in NK cells inhibits NK cell mediated lysis of DPSCs and MSCs. Monocytes co-cultured with either DPSCs or MSCs decrease lysis of stem cells by untreated or IL-2 treated NK cells. Monocytes also prevent NK cell apoptosis thereby raising the overall survival and function of NK cells, DPSCs or MSCs. Both total population of monocytes and those depleted of CD16(+) subsets were able to prevent NK cell mediated lysis of MSCs and DPSCs, and to trigger an increased secretion of IFN-gamma by IL-2 treated NK cells. Protection of stem cells from NK cell mediated lysis was also seen when monocytes were sorted out from stem cells before they were added to NK cells. However, this effect was not specific to monocytes since the addition of T and B cells to stem cells also protected stem cells from NK cell mediated lysis. NK cells were found to lyse monocytes, as well as T and B cells. CONCLUSION/SIGNIFICANCE: By increasing the release of IFN-gamma and decreasing the cytotoxic function of NK cells monocytes are able to shield stem cells from killing by the NK cells, resulting in an increased protection and differentiation of stem cells. More importantly studies reported in this paper indicate that anti-CD16 antibody can be used to prevent NK cell induced rejection of stem cells.

Dexamethasone induces dysferlin in myoblasts and enhances their myogenic differentiation.

Glucocorticoids are beneficial in many muscular dystrophies but they are ineffective in treating dysferlinopathy, a rare muscular dystrophy caused by loss of dysferlin. We sought to understand the molecular basis for this disparity by studying the effects of a glucocorticoid on differentiation of the myoblast cell line, C2C12, and dysferlin-deficient C2C12s. We found that pharmacologic doses of dexamethasone enhanced the myogenic fusion efficiency of C2C12s and increased the induction of dysferlin, along with specific myogenic transcription factors, sarcolemmal and structural proteins. In contrast, the dysferlin-deficient C2C12 cell line demonstrated a reduction in long myotubes and early induction of particular muscle differentiation proteins, most notably, myosin heavy chain. Dexamethasone partially reversed the defect in myogenic fusion in the dysferlin-deficient C2C12 cells. We hypothesize that a key therapeutic benefit of glucocorticoids may be the up-regulation of dysferlin as an important component of glucocorticoid-enhanced myogenic differentiation.