KD Cells Research Articles

LDH-A regulates the tumor microenvironment via HIF-signaling and modulates the immune response.

Previous studies show that LDH-A knockdown reduces orthotopic 4T1 breast tumor lactate and delays tumor growth and the development of metastases in nude mice. Here, we report significant changes in the tumor microenvironment (TME) and a more robust anti-tumor response in immune competent BALB/c mice. 4T1 murine breast cancer cells were transfected with shRNA plasmids directed against LDH-A (KD) or a scrambled control plasmid (NC). Cells were also transduced with dual luciferase-based reporter systems to monitor HIF-1 activity and the development of metastases by bioluminescence imaging, using HRE-sensitive and constitutive promoters, respectively. The growth and metastatic profile of orthotopic 4T1 tumors developed from these cell lines were compared and a primary tumor resection model was studied to simulate the clinical management of breast cancer. Primary tumor growth, metastasis formation and TME phenotype were significantly different in LDH-A KD tumors compared with controls. In LDH-A KD cells, HIF-1 activity, hexokinase 1 and 2 expression and VEGF secretion were reduced. Differences in the TME included lower HIF-1α expression that correlated with lower vascularity and pimonidazole staining, higher infiltration of CD3+ and CD4+ T cells and less infiltration of TAMs. These changes resulted in a greater delay in metastases formation and 40% long-term survivors (>20 weeks) in the LDH-A KD cohort following surgical resection of the primary tumor. We show for the first time that LDH-depletion inhibits the formation of metastases and prolongs survival of mice through changes in tumor microenvironment that modulate the immune response. We attribute these effects to diminished HIF-1 activity, vascularization, necrosis formation and immune suppression in immune competent animals. Gene-expression analyses from four human breast cancer datasets are consistent with these results, and further demonstrate the link between glycolysis and immune suppression in breast cancer.

Abstract 4144: The role of GABA metabolism in medulloblastoma spread

Abstract Central nervous system (CNS) tumors remain the leading cause of cancer-related childhood mortality. Amongst these, medulloblastoma (MB), a highly malignant cancer originating in the cerebellum, remains the most common pediatric CNS tumor. Patients are clinically stratified by age at diagnosis, extent of resection, and metastatic status. Patients with incomplete tumor resection or leptomeningeal spread at the time of diagnosis are considered high-risk. Standard of care for all patients consists of surgical resection followed by radiation and chemotherapy. MB tumors have recently been molecularly stratified into 4 groups, with each group associated with a particular effected pathway. Group 1 and 2 have deregulated WNT and SHH pathways, respectively. Group 3 and 4, however, are less characterized, but have been shown to be associated with a GABAergic and glutamanergic phenotype, respectively. While Group 1 and 2 MBs have an overall 5-year survival rate of 75%, Group 3 and 4 have a worse prognosis, with survival rates closer to 30-40%. This is correlated with the latter 2 groups having an increased rate of metastasis at diagnosis, as metastatic lesions are more advanced and therapeutically difficult to treat. Therefore, efforts must be made to elucidate mechanisms and treatments of metastasis, as well as finding subgroup specific therapeutic targets. Although canonical cancer therapy counters the tumors' effects on the surrounding environment, manipulating the effects of the tumor microenvironment on the tumor has also shown promise as a treatment regimen. Given that the cerebellum, the initiation site for MB, has high concentrations of GABA, and Group 3 tumors have upregulation of GABAergic properties, we explored GABA's role in this context. For these studies, we interrogated the GABA shunt pathway - the principal pathway for GABA metabolism - in two Group 3 MB cell lines. We utilized lentivirus mediated gene knockdown of GABA Transaminase (ABAT), the initial protein in the GABA shunt, to deduce phenotypic and genotypic changes in these cells. Cells supplemented with GABA had an increased ability to detach and form spheres. These spheres thrived in GABA supplemented floating conditions, while no similar phenomenon was seen in cells cultured in control medium or GABA supplemented ABAT knockdown cells. Genotypic analysis of these cells using RT-qPCR revealed an increase in the epithelial-to-mesenchymal transition profile of GABA supplemented spheres as compared to control conditions. To determine if knocking down ABAT changes tumor seeding capabilities in vivo, we transplanted MB cells into the lateral ventricle of athymic nude mice. Control MBL cells regardless of prior exposure to GABA or floating conditions, were able to seed and form tumors; While ABAT KD cells were unable to do so. These findings provide evidence that GABA metabolism in MB metastatic cells may be imperative for survivability in the CSF and leptomeningeal spread. Citation Format: Vahan Martirosian, Michelle Lin, Thomas C. Chen, Josh Neman. The role of GABA metabolism in medulloblastoma spread [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4144.

Abstract 1330: Role of epigenetic remodeling in sensitizing triple-negative breast cancer cells to treatment through enhanced chemotherapy-induced autophagy

Abstract Dysregulation of the epigenome is implicated in initiation and progression of variety of cancers and their acquired resistance to chemotherapy. As such targeting epigenetic regulators has the potential to modulate cancer cell biology and their sensitivity to chemotherapy and/or radiation. Our studies demonstrates that silencing of the epigenetic regulator Nucleosome Remodeling Factor (NURF) sensitizes breast tumor cells to chemotherapy and enhances the anti-tumor immune response. A screen of a variety of chemotherapeutic agents shows that NURF KD cells are selectively sensitized to Topo II inhibitors, which includes doxorubicin. NURF KD increases DNA damage (gamma H2AX staining) and autophagy (acridine orange staining) in breast tumor cells exposed to doxorubicin (Dox) and enhances growth inhibition as well as suppressing the ability of the cells to recover proliferative capacity. Increased autophagy as well as breast tumor sensitization to doxorubicin were observed using a small molecule inhibitor of NURF, suggesting that NURF can also be targeted pharmacologically. Our studies suggest that enhanced autophagy may be a primary contributor to chemo sensitivity in NURF KD cells. Studies are currently underway to confirm the role of autophagy in chemosensitization in- vitro and enhanced immune response in-vivo using autophagy deficient cells (through ATG silencing). Doxorubicin produces profound DNA damage and promotes immunogenic cell death enhanced by NURF KD. Increased cell autonomous antitumor effects by doxorubicin in concert with increased cell non-autonomous antigenicity could help to achieve tumor regression, reduce metastasis, and possibly promote long term remission in breast cancer. Citation Format: Liliya Tyutyunyk, Joseph Landry, David Gewirtz, Nga Dao. Role of epigenetic remodeling in sensitizing triple-negative breast cancer cells to treatment through enhanced chemotherapy-induced autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1330.

Dynamic localization of α-tubulin acetyltransferase ATAT1 through the cell cycle in human fibroblastic KD cells.

Acetylation of α-tubulin is a well-studied posttranscriptional modification, which is mostly catalyzed by α-tubulin N-acetyltransferase (ATAT1). ATAT1 possibly affects various cellular functions related with microtubules, such as intracellular transport, cell motility, cilia formation, and neuronal signaling. Here, we analyzed the subcellular localization of immunolabeled ATAT1 in human fibroblast KD cells through the cell cycle using confocal laser scanning microscopy. ATAT1 dramatically changed its localization through the cell cycle, depending on the mitotic phase. In interphase, immunolabeled ATAT1 was observed in centrioles, nuclei, and basal bodies if the cells projected primary cilia. ATAT1 was intensely detected as clusters in the nuclei in the G1-G2 phase. In telophase, ATAT1 colocalized with chromatids and spindle poles, and ultimately migrated to the daughter nucleus, newly synthesized centrioles, and midbody. The nucleolus is a core region of ribosomal RNA transcription, and the midbody is associated with severing and depolymerizing of microtubules in the stembody. The specific distributions of ATAT1 through the cell cycle suggest multiple functions of ATAT1, which could include acetylation of microtubules, RNA transcription activity, severing microtubules, and completion of cytokinesis.

Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

Plac1 is an X-linked trophoblast gene expressed at high levels in the placenta, but not in adult somatic tissues other than the testis. Plac1 however is re-expressed in several solid tumors and in most human cancer cell lines. To explore the role of Plac1 in cancer progression, Plac1 was reduced by RNA interference in EO771 mammary carcinoma cells. EO771 “knockdown” (KD) resulted in 50% reduction in proliferation in vitro and impaired tumor growth in syngeneic mice; however, tumor growth in SCID mice was equivalent to tumor cells expressing a non-silencing control RNA, suggesting that Plac1 regulated adaptive immunity. Gene expression profiling of Plac1 KD cells indicated reduction in several inflammatory and immune factors, including Cxcl1, Ccl5, Ly6a/Sca-1, Ly6c and Lif. Treatment of mice engrafted with wild-type EO771 cells with a Cxcr2 antagonist impaired tumor growth, reduced myeloid-derived suppressor cells and regulatory T cells, while increasing macrophages, dendritic cells, NK cells and the penetration of CD8+ T cells into the tumor bed. Cxcl1 KD phenocopied the effects of Plac1 KD on tumor growth, and overexpression of Cxcl1 partially rescued Plac1 KD cells. These results reveal that Plac1 modulates a tolerogenic tumor microenvironment in part by modulating the chemokine axis.

T‐Cell Protein Tyrosine Phosphatase Restricts Intestinal Epithelial Cell Expression of the Oncogene Annexin A4

Phosphotyrosine‐based signaling is regulated by the balanced actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) to control various cellular processes including growth, differentiation, metabolism, and motility. Aberrant activation of signaling pathways by mutations and/or overexpression of PTKs and PTPs can lead to various types of cancers including colon cancer. Evolving evidence indicates that expression levels of PTPs, mainly protein tyrosine phosphatase non‐receptor type‐2 (PTPN2, protein product called T cell protein tyrosine phosphatase ‐TCPTP), PTPN22, and PTPN11 are altered in actively inflamed intestinal tissue. Loss‐of‐function single nucleotide polymorphisms (SNPs) in PTPN2 are associated with inflammatory bowel disease (IBD). The aim of this study was to determine if TCPTP functions as a tumor suppressor in intestinal epithelial cells (IEC). In this study, we used previously validated control shRNA and PTPN2 knockdown (PTPN2‐KD) HT‐29 IECs to determine if TCPTP loss affects oncogene expression. We performed proteomic analysis using mass spectrometry (MS) with Tandem Mass Tags (TMT) on PTPN2‐KD and control IECs. The most striking difference between control and PTPN2‐KD IECs was a 2.9 fold increase in the peptide count of Annexin A4 in PTPN2‐KD cells compared with control cells. Earlier studies indicated that Annexin A4 modulates membrane permeability, membrane trafficking, participates in cellular growth and apoptosis, enhances tumor invasion and promotes anti‐tumor drug resistance. To confirm the MS result, we performed RT‐PCR and Western blotting to determine if PTPN2 deficiency increased Annexin A4 mRNA and protein levels respectively. Both Annexin A4 mRNA and protein levels were significantly increased in PTPN2 KD IECs above control cells (221.5±2.4%, p<0.0001, 202±16.2 p<0.01 respectively). Immunofluorescence studies also revealed that Annexin A4 staining intensity was increased 1.5 fold in PTPN2 KD cells compared to control cells analyzed using ImageJ software. Annexin A4 expression in PTPN2‐KD IECs was further increased by IFN‐g treatment (1000 U/ml; 24 hrs). In summary, TCPTP deficiency leads to elevated expression of the tumor promoter, Annexin A4. These data suggest that loss‐of‐function mutations in the PTPN2 gene may increase pro‐tumorigenic factors that predispose to colorectal cancer.Support or Funding InformationSupported by NIH 2R01DK091281 (DFM)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tofacitinib Inhibits Intestinal Epithelial JAK‐STAT Signaling and Prevents Barrier Dysfunction Induced by Interferon‐Gamma (IFN‐γ)

The orally‐administered Janus kinase (JAK)‐1,3 inhibitor, tofacitinib, is effective as an induction and maintenance therapy for ulcerative colitis (UC). Although tofacitinib promotes mucosal healing and remission in patients with UC, little is known about how it directly affects intestinal epithelial cells (IECs) and barrier function. The UC and Crohn's disease susceptibility gene protein tyrosine phosphatase non‐receptor type 2 (PTPN2) encodes the T cell protein tyrosine phosphatase (TCPTP), which dephosphorylates JAK‐STAT signaling molecules such as JAK1/3, STAT1/3. The aims of this study were to determine: (1) if tofacitinib reduces JAK‐STAT activation in IECs to prevent IFN‐γ‐induced barrier dysfunction; and (2) if tofacitinib corrects the JAK‐STAT signaling defects in IECs lacking TCPTP.MethodsT84 IECs were treated with tofacitinib at a concentration (50 μM) determined in a previous dose‐response study, or vehicle (0.5% DMSO), for 1 hr prior to treatment with IBD‐associated inflammatory cytokine IFN‐γ (1000 U/ml) for 1 hr. Effects of tofacitinib were also determined in HT29 IECs stably expressing control shRNA (CTL) or shRNA targeted against PTPN2 (PTPN2 KD) treated with tofacitinib (50 μM) or vehicle (0.5% DMSO) for 1 hr +/‐subsequent treatment of IFN‐γ (1000 U/ml) for 1 hr. Luciferase reporter assay was conducted in HT29 IECs to assess the claudin 2 gene promoter activity after treatment with tofacitinib (50 μM) or vehicle (0.5% DMSO) 1 hr prior to IFN‐γ (1000 U/ml; 24 hrs) treatment. Tight junction protein expression was analyzed in T84 IECs grown on transwells for 11–12 days followed by tofacitinib treatment as above or with tofacitinib or vehicle 6 hrs and 18 hrs post‐IFN‐γ challenge. Protein expression was determined by Western blotting and densitometry.ResultsTofacitinib pre‐treatment inhibited IFN‐γ‐induced JAK1 (n=4, p<0.05) and STAT1 (n=3, p<0.001) phosphorylation in T84 IECs. Tofacitinib reduced basal and IFN‐γ‐stimulated levels of phospho‐JAK1 (n=3, p<0.05; n=3, p<0.05, respectively) and phospho‐STAT1 (n=4, p<0.05; n=3, p<0.001, respectively) in HT29 PTPN2 KD cells to levels comparable to untreated or vehicle‐treated HT29 CTL cells. We previously showed that tofacitinib prevented decreases in TER caused by IFN‐γ (n=3, p<0.001), and that IFN‐γ increases claudin‐2 expression by JAK‐STAT activation. Here, we observed that tofacitinib pre‐treatment inhibited IFN‐γ induction of claudin 2 promoter activity (n=4, p<0.001) and protein expression (n=5, p<0.05). Tofacitinib also partially decreased claudin 2 protein levels after IFN‐γ induction (n=3, p<0.05). These data suggest that tofacitinib directly inhibits the JAK‐STAT signaling pathway in IECs to restrict intestinal barrier defects induced by IFN‐γ through claudin 2 regulation. This indicates the therapeutic potential of using tofacitinib to correct the consequences of PTPN2 genetic defects as a personalized medicine approach for patients harboring TCPTP loss‐of‐function mutations.Support or Funding InformationNIH 2R01DK091281 (DFM)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The siRNA silencing of DcR3 expression induces Fas ligand-mediated apoptosis in HepG2 cells.

Dysfunctional Fas ligand (FasL) may inhibit the apoptosis of tumor cells. FasL contains two receptors, Fas and Decoy Receptor 3 (DcR3). DcR3 competitively binds to FasL over Fas, resulting in the inhibition of FasL-mediated apoptosis. Therefore, it was suggested that the downregulation of DcR3 expression enhances FasL-mediated apoptosis. In the current study, the expression of DcR3 was silenced in liver cancer HepG2 cells in order to study the effect of FasL on HepG2 cell activity and invasiveness. DcR3 siRNA knockdown HepG2 cells (KD), DcR3 blank plasmid control HepG2 cells and wild-type HepG2 cells (WT) were treated with FasL (10 ng/ml). Flow cytometry was used to detect changes in the cell cycle and apoptosis. MTS, clonogenic, wound healing and Transwell assays were performed to examine changes in cell activity, proliferation, migration and invasiveness. Reverse transcription polymerase chain reaction and western blot analysis were performed to measure the expression of DcR3, matrix metallopeptidase 9 (MMP9), vascular endothelial growth factor (VEGF)-C and VEGF-D. The results demonstrated that, compared with WT cells, the proportion of KD cells in the G2/M phase decreased following treatment with FasL. KD cells were more sensitive to FasL-induced apoptosis. Following treatment with FasL, the activity and proliferation, migration and invasion of KD cells were reduced, and the expression of MMP9, VEGF-C and VEGF-D decreased. Furthermore, it was demonstrated that DcR3 is involved in the proliferation and invasion of HepG2 cells, and this mechanism may be associated with the regulatory effect of the expression of MMP9, VEGF-C and VEGF-D; however, the exact mechanism of action remains unclear. FollowingDcR3 silencing, FasL-mediated apoptosis increased in HepG2 cells. Therefore, DcR3 combined with FasL may be a potential target for the treatment of liver cancer.

Silencing of long isoforms of nuclear factor erythroid 2 like 1 primes macrophages towards M1 polarization.

Macrophages are a major component of the immune system and play an important role in regulating the magnitude, duration, and quality of the inflammatory response. Dissecting the functions of transcription factors regulating macrophage activation is important for understanding the inflammatory responses. Nuclear factor erythroid 2 like 1 (NFE2L1, also known as Nrf1) is a CNC-bZIP protein, which has multiple isoforms. While the exact physiological functions of various isoforms of NFE2L1 are still under investigation, accumulating evidence indicate that long isoforms of NFE2L1 (NFE2L1(L)) are important regulators in the antioxidant response, proteasome homeostasis and inflammation. In this study, we found that NFE2L1(L) was upregulated in response to LPS stimulation in RAW264.7 macrophages. Stable knockdown of Nfe2l1(L) (Nfe2l1(L)-KD) in RAW264.7 cells resulted in increased expression of multiple genes indicative of M1 polarization, including Il6, Il1β, Cox2, and Ccl2, under both resting and LPS-challenged conditions. In addition, lentiviral shRNA-mediated silencing of NFE2L1(L) in human monocytic SC and THP1 cells also significantly increased mRNA expression of IL6, IL1β, and TNFα. Furthermore, transient silence of NFE2L1(L) in primary human monocytes isolated from peripheral blood by nucleofection with small interfering RNA resulted in increased expression of IL6 and TNFα. Analysis of the key transcription factors involved in M1 polarization revealed that Nfe2l1(L)-KD RAW264.7 cells have increased mRNA and protein expression and phosphorylation of STAT1 and STAT3 under both resting and M1 polarized conditions. Activation of the NFκB, ERK1/2 and p38 pathways in response to LPS was not affected by the reduction of NFE2L1(L). Moreover, Nfe2l1(L)-KD cells were found to have elevated levels of intracellular ROS, but macrophage M1 polarization induced by Nfe2l1(L) silence was independent of ROS accumulation. Collectively, our results show that knockdown of Nfe2l1(L) leads macrophages to M1 polarization by disinhibition of STAT1/3, and not through the NFκB, ERK1/2 and/or p38 signaling pathways. These findings indicate that NFE2L1(L) functions as a negative regulator of M1 polarization and pro-inflammatory response in macrophages.

Perturbations of NAD+ salvage systems impact mitochondrial function and energy homeostasis in mouse myoblasts and intact skeletal muscle.

Nicotinamide adenine dinucleotide (NAD+) can be synthesized by nicotinamide phosphoribosyltransferase (NAMPT). We aimed to determine the role of NAMPT in maintaining NAD+ levels, mitochondrial function, and metabolic homeostasis in skeletal muscle cells. We generated stable Nampt knockdown (sh Nampt KD) C2C12 cells using a shRNA lentiviral approach. Moreover, we applied gene electrotransfer to express Cre recombinase in tibialis anterior muscle of floxed Nampt mice. In sh Nampt KD C2C12 myoblasts, Nampt and NAD+ levels were reduced by 70% and 50%, respectively, and maximal respiratory capacity was reduced by 25%. Moreover, anaerobic glycolytic flux increased by 55%, and 2-deoxyglucose uptake increased by 25% in sh Nampt KD cells. Treatment with the NAD+ precursor nicotinamide riboside restored NAD+ levels in sh Nampt cells and increased maximal respiratory capacity by 18% and 32% in control and sh Nampt KD cells, respectively. Expression of Cre recombinase in muscle of floxed Nampt mice reduced NAMPT and NAD+ levels by 38% and 43%, respectively. Glucose uptake increased by 40%, and mitochondrial complex IV respiration was compromised by 20%. Hypoxia-inducible factor (HIF)-1α-regulated genes and histone H3 lysine 9 (H3K9) acetylation, a known sirtuin 6 (SIRT6) target, were increased in shNampt KD cells. Thus, we propose that the shift toward glycolytic metabolism observed, at least in part, is mediated by the SIRT6/HIF1α axis. Our findings suggest that NAMPT plays a key role for maintaining NAD+ levels in skeletal muscle and that NAMPT deficiency compromises oxidative phosphorylation capacity and alters energy homeostasis in this tissue.

BIGH3 Promotes Osteolytic Lesions in Renal Cell Carcinoma Bone Metastasis by Inhibiting Osteoblast Differentiation

BACKGROUND: Bone metastasis is common in renal cell carcinoma (RCC), and the lesions are mainly osteolytic. The mechanism of bone destruction in RCC bone metastasis is unknown. METHODS: We used a direct intrafemur injection of mice with bone-derived 786-O RCC cells (Bo-786) as an in vivo model to study if inhibition of osteoblast differentiation is involved in osteolytic bone lesions in RCC bone metastasis. RESULTS: We showed that bone-derived Bo-786 cells induced osteolytic bone lesions in the femur of mice. We examined the effect of conditioned medium of Bo-786 cells (Bo-786 CM) on both primary mouse osteoblasts and MC3T3-E1 preosteoblasts and found that Bo-786 CM inhibited osteoblast differentiation. Secretome analysis of Bo-786 CM revealed that BIGH3 (Beta ig h3 protein), also known as TGFBI (transforming growth factor beta-induced protein), is highly expressed. We generated recombinant BIGH3 and found that BIGH3 inhibited osteoblast differentiation in vitro. In addition, CM from Bo-786 BIGH3 knockdown cells (786-BIGH3 KD) reduced the inhibition of osteoblast differentiation compared to CM from vector control. Intrafemural injection of mice with 786-BIGH3 KD cells showed a reduction in osteolytic bone lesions compared to vector control. Immunohistochemical staining of 18 bone metastasis specimens from human RCC showed strong BIGH3 expression in 11/18 (61%) and moderate BIGH3 expression in 7/18 (39%) of the specimens. CONCLUSIONS: These results suggest that suppression of osteoblast differentiation by BIGH3 is one of the mechanisms that enhance osteolytic lesions in RCC bone metastasis, and raise the possibilty that treatments that increase bone formation may improve therapy outcomes.

Down regulation of Peroxiredoxin-3 in 3T3-L1 adipocytes leads to oxidation of Rictor in the mammalian-target of rapamycin complex 2 (mTORC2)

Mitochondrially-derived oxidative stress has been implicated in the development of obesity-induced insulin resistance and is correlated with down regulation of Peroxiredoxin-3 (Prdx3). Prdx3 knockout mice exhibit whole-body insulin resistance, while Prdx3 transgenic animals remain insulin sensitive when placed on a high fat diet. To define the molecular events linking mitochondrial oxidative stress to insulin action, Prdx3 was silenced in 3T3-L1 adipocytes (Prdx3 KD) and the resultant cells evaluated for mitochondrial function, endoplasmic reticulum stress (ER stress), mitochondrial unfolded protein response (mtUPR) and insulin signaling. Prdx3 KD cells exhibit a two-fold increase in H2O2, reduced insulin-stimulated glucose transport and attenuated S473 phosphorylation of the mTORC2 substrate, Akt. Importantly, the decrease in glucose uptake can be rescued by pre-treatment with the antioxidant N-acetyl-cysteine (NAC). The changes in insulin sensitivity occur independently from activation of the ER stress or mtUPR pathways. Analysis of mTORC2, the complex responsible for phosphorylating Akt at S473, reveals increased cysteine oxidation of Rictor in Prdx3 KD cells that can be rescued with NAC. Taken together, these data suggest mitochondrial dysfunction in adipocytes may attenuate insulin signaling via oxidation of the mammalian-target of rapamycin complex 2 (mTORC2).

ELAS1 induces apoptotic death in adenocarcinoma DU145 and squamous-cell carcinoma SAS cancer cells, but not in normal KD cells.

We previously reported that an ELAS1 peptide containing 29 amino acids induces apoptotic death in U2OS human osteosarcoma cells following DNA double-strand break insults. Here, we show that ELAS1 also caused apoptosis in prostate adenocarcinoma DU145 cells and tongue squamous-cell carcinoma SAS cells. ELAS1 appears to be safe because it induced apoptosis only in cancer cells, not in normal KD cells. Because the effect of ELAS1 is dependent on increased stability of p53 and enhanced phosphorylation of p53-S46, we exogenously expressed wild-type p53 protein to fully promote ELAS1-mediated induction of apoptosis in SAS cells. Interestingly, simultaneous expression of Myc-ELAS1 and FLAG-p53 mediated by an internal ribosome entry site efficiently induced apoptosis in SAS cells. Moreover, we prepared a recombinant adenovirus that simultaneously expressed Myc-ELAS1 and FLAG-p53. This adenovirus also killed SAS cells, as determined by a cell viability assay, in the presence of camptothecin, an inducer of DNA double-strand breaks. Moreover, nude mice harboring Myc-ELAS1-expressing SAS cells lived longer than mice harboring Myc-vector-expressing SAS cells, suggesting the usefulness of ELAS1 in vivo. Notably, Cy5-tagged ELAS1-t, which contained only ten amino acids, also efficiently induced apoptosis in both DU145 and SAS cells, suggesting the usefulness of ELAS1-t as a peptide. Taken together, our results suggest that ELAS1 is therapeutically useful as a peptide drug.

TRIM28 epigenetic corepressor is indispensable for stable induced pluripotent stem cell formation

Cellular reprogramming proceeds in a stepwise pathway initiated by binding and transcription of pluripotency factors followed by genome-wide epigenetic changes. Priming events, such as erasure of DNA methylation and chromatin remodeling determines the success of pluripotency acquisition later. Therefore, growing efforts are made to understand epigenetic regulatory network that makes reprogramming possible and efficient. Here, we analyze the role of transcriptional corepressor TRIM28, involved in heterochromatin formation, during the process of reprogramming of mouse somatic cells into induced pluripotent stem cells (iPS cells). We demonstrate that Trim28 knockdown (Trim28 KD) causes that emerging iPS cells differentiate immediately back into MEFs therefore they fail to yield stable iPS cell colonies. To better comprehend the mechanism of TRIM28 action in reprogramming, we performed a reverse-phase protein array (RPPA) using in excess of 300 different antibodies and compared the proteomic profiles of wild-type and Trim28 KD cells during reprogramming. We revealed the differences in the dynamics of reprogramming of wild-type and Trim28 KD cells. Interestingly, proteomic profile of Trim28 KD cells at the final stage of reprogramming resembled differentiated state rather than maintenance of pluripotency and self-renewal, strongly suggesting spontaneous differentiation of Trim28 KD cells back to their parental cell type. We also observed that action of TRIM28 in reprogramming is accompanied by differential enrichment of proteins involved in cell cycle, adhesion and stemness. Collectively, these results suggest that regulation of epigenetic modifications coordinated by TRIM28 plays a crucial role in reprogramming process.

Abstract 3314: Synergistic effects of chemotherapy-induced autophagy and epigenetic remodeling

Abstract Cytotoxic chemotherapy has been shown to enhance tumor cell immunogenicity by promoting the secretion of damage-associated molecular pattern (DAMP) molecules which, in turn, stimulates the immune response. These effects can be further stimulated by combining chemotherapy with an immunotherapy designed to enhance effector cell (CD8 T cell or NK cell) anti-tumor activity. Our studies demonstrate that knockdown of the epigenetic regulator NURF increases DNA damage (gamma H2AX staining) and autophagy (acridine orange staining) in breast tumor cells exposed to doxorubicin and enhances growth inhibition as well as suppressing the capacity of the cells to recover and proliferate. Similar increases in autophagy were observed using a small molecule inhibitor of NURF, suggesting that NURF can be targeted therapeutically. Sensitization was not observed with ionizing radiation or cisplatin. Studies are in progress to assess the nature of autophagy in the NURF KD and NURF WT cells, distinguishing between the cytoprotective, cytotoxic, cytostatic and nonprotective forms. How NURF regulates doxorubicin induced DNA damage is being investigated by mapping sites of damage genome wide. It is anticipated that enhanced cell autonomous tumor cell sensitivity in concert with improvements in tumor cell antigenicity (cell non-autonomous sensitization) achieved by NURF depletion could improve anti-tumor immunogenicity, achieve tumor regression, reduce metastasis, and possibly achieve long term remission in breast cancer. Citation Format: Liliya Tyutyunyk, Joseph Landry, Tareq Saleh, David Gewirtz. Synergistic effects of chemotherapy-induced autophagy and epigenetic remodeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3314. doi:10.1158/1538-7445.AM2017-3314

Abstract 3521: Non-canonical activation of AR signaling by histone deacetylase SIRT1

Abstract SIRT1, a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, is a member of multigene family of Sirtuins. Traditionally SIRT1 is a known player in enhancing longevity. SIRT1 can target both histone and non-histone proteins to regulate diverse activities including cellular stress resistance, genomic stability, energy metabolism and tumorigenesis. Disruption of SIRT1 in the prostate results in the formation of prostatic intraepithelial neoplasia (PIN) lesions, suggesting a role for SIRT1 in the development of PIN, a precursor lesion for prostate cancer (PCA). Paradoxically, an oncogenic role for SIRT1 was reported as evidenced by development of micro-invasive prostate carcinomas in Pten+/-/Sirt1tg mice. Therefore, SIRT1 may possess both oncogenic and tumor suppressor activities in a context-dependent manner and its role in human cancer remains controversial. To address these paradoxical findings, we examined the role of SIRT1 in prostate pathogenesis using stably silenced for SIRT1 prostate cancer cell lines. Silencing SIRT1 in androgen responsive LNCaP cells resulted in (i) morphological changes indicative of clustering and epithelial mesenchymal transition (EMT); (ii) significant reduction in their ability to form colonies on soft agar; and (iii) increased autophagic flux. Similarly, silencing SIRT1 resulted in reduced proliferative, colony forming and migratory ability of androgen independent PC-3 cells. Furthermore, notably, genes involved in nuclear receptor (PXR/RXR/FXR/PPAR) and kinase (AMPK, MSP-RON, and RhoA) signaling are significantly altered as evidenced by RNA-seq analysis in these cells. Remarkably, SIRT1 KD cells showed decreased expression of AR and its target genes including PSA, TMPRSS-2, FKBP-5 and PMEPA1. Interestingly, SIRT1 KD reduced PSA-luciferase activity under both normal physiological and androgen deprivation growth conditions with no significant effect on nuclear or cytosolic levels of AR. More importantly, SIRT1 KD cells showed increased sensitivity to growth under androgen deprivation conditions. Intervention with resveratrol (RES, known SIRT1 activator) reduced incidence of HGPIN in prostate specific PTEN KO mouse model with no significant differences in the expression of AR or SIRT1. Consistent with these in vivo observations, SIRT1 had no significant impact on RES-induced growth inhibition. Taken together, these data indicate that SIRT1 could potentially contribute to androgen independence, independent of classical AR signaling. Our study demonstrates that SIRT1 may not play an essential role in mediating RES-induced tumor growth inhibition and that SIRT1 may confer resistance to androgen deprivation therapy and sustains cell survival, suggesting that combination of SIRT1 inhibitors and ADT might be a potential strategy for advanced PCA treatment. Supported by CPRIT RP 150166 and NCI CA 137518 (APK). Citation Format: Shih-Bo Huang, Dinesh Thapa, Suleman S. Hussain, Roble G. Bedolla, Guiming Li, Robert L. Reddick, Zhao Lai, Hung-I H. Chen, Yidong Chen, Rita Ghosh, Addanki P. Kumar. Non-canonical activation of AR signaling by histone deacetylase SIRT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3521. doi:10.1158/1538-7445.AM2017-3521

Deficiency of long isoforms of Nfe2l1 sensitizes MIN6 pancreatic β cells to arsenite-induced cytotoxicity

Increasing evidence indicates that chronic inorganic arsenic exposure is associated with type 2 diabetes (T2D), a disease of growing prevalence. Pancreatic β-cells were targeted and damaged by oxidative stress induced by arsenite. We previously showed that nuclear factor erythroid 2 like 2 (Nfe2l2)-deficient pancreatic β-cells were vulnerable to cell damage induced by oxidative stressors including arsenite, due to a muted antioxidant response. Like nuclear factor erythroid 2 like 2 (NFE2L2), NFE2L1 also belongs to the cap ‘n’ collar (CNC) basic-region leucine zipper (bZIP) transcription factor family, and regulates antioxidant response element (ARE) related genes. Our prior work showed NFE2L1 regulates glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells and isolated islets. In the current study, we demonstrated that MIN6 cells with a specific knockdown of long isoforms of Nfe2l1 (L-Nfe2l1) by lentiviral shRNA (Nfe2l1(L)-KD) were vulnerable to arsenite-induced apoptosis and cell damage. The expression levels of antioxidant genes, such as Gclc, Gclm and Ho-1, and intracellular reactive oxygen species (ROS) levels were not different in Scramble and Nfe2l1(L)-KD cells, while the expression of arsenic metabolism related-genes, such as Gsto1, Gstm1 and Nqo1, increased in Nfe2l1(L)-KD cells with or without arsenite treatment. The up-regulation of arsenic biotransformation genes was due to activated NFE2L2 in Nfe2l1(L)-KD MIN6 cells. Furthermore, the level of intracellular monomethylarsenic (MMA) was higher in Nfe2l1(L)-KD MIN6 cells than in Scramble cells. These results showed that deficiency of L-Nfe2l1 in pancreatic β-cells increased susceptibility to acute arsenite-induced cytotoxicity by promoting arsenic biotransformation and intracellular MMA levels.

ZNF131 suppresses centrosome fragmentation in glioblastoma stem-like cells through regulation of HAUS5.

Zinc finger domain genes comprise ∼3% of the human genome, yet many of their functions remain unknown. Here we investigated roles for the vertebrate-specific BTB domain zinc finger gene ZNF131 in the context of human brain tumors. We report that ZNF131 is broadly required for Glioblastoma stem-like cell (GSC) viability, but dispensable for neural progenitor cell (NPC) viability. Examination of gene expression changes after ZNF131 knockdown (kd) revealed that ZNF131 activity notably promotes expression of Joubert Syndrome ciliopathy genes, including KIF7, NPHP1, and TMEM237, as well as HAUS5, a component of Augmin/HAUS complex that facilitates microtubule nucleation along the mitotic spindle. Of these genes only kd of HAUS5 displayed GSC-specific viability loss. Critically, HAUS5 ectopic expression was sufficient to suppress viability defects of ZNF131 kd cells. Moreover, ZNF131 and HAUS5 kd phenocopied each other in GSCs, each causing: mitotic arrest, centrosome fragmentation, loss of Augmin/HAUS complex on the mitotic spindle, and loss of GSC self-renewal and tumor formation capacity. In control NPCs, we observed centrosome fragmentation and lethality only when HAUS5 kd was combined with kd of HAUS2 or HAUS4, demonstrating that the complex is essential in NPCs, but that GSCs have heightened requirement. Our results suggest that GSCs differentially rely on ZNF131-dependent expression of HAUS5 as well as the Augmin/HAUS complex activity to maintain the integrity of centrosome function and viability.

Intestinal Epithelial TCPTP Deficiency Reduces Expression of the Tight Junction Protein Tricellulin through MEK/ERK Signaling

Genome‐wide association studies identified protein tyrosine phosphatase non‐receptor type‐2 (PTPN2) as a susceptibility gene for inflammatory bowel disease (IBD), Diabetes and Celiac disease, while loss of the PTPN2 protein product T‐cell protein tyrosine phosphatase (TCPTP) compromises intestinal epithelial barrier function. The protein composition of tight junctions (TJ) determines the tightness of the barrier and alterations in select TJ proteins leads to increased paracellular permeability. Tricellulin (also called marvelD2) is a recently discovered tight junction (TJ) protein, which is preferentially localized at tricellular contacts. Tricellulin plays an important role in the maintenance of intestinal epithelial barrier function by restricting paracellular permeability to macromolecules. The aim of the present study was to identify the molecular mechanism underlying barrier defects in TCPTP‐deficient intestinal epithelial cells (IEC). For this purpose, we developed Caco2‐BBe and HT‐29 intestinal epithelial cell lines (IECs) expressing short hairpin RNA for TCPTP using a lentiviral system to stably knock down TCPTP expression (TCPTP‐KD). Western blot analysis indicated that TCPTP expression was significantly reduced (by 45 ± 4%, p<0.01, n=3) in TCPTP‐KD cells. FD4 permeability was increased in KD cells (p<0.001; n=3) compared to control cells. Western blot and immunofluorescence studies revealed that the expression of tricellulin expression was decreased (WB: 44 ± 8.8%, p<0.05; n=3) in unchallenged TCPTP‐KD cells compared to control shRNA transfected cells. This decrease was further enhanced by challenge with the pro‐inflammatory cytokine interferon‐gamma (IFN‐g) (n=2). In previous studies, we observed increased basal phosphorylation of the TCPTP substrate ERK1/2 in TCPTP‐KD cells. Moreover, an inverse relationship exists between mitogen‐activated protein kinase kinase kinase 1 (MEKK1) that lies upstream of ERK1/2, and MarvelD3, a member of the same protein family as tricellulin. To investigate whether tricellulin loss in TCPTP‐KD cells occurs through MEK/ERK signaling, TCPTP‐KD IEC were treated with the ERK1/2 inhibitor U0126 (10mM) for 14 hours. ERK inhibition rescued tricellulin expression in TCPTP‐KD cells. In conclusion, we report that tricellulin expression is down regulated, at least in part, by the MEK/ERK pathway and that decreased tricellulin levels could contribute to the increased macromolecule permeability in cells lacking TCPTP.Support or Funding InformationNIH‐R01‐DK‐091281 and Crohn's & Colitis Foundation of America(CCFA) to Declan F. McCole

The role of ELK3 to regulate peritumoral lymphangiogenesis and VEGF-C production in triple negative breast cancer cells

Tumor-induced lymphangiogenesis, a major conduit for cancer cell dissemination from the primary tumor site to lymph nodes and beyond, eventually leads to metastasis in cancer patients. Given the recent evidence revealing that the suppression of ELK3 inhibits the metastasis of triple-negative breast cancer cells, we aimed to study the underlying mechanism of impaired metastasis in ELK3-suppressed MDA-MB-231 cells (ELK3 KD) with regard to lymphangiogenesis. We found that the secretome of ELK3 KD cells inhibited tube formation, whereas it promoted the migration and invasion of lymphatic endothelial cells (LECs) in vitro. In vivo analysis revealed that peritumoral lymphatic vessels were not developed around the xenografted tumors of ELK3 KD. We further revealed that the suppression of NF-κB signaling in ELK3 KD was the primary cause of the reduced VEGFC expression. Taken together, we suggest that ELK3 is an upstream regulator of the NF-κB signaling pathway, the inhibition of which leads to the suppression of peritumoral lymphatic vessel development, possibly due to a low VEGFC expression.