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Mitigation of IL-17 response to ETBF in absence of Tregs is not restricted to CD4+ (Th17) cells.. B6.Foxp3DTR mice were inoculated with ETBF. Either 150 μl purified sterile H2O or 50 ng/g DT was administered ip on days -2, -1, 1, 3, & 5. Colonic LPLs from each mouse were harvested on day 7. Cells were stimulated ex vivo, followed by ICS. Dot plots show viable LPLs from 1 mouse per group and are representative of 2 experiments.

PDF file - 7618K, S1. Analysis of the genetic interaction between 53BP1 and ATM in organismal growth and development. S2. Analysis of ploidy in Trp53bp1-/-/Atm-/- lymphomas. S3. Analysis of Trp53bp1-/-/Atm-/- thymic lymphomas by TCRα/δ locus FISH. S4. SKY analysis of two Trp53bp1-/-/Atm-/- lymphomas. S5. Cell cycle analysis of resting lymphocytes after IR. S6. Aberrant Vd2-Dd1/Dd2-Jd1 coding junctions are not detected in Trp53bp1-/-/Atm-/- mice. S7. Aberrant Vd5-Dd2 Recombination Signal Junctions are not detected in Trp53bp1-/-/Atm-/- mice. S8. Histograms showing the frequency distribution of deletions from each coding end (A-D) and signal end (E-F) analyzed. S9. Cell cycle analysis of activated (cycling) lymphocytes after IR. S10. Analysis of genomic stability in splenic B or T cells treated with olaparib. S11. Analysis of the G2/M checkpoint. α-CD40/IL-4-activated B cells were harvested one hour after exposure to 2 Gy of IR, stained with a FITC-labeled antibody to phospho(P)-histone H3 (Ser10) and propidium iodide (PI) and analyzed by flow cytometry. S12. A second primer set detects deletions at hybrid V(D)J recombination junctions in Trp53bp1-/-/Atm-/- thymic DNA from 7 day-old mice. Table S1. Mendelian ratios in liveborn mice from 53BP1+/-/ATM+/- intercrosses (n=19 litters) Table S2. Analysis of genomic stability in HU-treated B lymphocytes deficient for 53BP1 and/or ATM. Table S3. Analysis of genomic stability in α-CD40+IL-4activated B lymphocytes deficient for 53BP1 and/or ATM. Table S4. Analysis of genomic stability in α-CD40/IL-4 activated B cells deficient for 53BP1 and/or ATM. Metaphase spreads were obtained 24 hr after IR and stained with a telomere probe. Table S5. Analysis of genomic stability in LPS-activated B cells deficient for 53BP1 and/or ATM. Metaphases were obtained 24 hr after exposure to IR and stained with a telomere probe. Table S6. Analysis of genomic stability in T lymphocytes deficient for 53BP1 and/or ATM.

The DNA damage response (DDR) factors ataxia telangiectasia mutated (ATM) and p53 binding protein 1 (53BP1) function as tumor suppressors in humans and mice, but the significance of their mutual interaction to the suppression of oncogenic translocations in vivo has not been investigated. To address this question, the phenotypes of compound mutant mice lacking 53BP1 and ATM (Trp53bp1−/−/Atm−/−), relative to single mutants, were examined. These analyses revealed that loss of 53BP1 markedly decreased the latency of T-lineage lymphomas driven by RAG-dependent oncogenic translocations in Atm−/− mice (average survival, 14 and 23 weeks for Trp53bp1−/−/Atm−/− and Atm−/− mice, respectively). Mechanistically, 53BP1 deficiency aggravated the deleterious effect of ATM deficiency on nonhomologous end-joining (NHEJ)—mediated double-strand break repair. Analysis of V(D)J recombinase-mediated coding joints and signal joints in Trp53bp1−/−/Atm−/− primary thymocytes is, however, consistent with canonical NHEJ-mediated repair. Together, these findings indicate that the greater NHEJ defect in the double mutant mice resulted from decreased efficiency of rejoining rather than switching to an alternative NHEJ-mediated repair mechanism. Complementary analyses of irradiated primary cells indicated that defects in cell-cycle checkpoints subsequently function to amplify the NHEJ defect, resulting in more frequent chromosomal breaks and translocations in double mutant cells throughout the cell cycle. Finally, it was determined that 53BP1 is dispensable for the formation of RAG-mediated hybrid joints in Atm−/− thymocytes but is required to suppress large deletions in a subset of hybrid joints.Implications: The current study uncovers novel ATM-independent functions for 53BP1 in the suppression of oncogenic translocations and in radioprotection.Visual Overview: http://mcr.aacrjournals.org/content/11/10/1223/F1.large.jpg.Mol Cancer Res; 11(10); 1223–34. ©2013 AACR.

IL-17+ Foxp3+ Tregs infiltrate ETBF-infected colons. Min mice were inoculated with ETBF on day 0. At 10 weeks, colonic LPLs were harvested from colon tumors (left) and adjacent non-tumor colon tissue (right). Cells were stimulated ex vivo, followed by ICS. Dot plots show viable CD4+ LPLs. Percentage of IL-17+ within Foxp3+ gate is indicated.

Uterine cancer is a common cause for cancer death in women and there is no effective therapy for metastatic disease. Thus, research is urgently needed to identify new therapeutic agents. We showed previously that all female HMGA1a transgenic mice develop malignant uterine tumors, indicating that HMGA1a causes uterine cancer in vivo. We also demonstrated that HMGA1a up-regulates cyclooxygenase-2 (COX-2) during tumorigenesis in this model. Similarly, we found that HMGA1a and COX-2 are overexpressed in human leiomyosarcomas, a highly malignant uterine cancer. Although epidemiologic studies indicate that individuals who take COX inhibitors have a lower incidence of some tumors, these inhibitors have not been evaluated in uterine cancer. Here, we show that HMGA1a mice on sulindac (a COX-1/COX-2 inhibitor) have significantly smaller uterine tumors than controls. To determine if COX inhibitors are active in human uterine cancers that overexpress HMGA1a, we treated cultured cells with sulindac sulfide or celecoxib (a specific COX-2 inhibitor). Both drugs block anchorage-independent growth in high-grade human uterine cancer cells that overexpress HMGA1a (MES-SA cells). In contrast, neither inhibitor blocked transformation in cells that do not overexpress HMGA1a. Moreover, xenograft tumors from MES-SA cells were significantly inhibited in mice on sulindac. More strikingly, no tumors formed in mice on celecoxib. These preclinical studies suggest that COX inhibitors could play a role in preventing tumor onset or progression in uterine cancers with dysregulation of the HMGA1a-COX-2 pathway. Importantly, these drugs have lower toxicity than chemotherapeutic agents used to treat advanced-stage uterine cancers. [Mol Cancer Ther 2008;7(7):2090–5]

Supplementary Fig from Cyclooxygenase inhibitors block uterine tumorigenesis in HMGA1a transgenic mice and human xenografts

Supplementary Figure Legends. Legend for Supplementary Figures S1-S10.

Supplementary Table S4. Histology scores for CD4Stat3-/- and WT (CD4Stat3+/+) C57BL/6 mice colonized 7 days with ETBF.

Supplemental Materials and Methods and Supplemental References

Supplementary Table S1. List of antibody clones used for flow cytometry analysis.

Supplementary Figure S1. IL-17-producing cells in C57BL/6 WT mice 7 days after ETBF colonization. Supplementary Figure S2. CD3+ cell recovery from distal colon of WT (CD4Stat3+/+), and CD4Stat3-/- C57BL/6 mice one week after ETBF colonization. Supplementary Figure S3. pStat3 scores in immune cells (IC) and colon epithelial cells do not differ between C57BL/6-CD4Stat3-/- and C57BL/6-CD4Stat3+/+ 3 and 7 days after ETBF colonization . Supplementary Figure S4. Gene expression analysis in distal colon tissue collected from WT (CD4Stat3+/+) (, CD4Stat3-/- and γδ-- - CD4Stat3-/- C57BL/6 mice 7 days after ETBF colonization. Supplementary Figure S5. Difference in IL-17 expression by CD4+ T cells (Th17), γδ+ T cells (γδ T17) and non Th17, non γδT17 cells in the distal colon of WT (CD4Stat3+/+) C57BL/6 mice 7 days after ETBF colonization. Supplementary Figure S6. Macroscopic tumor size distribution in 3 month-ETBF colonized parental Min (Min-CD4Stat3+/+) versus Min-CD4Stat3-/- mice. Supplementary Figure S7. Parental (CD4Stat3+/+) and CD4Stat3-/- Min mice were treated with anti-IL17 mAb between week 6 and 12 following ETBF colonization. Supplementary Figure S8. Myeloid cells accumulation (CD11b+) in colon tumors compared to whole distal colons (tumor and normal tissues) of 3 month ETBF-colonized Min mice. Supplementary Figure S9. Innate lymphoid cells (ILC) reconstitute the lamina propria of BM chimera mice.

Supplementary Figures 1-3 from The High-Mobility Group A1 Gene Up-Regulates Cyclooxygenase 2 Expression in Uterine Tumorigenesis

Supplementary Methods. Description of additional methods and procedures used in the study.

Supplementary Figures S1-S10. Tests for specificity of the anti-HEYL antibody (S1); Notch pathway induces HEYL expression (S2); In vitro analysis of direct interaction of HEYL and Smad3 in Cos 1 cells (S3); Transcriptional control of TGF-ß responsive reporter consisting of tandem synthetic Smad-binding elements by luciferase assays (S4); Hematoxylin and eosin (H&E) staining of mammary glands of wild-type or transgenic HEYL+ virgin mice, 13 weeks of age (S5); Mammary gland H&E staining of wild-type or transgenic mice at day 10 of pregnancy (S6); Mammary gland hyperplasia was commonly observed in HEYL+ transgenic mice, Representative photomicrographs from mouse numbers, 1-4 are shown (S7); Expression of HeyL in mammary tumors from two HEYL+ transgenic mice measured by RT-PCR and IHC staining (S8); Expression of downstream effectors of TGFß, PAI-1 and P15 in HEYL+ transgenic mammary tumors in comparison to normal mammary glands of WT mice (S9); The antagonistic network between Notch and TGF-ß signaling through the interactions between HEY genes and Smads (S10).

Efforts to induce the differentiation of cancer stem cells through treatment with all-trans retinoic acid (ATRA) have yielded limited success, partially due to the epigenetic silencing of the retinoic acid receptor (RAR)-β. The histone deacetylase inhibitor entinostat is emerging as a promising antitumor agent when added to the standard-of-care treatment for breast cancer. However, the combination of epigenetic, cellular differentiation, and chemotherapeutic approaches against triple-negative breast cancer (TNBC) has not been investigated. In this study, we found that combined treatment of TNBC xenografts with entinostat, ATRA, and doxorubicin (EAD) resulted in significant tumor regression and restoration of epigenetically silenced RAR-β expression. Entinostat and doxorubicin treatment inhibited topoisomerase II-β (TopoII-β) and relieved TopoII-β-mediated transcriptional silencing of RAR-β. Notably, EAD was the most effective combination in inducing differentiation of breast tumor–initiating cells in vivo. Furthermore, gene expression analysis revealed that the epithelium-specific ETS transcription factor-1 (ESE-1 or ELF3), known to regulate proliferation and differentiation, enhanced cell differentiation in response to EAD triple therapy. Finally, we demonstrate that patient-derived metastatic cells also responded to treatment with EAD. Collectively, our findings strongly suggest that entinostat potentiates doxorubicin-mediated cytotoxicity and retinoid-driven differentiation to achieve significant tumor regression in TNBC. Cancer Res; 76(7); 2013–24. ©2016 AACR.

Supplementary Table S3. Demographic and pathologic characteristics of the Malaysian CRC patients.

IL17-producing Th17 cells, generated through a STAT3-dependent mechanism, have been shown to promote carcinogenesis in many systems, including microbe-driven colon cancer. Additional sources of IL17, such as γδ T cells, become available under inflammatory conditions, but their contributions to cancer development are unclear. In this study, we modeled Th17-driven colon tumorigenesis by colonizing MinApc+/− mice with the human gut bacterium, enterotoxigenic Bacteroides fragilis (ETBF), to investigate the link between inflammation and colorectal cancer. We found that ablating Th17 cells by knocking out Stat3 in CD4+ T cells delayed tumorigenesis, but failed to suppress the eventual formation of colonic tumors. However, IL17 blockade significantly attenuated tumor formation, indicating a critical requirement for IL17 in tumorigenesis, but from a source other than Th17 cells. Notably, genetic ablation of γδ T cells in ETBF-colonized Th17-deficient Min mice prevented the late emergence of colonic tumors. Taken together, these findings support a redundant role for adaptive Th17 cell- and innate γδT17 cell-derived IL17 in bacteria-induced colon carcinogenesis, stressing the importance of therapeutically targeting the cytokine itself rather than its cellular sources. Cancer Res; 76(8); 2115–24. ©2016 AACR.

Supplemental Figures: Supplementary Figure 1 - Paclitaxel or carboplatin is less effective than doxorubicin in combination with entinostat and ATRA on induction of cell death. Supplementary Figure 2 - Paclitaxel or carboplatin in the combination is less effective than doxorubicin on induction of RAR-β expression; depletion of RAR-β decreases sensitivity to EAD; and transcriptional regulation of RAR-β by TopoII-β Supplementary Figure 3 - EAD decreases the number of CD44+/CD24-/Epcam+ cells and induces expression of differentiation markers Supplementary Figure 4 - EAD potentiates differentiation in vivo. Supplementary Figure 5 - ELF-3 is increased in differentiated stem cell and mediates the entinostat-differentiation effect. Supplementary Figure 6 - EAD targets metastatic samples.

Supplementary Table S2. Demographic and pathologic characteristics of the US CRC patients.

Uterine cancer is the most common cancer of the female genital tract and is the fourth most frequent cause of cancer death in women in the U.S. Despite the high prevalence of uterine cancers, the molecular events that lead to neoplastic transformation in the uterus are poorly understood. Moreover, there are limited mouse models to study these malignancies. We generated transgenic mice with high-mobility group A1 gene (HMGA1a) expression targeted to uterine tissue and all female mice developed tumors by 9 months of age. Histopathologically, the tumors resemble human uterine adenosarcoma and are transplantable. To determine whether these findings are relevant to human disease, we evaluated primary human uterine neoplasms and found that HMGA1a mRNA and protein levels are increased in most high-grade neoplasms but not in normal uterine tissue, benign tumors, or most low-grade neoplasms. We also found that HMGA1a up-regulates cyclooxygenase 2 (COX-2) expression in transgenic tumors. Moreover, both HMGA1a and COX-2 expression are up-regulated in high-grade human leiomyosarcomas. Using chromatin immunoprecipitation, HMGA1a binds directly to the COX-2 promoter in human uterine cancer cells in vivo and activates its expression in transfection experiments. We also show that blocking either HMGA1a or COX-2 in high-grade human uterine cancer cells blocks anchorage-independent cell growth in methylcellulose. These findings show that HMGA1a functions as an oncogene when overexpressed in the uterus and contributes to the pathogenesis of human uterine cancer by activating COX-2 expression. Although a larger study is needed to confirm these results, HMGA1a may be a useful marker for aggressive human uterine cancers. [Cancer Res 2007;67(9):3998–4004]

Supplemental Tables: Supplementary Table S1A. Estimated tumor volume of MDA-MB-231 xenografts treated with entinostat, ATRA, and doxorubicin as single and combined therapies. Supplementary Table S1B. Comparison of the estimated tumor volume of MDA-MB-231 xenografts treated with entinostat, ATRA, and doxorubicin as single and combined therapies. Supplementary Table S2A. Estimated tumor volume of SUM-159 xenografts treated with entinostat, ATRA, and doxorubicin as single and combined therapy. Supplementary Table S2B. Comparison of the estimated tumor volume of SUM-159 xenografts treated with entinostat, ATRA, and doxorubicin as single and combined therapy. Supplementary Table S3A. Estimated tumor volume of MDA-MB-231 xenografts treated with entinostat, ATRA, and carboplatin as single and combined therapy. Supplementary Table S3B. Comparison of the estimated tumor volume of MDA-MB-231 xenografts treated with entinostat, ATRA, and carboplatin as single and combined therapies. Supplementary Table S4A. Estimated tumor volume of SUM-159 xenografts treated with entinostat, ATRA, and carboplatin as single and combined therapy. Supplementary Table S4B. Comparison of the estimated tumor volume of SUM-159 xenografts treated with entinostat, ATRA, and carboplatin as single and combined therapy. Supplementary Table S5. Necrosis scores in mouse xenografts. Supplementary Table S6. EAD treatment shows reduced tumor incidence at limiting dilution. Supplementary Table S7. Characteristics of primary patient samples.

Legends of figures S1 to S9

Supplemental Figure S1 Legend Supplemental Figure S2 Legend Supplemental Figure S3 Legend Supplemental Figure S4 Legend Supplemental Figure S5 Legend Supplemental Figure S6 Legend

G protein-coupled receptor (GPR)35 is highly expressed in the gastro-intestinal tract, predominantly in colon epithelial cells (CEC), and has been associated with inflammatory bowel diseases (IBD), suggesting a role in gastrointestinal inflammation. The enterotoxigenic Bacteroides fragilis (ETBF) toxin (BFT) is an important virulence factor causing gut inflammation in humans and animal models. We identified that BFT signals through GPR35. Blocking GPR35 function in CECs using the GPR35 antagonist ML145, in conjunction with shRNA knock-down and CRISPRcas-mediated knock-out, resulted in reduced CEC-response to BFT as measured by E-cadherin cleavage, beta-arrestin recruitment and IL-8 secretion. Importantly, GPR35 is required for the rapid onset of ETBF-induced colitis in mouse models. GPR35-deficient mice showed reduced death and disease severity compared to wild-type C57Bl6 mice. Our data support a role for GPR35 in the CEC and mucosal response to BFT and underscore the importance of this molecule for sensing ETBF in the colon., Boleij et al. show that G protein-coupled receptor 35 (GPR35) regulates the responses to enterotoxigenic Bacteroides fragilis (ETBF) in colonic epithelial cells. They find that GPR35-deficiency nearly protected mice from ETBF-induced death, suggesting the importance of GPR35 in sensing ETBF in the colon.

Biofilms provide refuge for cancerous bacteria Familial adenomatous polyposis (FAP) causes benign polyps along the colon. If left untreated, FAP leads to a high incidence of colon cancer. To understand how polyps influence tumor formation, Dejea et al. examined the colonic mucosa of FAP patients. They discovered biofilms containing the carcinogenic versions of the bacterial species Escherichia coli and Bacteroides fragilis . Colon tissue from FAP patients exhibited greater expression of two bacterial genes that produce secreted oncotoxins. Studies in mice showed that specific bacteria could work together to induce colon inflammation and tumor formation. Science , this issue p. 592

The stromal signals that promote B lymphopoiesis remain poorly understood. Hedgehog (Hh) signaling promotes B lymphopoiesis in a non-cell-autonomous fashion in vitro, and depletion of the Hh effector Smoothened (Smo) from stromal cells is associated with the loss of osteoblastoid markers. These observations suggested that Hh signaling in the osteoblastoid lineage promotes B lymphopoiesis in vivo. To test this, we employed a mouse model for conditional ablation of Smo in the osteoblastoid lineage. Depletion of Smo from osteoblastoid cells is associated with profound and selective reductions in the number and proportion of bone marrow B-lymphoid progenitors. Upon partial bone marrow ablation, mutant animals exhibit delayed repopulation of the B-lymphoid compartment after the early lymphoid progenitor stage. Primary osteoblasts from mutant mice are defective in supporting B lymphopoiesis in vitro, whereas hematopoietic progenitors from mutant mice exhibit normal differentiation. We conclude that efficient B lymphopoiesis in vivo is dependent on the maintenance of Hh signaling in the osteoblastoid lineage.

The standard treatment for ductal carcinoma in situ (DCIS) of the breast is surgical resection, followed by radiation. Here, we tested localized therapy of DCIS in mice using the immunoconjugate 225Ac linked-trastuzumab delivered through the intraductal (i.duc) route. Trastuzumab targets HER-2/neu, while the alpha-emitter 225Ac (half-life, 10 days) delivers highly cytotoxic, focused doses of radiation to tumors. Systemic 225Ac, however, elicits hematologic toxicity and at high doses free 213Bi, generated by its decay, causes renal toxicity. I.duc delivery of the radioimmunoconjugate could bypass its systemic toxicity. Bioluminescent imaging showed that the therapeutic efficacy of intraductal 225Ac-trastuzumab (10-40 nCi per mammary gland; 30-120 nCi per mouse) in a DCIS model of human SUM225 cancer cells in NSG mice was significantly higher (p

Efforts to induce the differentiation of cancer stem cells through treatment with all-trans retinoic acid (ATRA) have yielded limited success, partially due to the epigenetic silencing of the retinoic acid receptor (RAR)-β. The histone deacetylase inhibitor entinostat is emerging as a promising antitumor agent when added to the standard-of-care treatment for breast cancer. However, the combination of epigenetic, cellular differentiation, and chemotherapeutic approaches against triple-negative breast cancer (TNBC) has not been investigated. In this study, we found that combined treatment of TNBC xenografts with entinostat, ATRA, and doxorubicin (EAD) resulted in significant tumor regression and restoration of epigenetically silenced RAR-β expression. Entinostat and doxorubicin treatment inhibited topoisomerase II-β (TopoII-β) and relieved TopoII-β-mediated transcriptional silencing of RAR-β. Notably, EAD was the most effective combination in inducing differentiation of breast tumor–initiating cells in vivo. Furthermore, gene expression analysis revealed that the epithelium-specific ETS transcription factor-1 (ESE-1 or ELF3), known to regulate proliferation and differentiation, enhanced cell differentiation in response to EAD triple therapy. Finally, we demonstrate that patient-derived metastatic cells also responded to treatment with EAD. Collectively, our findings strongly suggest that entinostat potentiates doxorubicin-mediated cytotoxicity and retinoid-driven differentiation to achieve significant tumor regression in TNBC. Cancer Res; 76(7); 2013–24. ©2016 AACR.

Despite dramatic improvements in treatment and survival, acute lymphoid malignancies remain the leading cause of death in children with cancer and a highly lethal malignancy in adults.[1] Acute lym...

UNLABELLED Wilson disease (WD) is a hepatoneurological disorder caused by mutations in the copper-transporter, ATP7B. Copper accumulation in the liver is a hallmark of WD. Current therapy is based on copper chelation, which decreases the manifestations of liver disease, but often worsens neurological symptoms. We demonstrate that in Atp7b(-/-) mice, an animal model of WD, liver function can be significantly improved without copper chelation. Analysis of transcriptional and metabolic changes in samples from WD patients and Atp7b(-/-) mice identified dysregulation of nuclear receptors (NRs), especially the liver X receptor (LXR)/retinoid X receptor heterodimer, as an important event in WD pathogenesis. Treating Atp7b(-/-) mice with the LXR agonist, T0901317, ameliorated disease manifestations despite significant copper overload. Genetic markers of liver fibrosis and inflammatory cytokines were significantly decreased, lipid profiles normalized, and liver function and histology were improved. CONCLUSIONS The results demonstrate the major role of an altered NR function in the pathogenesis of WD and suggest that modulation of NR activity should be explored as a supplementary approach to improving liver function in WD. (Hepatology 2016;63:1828-1841).

Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors characterized by a dense desmoplastic stroma comprised of cancer associated fibroblasts (CAFs) and fibrotic scar tissue. While stromal CAFs provide a barrier to therapy and release signals that foster tumor growth and invasion, the stroma also restrains tumor growth in murine models. The High Mobility Group A1 (HMGA1) chromatin remodeling gene encodes an oncofetal protein and epigenetic regulator that amplifies signals from the microenvironment to foster stem cell properties within intestinal epithelium. HMGA1 is also highly expressed during embryogenesis and in adult stem cells, but silenced postnatally in most differentiated cells. In diverse, aggressive cancers, HMGA1 becomes aberrantly re-expressed where high levels portend adverse clinical outcomes. In PDAC, HMGA1 protein is detected in late stage precursor lesions and invasive tumors, but not in normal pancreas nor in early precursor lesions. Furthermore, high HMGA1 levels correlate with poor differentiation status and decreased patient survival. Here, we discovered a novel epigenetic program mediated by HMGA1 that recruits CAFs to drive PDAC progression. We discovered that silencing HMGA1 in multiple PDAC cell lines halts proliferation and disrupts oncogenic properties, including migration, invasion, clonogenicity, and xenograft tumorigenesis. HMGA1 silencing also impairs three-dimensional sphere formation and depletes tumor initiator cells in limiting dilution assays. Through RNA sequencing analysis comparing PDAC cells overexpressing HMGA1 to those with HMGA1 silencing, we identified FGF-19 as a potential transcriptional target of HMGA1. HMGA1 binds specifically to the FGF-19 promoter and recruits the active histone mark, H3K4me3, to activate its expression. HMGA1 is also required for FGF-19 secretion from PDAC cells. Similar to HMGA1, silencing FGF-19 blocks oncogenic and cancer stem cell properties in vitro while disrupting tumorigenesis and depleting tumor initiator cells in vivo. In co-culture experiments, HMGA1 expressed in PDAC cells amplifies FGF-19 secretion, thereby stimulating CAF migration to tumor cells across a membrane. Silencing HMGA1 or FGF-19 prevents CAF recruitment to PDAC tumor cells. Furthermore, CAF recruitment is blocked by either FGF-19 blocking antibodies or an inhibitor to the FGF-19 receptor (FGR4). In murine models, silencing HMGA1 also decreases formation of a fibroblastic stroma. Moreover, overexpression of HMGA1 together with that of FGF-19 predict decreased survival in primary human PDAC. Our results reveal a novel paradigm whereby PDAC cells collaborate with stromal CAFs via HMGA1 and FGF-19 to drive tumor progression. These data also provide insight into mechanisms for tumor progression and illuminate FGF-19 as a rational therapeutic target in PDACs with up-regulation of HMGA1 and FGF-19. Citation Format: Lionel Chia, Shuai Shuai, Lingling Xian, Jung-Hyun Kim, Ruitao Zhang, Tait Huso, David Huso, Leslie Cope, Karen Reddy, Linda Resar. HMGA1 induces FGF-19 to foster tumor-stromal cell crosstalk and drive tumor progression in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 297.

Women with Marfan syndrome (MFS) are at high risk for pregnancy-associated aortic dissection. Pathogenic models that singularly invoke hemodynamic stress are difficult to reconcile with predominant postnatal occurrence of aortic tear, often occurring weeks to months after delivery. In consideration of events that peak at term, are sustained after delivery, and might synergize with previously defined signaling pathways implicated in aneurysm progression, we examined the hormone oxytocin, which initiates uterine contraction and milk letdown for the duration of lactation through phosphorylation of extracellular signal-regulated kinase (ERK). In a mouse model of MFS that shows highly penetrant postnatal aortic dissection, risk was strongly attenuated by preventing lactation or use of an oxytocin receptor antagonist. Survival correlated inversely with the extent of ERK activation in the aortic wall, and strong protection was observed upon attenuation of ERK phosphorylation using an inhibitor of ERK kinase (MEK) or the U.S. Food and Drug Administration-approved medication hydralazine, offering potential therapeutic strategies for pregnancy-associated vascular catastrophe in the setting of MFS.

Pro-carcinogenic bacteria have the potential to initiate and/or promote human colon cancer. However, the mechanism(s) by which a bacterium may trigger carcinogenesis remains unclear. Herein, we show Bacteroides fragilis toxin (BFT) secretion by the human pathobiont enterotoxigenic Bacteroides fragilis (ETBF) triggers in Min Apc /- mice a ETBF pro- carcinogenic inflammatory cascade requiring IL-17R, NF-κB, and Stat3 signaling in colonic epithelial cells (CEC). This signaling promotes the chemokine-dependent recruitment of pro-tumoral myeloid cells. Although necessary, Stat3 activation in CEC upon ETBF colonization is not sufficient to trigger colon tumorigenesis. Notably, IL-17-dependent NF-κB activation in CECs induces a proximal to distal mucosal gradient of C-X-C chemokines, including CXCL1 that mediates CXCR2-expressing polymorphonuclear immature myeloid cell recruitment with parallel onset of ETBF tumorigenesis in the distal colon, explaining the restricted localization of ETBF tumorigenesis. Inhibitors of pathway drivers linking specific microbial mediators to early colon tumorigenesis could disrupt a myeloid/epithelial pro-tumoral loop.

Many epithelial cancers are associated with chronic inflammation. However, the features of inflammation that are procarcinogenic are not fully understood. Regulatory T cells (Treg) typically restrain overt inflammatory responses and maintain intestinal immune homeostasis. Their immune-suppressive activity can inhibit inflammation-associated cancers. Paradoxically, we show that colonic Tregs initiate IL17-mediated carcinogenesis in multiple intestinal neoplasia mice colonized with the human symbiote enterotoxigenic Bacteroides fragilis (ETBF). Depletion of Tregs in ETBF-colonized C57BL/6 FOXP3DTR mice enhanced colitis but diminished tumorigenesis associated with shifting of mucosal cytokine profile from IL17 to IFNγ; inhibition of ETBF-induced colon tumorigenesis was dependent on reduced IL17 inflammation and was independent of IFNγ. Treg enhancement of IL17 production is cell-extrinsic. IL2 blockade restored Th17 responses and tumor formation in Treg-depleted animals. Our findings demonstrate that Tregs limit the availability of IL2 in the local microenvironment, allowing the Th17 development necessary to promote ETBF-triggered neoplasia, and thus unveil a new mechanism whereby Treg responses to intestinal bacterial infection can promote tumorigenesis. Significance: Tregs promote an oncogenic immune response to a common human symbiote associated with inflammatory bowel disease and colorectal cancer. Our data define mechanisms by which mucosal Tregs, despite suppressing excessive inflammation, promote the earliest stages of immune procarcinogenesis via enhancement of IL17 production at the expense of IFNγ production. Cancer Discov; 5(10); 1098–109. ©2015 AACR. See related commentary by Irrazabal and Martin, p. 1021. This article is highlighted in the In This Issue feature, p. 1005

Background: Tissue fibrosis and microvascular rarefaction are hallmarks of progressive renal disease. CD248 is a transmembrane glycoprotein expressed by key effector cells within the stroma of fibrotic kidneys including pericytes, myofibroblasts and stromal fibroblasts. In human disease, increased expression of CD248 by stromal cells predicts progression to end-stage renal failure. We therefore, hypothesized that the genetic deletion of the CD248 gene would protect against fibrosis following kidney injury. Methods: Using the unilateral ureteral obstruction (UUO) model of renal fibrosis, we investigated the effect of genetic deletion of CD248 on post obstructive kidney fibrosis. Results: CD248 null mice were protected from fibrosis and microvascular rarefaction following UUO. Although the precise mechanism is not known, this may to be due to a stabilizing effect of pericytes with less migration and differentiation of pericytes toward a myofibroblast phenotype in CD248-/- mice. CD248-/- fibroblasts also proliferated less and deposited less collagen in vitro. Conclusion: These studies suggest that CD248 stromal cells have a pathogenic role in renal fibrosis and that targeting CD248 is effective at inhibiting both microvascular rarefaction and renal fibrosis through modulation of pericyte and stromal cell function.

Autosomal-dominant polycystic kidney disease (ADPKD) is a progressive, proliferative renal disease. Kidneys from ADPKD patients are characterized by the presence of cysts that are marked by enhanced proliferation and apoptosis of renal tubular epithelial cells. Current treatment of this disease is supportive, as there are few if any clinically validated targeted therapeutics. Given the parallels between cystic disease and cancer, and in light of our findings of the efficacy of the nuclear transport inhibitors in kidney cancer, which has similarities to ADPKD, we asked whether such inhibitors show utility in ADPKD. In this study, we tested selective inhibitors of nuclear export (SINE) in two human ADPKD cell lines and in an in vivo mouse model of ADPKD. After effective downregulation of a nuclear exporter, exportin 1 (XPO1), with KPT-330, both cell lines showed dose-dependent inhibition of cell proliferation through G0/G1 arrest associated with downregulation of CDK4, with minimal apoptosis. To analyze mechanisms of CDK4 decrease by XPO1 inhibition, localization of various XPO1 target proteins was examined, and C/EBPβ was found to be localized in the nucleus by XPO1 inhibition, resulting in an increase of C/EBPα, which activates degradation of CDK4. Furthermore, inhibition of XPO1 with the parallel inhibitor KPT-335 attenuated cyst growth in vivo in the PKD1 mutant mouse model Pkd1v/v. Thus, inhibition of nuclear export by KPT-330, which has shown no adverse effects in renal serum chemistries and urinalyses in animal models, and which is already in phase 1 trials for cancers, will be rapidly translatable to human ADPKD.

High-mobility group A1 (Hmga1) chromatin remodelling proteins are enriched in intestinal stem cells (ISCs), although their function in this setting was unknown. Prior studies showed that Hmga1 drives hyperproliferation, aberrant crypt formation and polyposis in transgenic mice. Here we demonstrate that Hmga1 amplifies Wnt/β-catenin signalling to enhance self-renewal and expand the ISC compartment. Hmga1 upregulates genes encoding both Wnt agonist receptors and downstream Wnt effectors. Hmga1 also helps to ‘build' an ISC niche by expanding the Paneth cell compartment and directly inducing Sox9, which is required for Paneth cell differentiation. In human intestine, HMGA1 and SOX9 are positively correlated, and both become upregulated in colorectal cancer. Our results define a unique role for Hmga1 in intestinal homeostasis by maintaining the stem cell pool and fostering terminal differentiation to establish an epithelial stem cell niche. This work also suggests that deregulated Hmga1 perturbs this equilibrium during intestinal carcinogenesis., The function of high mobility group A1 (Hmga1) chromatin remodelling proteins in intestinal stem cells (ISC) is unknown. Here, the authors show that Hmga1 amplifies Wnt/β-catenin signalling to enhance self-renewal and induces Sox9 to expand the Paneth cell compartment and enrich the ISC niche.

CASE DESCRIPTION A 9-year-old spayed female Rottweiler with hind limb ataxia was examined because of anorexia and an acute onset of hind limb paresis. CLINICAL FINDINGS Neurologic evaluation revealed hind limb ataxia and symmetric paraparesis with bilaterally abnormal hind limb postural reactions including hopping, hemiwalking, hemistanding, and delayed proprioception, which were suggestive of a lesion somewhere in the T3-L3 segment of the spinal cord. Thoracolumbar radiography revealed an abnormal radiopacity suggestive of a mass at T11. Two 3.5-cm-long osseous core biopsy specimens of the mass were obtained by MRI guidance. Histologic appearance of the specimens was consistent with osteosarcoma. TREATMENT AND OUTCOME The owners of the dog declined further treatment owing to a poor prognosis. The dog was euthanized within 12 months after diagnosis because of a declining quality of life. CLINICAL RELEVANCE The acquisition of biopsy specimens by MRI guidance is an emerging technique in veterinary medicine. As evidenced by the dog of this report, MRI-guided biopsy can be used to safely obtain diagnostic biopsy specimens from tissues at anatomic locations that are difficult to access. This technique can potentially be used to facilitate early diagnosis and treatment of disease, which could improve patient outcome. The MRI guidance technique described may also be useful for local administration of chemotherapeutics or radiofrequency ablation or cryoablation of various neoplasms of the vertebral column.

Acquired resistance to TGFβ is a key step in the early stages of tumorigenesis. Mutations in TGFβ signaling components are rare, and little is known about the development of resistance in breast cancer. On the other hand, an activated Notch pathway is known to play a substantial role in promoting breast cancer development. Here, we present evidence of crosstalk between these two pathways through HEYL. HEYL, a basic helix–loop–helix transcription factor and a direct target of Notch signaling, is specifically overexpressed in breast cancer. HEYL represses TGFβ activity by binding to TGFβ-activated Smads. HeyL−/− mice have defective mammary gland development with fewer terminal end buds. On the other hand, HeyL transgenic mice show accelerated mammary gland epithelial proliferation and 24% of multiparous mice develop mammary gland cancer. Therefore, repression of TGFβ signaling by Notch acting through HEYL may promote initiation of breast cancer. Cancer Res; 74(22); 6509–18. ©2014 AACR.

Somatic activation of the KRAS proto-oncogene is evident in almost all pancreatic cancers, and appears to represent an initiating event. These mutations occur primarily at codon 12 and less frequently at codons 13 and 61. Although some studies have suggested that different KRAS mutations may have variable oncogenic properties, to date there has been no comprehensive functional comparison of multiple KRAS mutations in an in vivo vertebrate tumorigenesis system. We generated a Gal4/UAS-based zebrafish model of pancreatic tumorigenesis in which the pancreatic expression of UAS-regulated oncogenes is driven by a ptf1a:Gal4-VP16 driver line. This system allowed us to rapidly compare the ability of 12 different KRAS mutations (G12A, G12C, G12D, G12F, G12R, G12S, G12V, G13C, G13D, Q61L, Q61R and A146T) to drive pancreatic tumorigenesis in vivo. Among fish injected with one of five KRAS mutations reported in other tumor types but not in human pancreatic cancer, 2/79 (2.5%) developed pancreatic tumors, with both tumors arising in fish injected with A146T. In contrast, among fish injected with one of seven KRAS mutations known to occur in human pancreatic cancer, 22/106 (20.8%) developed pancreatic cancer. All eight tumorigenic KRAS mutations were associated with downstream MAPK/ERK pathway activation in preneoplastic pancreatic epithelium, whereas nontumorigenic mutations were not. These results suggest that the spectrum of KRAS mutations observed in human pancreatic cancer reflects selection based on variable tumorigenic capacities, including the ability to activate MAPK/ERK signaling.

SummaryAutosomal dominant polycystic kidney disease is a common form of inherited kidney disease that is caused by mutations in two genes, PKD1 (polycystin-1) and PKD2 (polycystin-2). Mice with germline deletion of either gene die in midgestation with a vascular phenotype that includes profound edema. Although an endothelial cell defect has been suspected, the basis of this phenotype remains poorly understood. Here, we demonstrate that edema in Pkd1- and Pkd2-null mice is likely to be caused by defects in lymphatic development. Pkd1 and Pkd2 mutant embryos exhibit reduced lymphatic vessel density and vascular branching along with aberrant migration of early lymphatic endothelial cell precursors. We used cell-based assays to confirm that PKD1- and PKD2-depleted endothelial cells have an intrinsic defect in directional migration that is associated with a failure to establish front-rear polarity. Our studies reveal a role for polycystin signaling in lymphatic development.

Inflammatory bowel disease (IBD) is postulated to result from a dysregulated immune response to the host microbiota in genetically susceptible individuals.1,2 Although no specific pathogen(s) has been clearly implicated, enteric bacteria seem to play a significant role in IBD pathogenesis.1,3 Increased epithelial permeability in IBD patients is believed to enhance direct contact between colonic flora and the mucosal immune system, possibly serving as a critical factor in disease initiation. In fact, IBD patients have increased the secretion of IgG antibodies against their microbiota into the intestinal lumen.4 Furthermore, there seems to be diminished intestinal mucosal tolerance because there is an increase in systemic adaptive immune responses against antigens, like flagellin, of the bacteria.5,6 Signal transducers and activators of transcription (Stat) proteins are transcription factors that once activated play a critical role in the regulation of immune responses.7 This family of transcription factors (Stats 1–6) undergoes activation in the cytosol by tyrosine phosphorylation before nuclear translocation. The contribution of Stat protein activation to epithelial and immune cell function is complex. Within T cells, activated Stat proteins (pStats) induce and maintain T-cell subset identity through tran-scriptional activation of subset-specific transcription factors and cotranscriptional regulation of subset-specific cytokine genes. For example, Stat1, Stat2, and Stat4 are required for the generation of Th1 cells, Stat6 for Th2 cells, and Stat3 for Th17 cells. Recent studies have highlighted the contribution of Th17-mediated inflammation in the pathogenesis of IBD.8 In genome-wide analyses, single-nucleotide polymorphisms of the Stat3 protein have been identified in patients with Crohn’s disease and ulcerative colitis. Furthermore, mutations in the IL-23 receptor gene are some of the host mutations most highly associated with IBD.9 Although IL-23 has several roles in immune regulation, one of its actions is to promote the expansion and stabilization of Th17 cell populations.10 A dense, adherent biofilm mass predominately consisting of Bacteroides fragilis group of organisms has been reported to adhere to the inflamed mucosal surface of IBD patients.11 B. fragilis sensu stricto is a common colonic symbiote estimated to colonize up to 90% of individuals.12 Colonization with B. fragilis is proposed to play a pivotal role in the regulation of mucosal and systemic immunity.13 However, B. fragilis can act as opportunistic pathogens being the leading anaerobe in bloodstream infections and intra-abdominal abscesses.14 Enterotoxigenic B. fragilis (ETBF) is a molecular subset of B. fragilis that secrete a zinc-dependent metalloprotease toxin termed the B. fragilis toxin (BFT, also known as fragilysin).15 ETBF is associated globally with pediatric and adult diarrheal illnesses and, in limited data, with active IBD and colon cancer.16–19 Previously, we reported that ETBF induce symptomatic acute and asymptomatic chronic colitis in conventional C57Bl/6 mice.20,21 Furthermore, in multiple intestinal neoplasia (Min) mice that are heterozygous for the adenomatous polyposis coli (Apc) gene, ETBF colonization induces colonic inflammation with rapid-onset colonic tumor formation. This process is characterized by the selective activation of Stat3 by day 2 after colonization with a subsequent colonic mucosal Th17 response. Blockade of IL-17 alone or IL-17 and the IL-23 receptor in this model diminishes colon tumor formation, indicating that Stat3/Th17 adaptive immunity contributes to ETBF carcinogenesis. In this study, we further characterized Stat3 activation in ETBF-induced murine colitis in C57Bl/6, C57BL/6Stat3ΔIEC, and Rag-1 mice. We also tested whether BFT, the only known ETBF virulence factor, activates colonic epithelial cell Stat3. Our results show that ETBF rapidly activates mucosal immune cell Stat3. Subsequently, increased mucosal permeability occurs, together with increased mucosal immune cell Stat3 activation and Stat3 activation in colonic epithelial cells. In ETBF-colonized mice, Stat3 activation persists for months along with a mucosal Th17 response. Our findings indicate that ETBF-induced Stat3 activation in mucosal immune cells precedes detectable increased mucosal permeability or colonic epithelial Stat3 activation, suggesting that ETBF, likely through BFT, triggers rapid release of early epithelial cell mediators that contribute to early immune Stat3 activation. Peak mucosal Stat3 activation (immune and epithelial cells) occurs later when other colonic bacteria may contribute to the immune response due to barrier dysfunction.

Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of renal failure that is due to mutations in two genes, PKD1 and PKD2. Vascular complications, including aneurysms, are a well recognized feature of ADPKD, and a subgroup of families exhibits traits reminiscent of Marfan syndrome (MFS). MFS is caused by mutations in fibrillin-1 (FBN1), which encodes an extracellular matrix protein with homology to latent TGF-β binding proteins. It was recently demonstrated that fibrillin-1 deficiency is associated with upregulation of TGF-β signaling. We investigated the overlap between ADPKD and MFS by breeding mice with targeted mutations in Pkd1 and Fbn1. Double heterozygotes displayed an exacerbation of the typical Fbn1 heterozygous aortic phenotype. We show that the basis of this genetic interaction results from further upregulation of TGF-β signaling caused by Pkd1 haploinsufficiency. In addition, we demonstrate that loss of PKD1 alone is sufficient to induce a heightened responsiveness to TGF-β. Our data link the interaction of two important diseases to a fundamental signaling pathway.

Kabuki syndrome is a Mendelian intellectual disability syndrome caused by mutations in either of two genes (KMT2D and KDM6A) involved in chromatin accessibility. We previously showed that an agent that promotes chromatin opening, the histone deacetylase inhibitor (HDACi) AR-42, ameliorates the deficiency of adult neurogenesis in the granule cell layer of the dentate gyrus and rescues hippocampal memory defects in a mouse model of Kabuki syndrome (Kmt2d+/βGeo). Unlike a drug, a dietary intervention could be quickly transitioned to the clinic. Therefore, we have explored whether treatment with a ketogenic diet could lead to a similar rescue through increased amounts of beta-hydroxybutyrate, an endogenous HDACi. Here, we report that a ketogenic diet in Kmt2d+/βGeo mice modulates H3ac and H3K4me3 in the granule cell layer, with concomitant rescue of both the neurogenesis defect and hippocampal memory abnormalities seen in Kmt2d+/βGeo mice; similar effects on neurogenesis were observed on exogenous administration of beta-hydroxybutyrate. These data suggest that dietary modulation of epigenetic modifications through elevation of beta-hydroxybutyrate may provide a feasible strategy to treat the intellectual disability seen in Kabuki syndrome and related disorders.

Nanoparticle gene therapy holds great promise for the treatment of malignant disease in light of the large number of potent, tumor-specific therapeutic payloads potentially available for delivery. To be effective, gene therapy vehicles must be able to deliver their therapeutic payloads to metastatic lesions after systemic administration. Here we describe nanoparticles comprised of a core of high molecular weight linear polyethylenimine (LPEI) complexed with DNA and surrounded by a shell of polyethyleneglycol-modified (PEGylated) low molecular weight LPEI. Compared with a state-of-the-art commercially available in vivo gene delivery formulation, i.v. delivery of the core/PEGylated shell (CPS) nanoparticles provided more than a 16,000-fold increase in the ratio of tumor to nontumor transfection. The vast majority of examined liver and lung metastases derived from a colorectal cancer cell line showed transgene expression after i.v. CPS injection in an animal model of metastasis. Histological examination of tissues from transfected mice revealed that the CPS nanoparticles selectively transfected neoplastic cells rather than stromal cells within primary and metastatic tumors. However, only a small fraction of neoplastic cells (

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia with high relapse rates compared to B-lineage ALL. We previously showed that HMGA1a transgenic mice develop aggressive T-ALL, indicating that HMGA1 causes leukemic transformation in vivo. HMGA1 is also highly expressed in embryonic stem cells, hematopoietic stem cells and diverse, refractory human cancers. Disruption of the CDKN2A tumor suppressor locus occurs in most cases of T-ALL and is thought to contribute to leukemic transformation. To determine whether loss of function of CDKN2A cooperates with HMGA1 in T-ALL, we crossed HMGA1a transgenics onto a Cdkn2a null background. We discovered that T-ALL is markedly accelerated in HMGA1a transgenic Cdkn2a null mice. In addition, these mice recapitulate salient clinical and pathologic features of human T-ALL. HMGA1 is also highly overexpressed in human T-ALL. These findings suggest that HMGA1 plays a causative role in T-ALL and could represent a rational therapeutic target.

Because colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, more accessible screening tests are urgently needed to identify early stage lesions. We hypothesized that highly sensitive, metabolic profile analysis of stool samples will identify metabolites associated with early stage lesions and could serve as a noninvasive screening test. We therefore applied traveling wave ion mobility mass spectrometry (TWIMMS) coupled with ultraperformance liquid chromatography (UPLC) to investigate metabolic aberrations in stool samples in a transgenic model of premalignant polyposis aberrantly expressing the gene encoding the high mobility group A (Hmga1) chromatin remodeling protein. Here, we report for the first time that the fecal metabolome of Hmga1 mice is distinct from that of control mice and includes metabolites previously identified in human CRC. Significant alterations were observed in fatty acid metabolites and metabolites associated with bile acids (hypoxanthine xanthine, taurine) in Hmga1 mice compared to controls. Surprisingly, a marked increase in the levels of distinctive short, arginine-enriched, tetra-peptide fragments was observed in the transgenic mice. Together these findings suggest that specific metabolites are associated with Hmga1-induced polyposis and abnormal proliferation in intestinal epithelium. Although further studies are needed, these data provide a compelling rationale to develop fecal metabolomic analysis as a noninvasive screening tool to detect early precursor lesions to CRC in humans.

To determine the temporal expression and pattern of Rel/nuclear factor (NF)-κB proteins in renal tissue in polycystic kidney disease (PKD).The renal expression of Rel/NF-κB proteins was determined by immunohistochemistry, immunofluorescence and immunoblot analysis in Lewis polycystic kidney rats (LPK, a genetic ortholog of human nephronopthsis-9) from postnatal weeks 3 to 20. At each timepoint, renal disease progression and the mRNA expression of NF-κB-dependent genes (TNFα and CCL2) were determined. NF-κB was also histologically assessed in human PKD tissue.Progressive kidney enlargement in LPK rats was accompanied by increased renal cell proliferation and interstitial monocyte accumulation (peaking at weeks 3 and 10 respectively), and progressive interstitial fibrosis (with α smooth muscle actin and Sirius Red deposition significantly increased compared to Lewis kidneys from weeks 3 to 6 onwards). Rel/NF-κB proteins (phosphorylated-p105, p65, p50, c-Rel and RelB) were expressed in cystic epithelial cells (CECs) of LPK kidneys as early as postnatal week 3 and sustained until late-stage disease at week 20. From weeks 10 to 20, nuclear p65, p50, RelB and cytoplasmic IκBα protein levels, and TNFα and CCL2 expression, were upregulated in LPK compared to Lewis kidneys. NF-κB proteins were consistently expressed in CECs of human PKD. The DNA damage marker γ-H2AX was also identified in the CECs of LPK and human polycystic kidneys.Several NF-κB proteins are consistently expressed in CECs in human and experimental PKD. These data suggest that the upregulation of both the canonical and non-canonical pathways of NF-κB signaling may be a constitutive and early pathological feature of cystic renal diseases.