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Background & Aims Self-renewal and multipotent differentiation are cardinal properties of intestinal stem cells (ISCs), mediated in part by WNT and NOTCH signaling. Although these pathways are well characterized, the molecular mechanisms that control the ‘stemness’ of ISCs are still not well defined. Here, we investigated the role of Kruppel-like factor 5 (KLF5) in regulating ISC functions. Methods We performed studies in adult Lgr5EGFP-IRES-creERT2;Rosa26LSLtdTomato (Lgr5Ctrl) and Lgr5EGFP-IRES-creERT2;Klf5fl/fl;Rosa26LSLtdTomato (Lgr5ΔKlf5) mice. Mice were injected with tamoxifen to activate Cre recombinase, which deletes Klf5 from the intestinal epithelium in Lgr5ΔKlf5 but not Lgr5Crtl mice. In experiments involving irradiation, mice were subjected to 12 Gy total body irradiation (TBI). Tissues were collected for immunofluorescence (IF) analysis and next generation sequencing. Oganoids were derived from fluoresecence activated cell sorted- (FACS-) single cells from tamoxifen-treated Lgr5ΔKlf5 or Lgr5Crtl mice and examined by immunofluorescence stain. Results Lgr5+ ISCs lacking KLF5 proliferate faster than control ISCs but fail to self-renew, resulting in a depleted ISC compartment. Transcriptome analysis revealed that Klf5-null Lgr5+ cells lose ISC identity and prematurely differentiate. Following irradiation injury, which depletes Lgr5+ ISCs, reserve Klf5-null progenitor cells fail to dedifferentiate and regenerate the epithelium. Absence of KLF5 inactivates numerous selected enhancer elements and direct transcriptional targets including canonical WNT- and NOTCH-responsive genes. Analysis of human intestinal tissues showed increased levels of KLF5 in the regenerating epithelium as compared to those of healthy controls. Conclusion We conclude that ISC self-renewal, lineage specification, and precursor dedifferentiation require KLF5, by its ability to regulate epigenetic and transcriptional activities of ISC-specific gene sets. These findings have the potential for modulating ISC functions by targeting KLF5 in the intestinal epithelium.

We explored the transcriptional and epigenetic programs underlying the differentiation of hair cells from postnatal progenitor cells in cochlear organoids. Heterogeneity in the cells including cells with the transcriptional signatures of mature hair cells allowed a full picture of possible cell fates. Construction of trajectories identified Lgr5+ cells as progenitors for hair cells and the genomic data revealed gene regulatory networks leading to hair cells. We validated these networks, demonstrating dynamic changes both in expression and predicted binding sites of these transcription factors during organoid differentiation. We identified known regulators of hair cell development, Atoh1, Pou4f3, and Gfi1, and predicted novel regulatory factors, Tcf4, an E-protein and heterodimerization partner of Atoh1, and Ddit3, a CCAAT/enhancer-binding protein (C/EBP) that represses Hes1 and activates transcription of Wnt signaling-related genes. Deciphering the signals for hair cell regeneration from mammalian cochlear supporting cells reveals candidates for HC regeneration which is limited in the adult.

Cancer is a multistep process that requires cells to respond appropriately to the tumor microenvironment, both in early proliferative stages and in later invasive disease. Arl8b is a lysosome localized Arf-like GTPase that controls the spatial distribution of lysosomes via recruitment of kinesin motors. Common features of the tumor microenvironment such as acidic extracellular pH and various growth factors stimulate lysosome trafficking to the cell periphery (anterograde), which is critical for tumor invasion by facilitating the release of lysosomal proteases to promote matrix remodeling. Herein we report for the first time that Arl8b regulates anterograde lysosome trafficking in response to hepatocyte growth factor, epidermal growth factor, and acidic extracellular pH. Depletion of Arl8b results in juxtanuclear lysosome aggregation, and this effect corresponds with both diminished invasive growth and proteolytic extracellular matrix degradation in a three-dimensional model of prostate cancer. Strikingly, we found that depletion of Arl8b abolishes the ability of prostate cancer cells to establish subcutaneous xenografts in mice. We present evidence that Arl8b facilitates lipid hydrolysis to maintain efficient metabolism for a proliferative capacity in low nutrient environments, suggesting a likely explanation for the complete inability of Arl8b-depleted tumor cells to grow in vivo. In conclusion, we have identified two mechanisms by which Arl8b regulates cancer progression: 1) through lysosome positioning and protease release leading to an invasive phenotype and 2) through control of lipid metabolism to support cellular proliferation. These novel roles highlight that Arl8b is a potential target for the development of novel anti-cancer therapeutics.

After acquiring competence for selected cell fates, embryonic primordia may remain plastic for variable periods before tissue identity is irrevocably determined (commitment). We investigated the chromatin basis for these developmental milestones in mouse endoderm, a tissue with recognizable rostro–caudal patterning and transcription factor (TF)-dependent interim plasticity. Foregut-specific enhancers are as accessible and active in early midgut as in foregut endoderm, and intestinal enhancers and identity are established only after ectopic cis-regulatory elements are decommissioned. Depletion of the intestinal TF CDX2 before this cis element transition stabilizes foregut enhancers, reinforces ectopic transcriptional programs, and hence imposes foregut identities on the midgut. Later in development, as the window of chromatin plasticity elapses, CDX2 depletion weakens intestinal, without strengthening foregut, enhancers. Thus, midgut endoderm is primed for heterologous cell fates, and TFs act on a background of shifting chromatin access to determine intestinal at the expense of foregut identity. Similar principles likely govern other fate commitments.

Breast cancer remains one of the leading causes of death in women diagnosed with cancer. In breast cancer, aberrant expression of the CYP19A1 gene, which encodes the aromatase enzyme, contributes to increased intratumoral levels of estradiol. Regardless of whether this estrogen is produced by peripheral tissues or within specific subpopulations of cells within the breast tumor, it is clear that the aromatase enzymatic activity is critical for the growth of estrogen-dependent tumors. Currently, aromatase inhibitors have proven to be highly effective in blocking the growth of estrogen-dependent forms of breast cancer. CYP19A1 transcription is tightly controlled by 10 tissue-specific promoters. In breast cancer, however, aromatase transcription is driven by multiple promoters that somehow override the tissue-specific regulation of normal tissue. Here, we explore the role that the deacetylase, sirtuin-1 (SIRT1), plays in positively regulating aromatase in breast cancer. We demonstrate that the use of cambinol and the SIRT1/2 inhibitor VII, 2 small molecule inhibitors of SIRT1 and SIRT2, as well as small molecule inhibitors and small interfering RNA specific to SIRT1, all reduce the levels of aromatase mRNA. We further demonstrate that pharmacologic inhibition causes a marked reduction in aromatase protein levels. Additionally, by chromatin immunoprecipitation, we demonstrate that SIRT1 occupies the promoter regions PI.3/PII and PI.4, and its inhibition leads to increased acetylation of estrogen-related receptorα, a transcription factor that positively regulates CYP19A1 transcription in epithelial cells. Finally, we demonstrate by immunohistochemistry that SIRT1 is significantly up-regulated in invasive ductal carcinoma relative to normal tissue adjacent to tumor, further suggesting a role of SIRT1 in breast cancer. This work uncovers a new mechanism for the regulation of aromatase and provides rationale for further investigation of how the inhibition of specific sirtuins may provide a unique strategy for inhibiting aromatase that may complement or synergize with existing therapies.

Replicating Lgr5+ stem cells and quiescent Bmi1+ cells behave as intestinal stem cells (ISCs) in vivo. Disrupting Lgr5+ ISCs triggers epithelial renewal from Bmi1+ cells, from secretory or absorptive progenitors, and from Paneth cell precursors, revealing a high degree of plasticity within intestinal crypts. Here, we show that GFP+ cells from Bmi1GFP mice are preterminal enteroendocrine cells and we identify CD69+CD274+ cells as related goblet cell precursors. Upon loss of native Lgr5+ ISCs, both populations revert toward an Lgr5+ cell identity. While active histone marks are distributed similarly between Lgr5+ ISCs and progenitors of both major lineages, thousands of cis elements that control expression of lineage-restricted genes are selectively open in secretory cells. This accessibility signature dynamically converts to that of Lgr5+ ISCs during crypt regeneration. Beyond establishing the nature of Bmi1GFP+ cells, these findings reveal how chromatin status underlies intestinal cell diversity and dedifferentiation to restore ISC function and intestinal homeostasis.

Tamoxifen is a synthetic non-steroidal Selective Estrogen Receptor Modulator used in the treatment of breast cancer and in treatment of male fertility. Earlier studies from our laboratory had demonstrated an increase in post-implantation embryo loss following tamoxifen treatment to adult male rats at a dose of 0.4mg/kg/day for 60 days. The post-implantation loss occurred at around 9-10 days of gestation suggesting that paternal factors involved in embryo development were affected by tamoxifen treatment. The present study was done to determine if any chromosomal aberrations occurred in the embryos sired by tamoxifen treated male rats. Chromosomal aberrations induced by tamoxifen treatment to adult male rats in the bone marrow (F(0) males) and in the embryos sired by these males (F(1) progeny) were determined. In addition, the reproductive performance of the F(1) progeny was assessed. A significant dose dependent reduction in mitotic activity in the bone marrow and embryonic cells was observed after tamoxifen treatment. In addition, tamoxifen also induced a significant dose dependent increase in the frequency of chromosomal aberrations, mainly gaps and breaks in bone marrow and embryonic cells. However, the embryos sired by the tamoxifen treated males had no effect on developmental milestones achieved and on their reproductive performance. The present study suggests that chromosomal aberrations observed in the embryos did not the affect their development until adulthood but could make the progeny of the tamoxifen treated males vulnerable to the development of adult onset diseases later in life.

Objective: To determine the effect of tamoxifen treatment on global and insulin-like growth factor 2-H19 imprinting control region (Igf2-H19 ICR)-specific DNA methylation in rat spermatozoa and analyze its association with postimplantation loss. Design: Experimental prospective study. Setting: Animal research and academic research facility. Subject(s): Male and female 75-day-old Holtzman rats. Intervention(s): Global and Igf2-H19 ICR-specific DNA methylation was analyzed in an epididymal sperm sample in control and tamoxifen-treated rats at a dose of 0.4 mg tamoxifen/kg/day. DNA methylation status was correlated to postimplantation loss in females mated with tamoxifen-treated males. Main Outcome Measure(s): Global sperm DNA methylation level, methylation status of Igf2-H19 ICR in sperm, postimplantation loss. Result(s): Tamoxifentreatmentsignificantlyreducedmethylationat Igf2-H19 ICRinepididymalsperm.However, the global methylation level was not altered. A mating experiment confirmed a significant increase in postimplantation loss upon tamoxifen treatment and showed significant correlation with methylation at Igf2-H19 ICR. Conclusion(s): Reduced DNA methylation at Igf2-H19 ICR in rat spermatozoa upon tamoxifen treatment indicated a role of estrogen-associated signaling in the acquisition of paternal-specific imprints during spermatogenesis. In addition, association between DNA methylation and postimplantation loss suggests that errors in paternal imprints at Igf2-H19 ICR could affect embryo development. (Fertil Steril� 2009;91:2253‐63. � 2009 by American

SIRT1 deacetylase is overexpressed in different types of cancers and we and a few others have described a role for SIRT1 in regulating cancer cell migration. Rho GTPases are classic regulators of cell motility and are known to aberrantly enhance cancer cell migration in response to several anomalous cues. We hypothesized that one of mechanisms employed by SIRT1 to regulate cell motility is through regulation of Rac1-GTPase activation. To address this we utilized two cancer cell lines, CFPAC-1 pancreatic and MDA-MB-231 breast cancer cells, as our model. A significant decrease was observed in active Rac1 (GTP-bound Rac1) levels, in a GST-PAK-binding domain pull-down assay, upon inhibition of SIRT1 by small molecule inhibitors or knockdown of SIRT1 by shRNA as compared to vehicle-treated or non-targeting shRNA transfected cells. Subsequent analysis revealed that SIRT1 co-immunoprecipitated with the Rac-guanine nucleotide exchange factor (GEF), TIAM1. To test whether TIAM1 is acetylated and if SIRT1 regulates its deacetylation we performed antibody-based pull-down of acetylated lysines from whole cell protein extracts and observed TIAM1 acetylation. Inhibition of deacetylase activity by small molecule inhibitors or overexpression of acetyltransferases caused an enhancement of acetylated TIAM1 signal. While a change in TIAM1 localization was not observed with SIRT1 knockdown, its ability to activate Rac1 was compromised. Several studies have demonstrated SIRT1's pro-tumorigenic potential. In this study we elucidate a mechanism by which SIRT1 regulates Rac-GTPase activation via TIAM1 acetylation. Controlling the “switch-mechanism” that the small GTPases utilize for inducing downstream activity can be crucial from a drug-development perspective. Our findings not only provide a new mechanism for regulation of Rac-GTPase activity but also emphasize SIRT1's potential as a targetable molecule in cancer. This work is supported by CA155223 and the graduate stipend for Madhurima Saxena is provided by a Carroll-Feist Pre-doctoral Fellowship awarded by the Feist-Weiller Cancer Center at LSU Health Shreveport, Shreveport, LA. Citation Format: Madhurima Saxena, Samantha S. Dykes, Allison E. Wang, James A. Cardelli2,3, Kevin Pruitt1,3. SIRT1 regulates Rac1-GTPase activation via the guanine nucleotide exchange factor TIAM1. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A86.