Terminal Epithelial Differentiation Research Articles

Downregulation of p38 MAPK Signaling Pathway Ameliorates Tissue-Engineered Corneal Epithelium.

Tissue-engineered corneal epithelium transplantation is effective treatment for severe limbal stem cell deficiency (LSCD), while epithelial terminal differentiation, tans-differentiation, and insufficient stem cell during construction affect the quality of tissue-engineered corneal epithelium. In this study, we applied SB203580 in the culture medium to downregulate the p38 mitogen-activated protein kinase (MAPK) signaling pathway during construction of tissue-engineered corneal epithelium. With application of SB203580, tissue-engineered corneal epithelium showed enhanced strength and condensed structure. The expression of progenitor cell markers ATP-binding cassette sub-family G member 2, tumor protein p63, keratin 14, and Wnt family member 7A was increased, differentiation markers keratin 12, paired box 6, keratin 10, and keratin 13 and trans-differentiation markers actin alpha 2, smooth muscle and snail family transcriptional repressor 1 was decreased, while cell junction markers claudin 1 and cadherin 1 was increased in the tissue-engineered corneal epithelium. The Wnt/catenin beta 1 signaling pathway was upregulated in the epithelium after p38 MAPK inhibition. Transplantation of tissue-engineered corneal epithelium treated with SB203580 to rabbit LSCD model showed faster wound healing and improved epithelial quality. We conclude that downregulation of p38 MAPK signaling pathway helps maintain the stemness and prevent terminal differentiation and abnormal differentiation of corneal epithelial cells during epithelium construction process, and thus can improve the quality of tissue-engineered corneal epithelium. Impact statement Downregulation of p38 MAPK signaling pathway helps maintain the self-renewal of limbal stem cells and prevents terminal differentiation and abnormal differentiation of corneal epithelial cells. Small molecules modulating p38 MAPK signaling pathway ameliorate tissue-engineered corneal epithelium.

Abstract A20: Disruption and restoration of pioneer transcription factor function in bladder cancer

Abstract Urothelial bladder cancer (BLCA) is characterized by the failure to complete epithelial-differentiation. The mechanisms underlying this failure are unknown. To address this knowledge gap, we used unbiased analyzes to identify 1,000 genes significantly suppressed in BLCA vs normal bladder (p<0.001, FDR<0.01) (n=426, TCGA RNA-seq). Remarkably, these genes are located in compacted chromatin in embryonic stem cells (ESC) (decreased H3K4me3, increased CpG methylation), while 1,000 genes upregulated in BLCA vs normal bladder are located in open chromatin at this same ultimate baseline. This striking pattern in unbiased analyses suggested pioneer transcription factor (TF) loss of function in BLCA: pioneer TF access compacted chromatin to initiate the remodeling necessary for gene activation. The pioneer TF hub containing FOXA1, GATA4 is highly expressed in normal bladder, and these pioneer TFs are frequently haploinsufficient in BLCA, 32% and 56%, respectively. We pulled down endogenous FOXA1, the most expressed TF, from UM-UC-6 BLCA cells. Proteomic analyzes by mass spectrometry revealed enrichment for corepressors (DNMT1) over coactivators (ARID1A) in this pioneer TF hub. Genetic analyses showed frequent mutation/deletion of the coactivators: 43% bi-allelic ARID1A inactivation, while corepressors, e.g., CHD4 and DNMT1, were amplified or unaltered. Restoration of ARID1A into UM-UC-6 upregulated epithelial genes, downregulated MYC, upregulated CDKN1B, and terminated proliferation. To translate into therapy, we confirmed using immunoprecipitation LCMS/MS that DNMT1-depletion using noncytotoxic concentrations of the clinical drug decitabine (0.2-0.5 μM) shifted hub composition to coactivators, to thereby induce terminal epithelial-differentiation of BLCA cells. This was also seen in vivo in UM-UC-6 xenograft: very low dose non-cytotoxic decitabine (0.1 mg/kg) combined with an inhibitor of its degradation, THU, decreased tumor size more than standard high-dose cytotoxic gemcitabine/cisplatinum. Thus, BLCA suppresses hundreds of epithelial-differentiation genes via corepressor/coactivator imbalance in the bladder pioneer TF hub. Sensitivity of the pioneer TF hub to corepressor/coactivator stoichiometry, however, also enables pharmacologic reduction of corepressors to compensate for genetic reduction of coactivators, to thereby release BLCA cells to destined terminal epithelial fates. Citation Format: Caroline Schuerger, Xiaorong Gu, Yvonne Parker, Daniel Lindner, Babal Jha. Disruption and restoration of pioneer transcription factor function in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A20.

Newly recognized non-adenomatous lesions associated with enteric carcinomas in inflammatory bowel disease – Report of six rare and unique cases

It is the current view that the inflammatory bowel disease (IBD)-associated precancerous lesions may be adenomatous (with classic cytologic dysplasia) and non-adenomatous (without frank cytologic dysplasia), and the latter ones are in various histomorphologies including serrated, mucinous, eosinophilic (goblet cell deficient), and differentiated (dysplasia with terminal epithelial differentiation) types. By retrospectively reviewing the surgically resected IBD-associated colorectal and ileal carcinomas (×53), analyzing the background epithelial changes/lesions in the mucosa surrounding and adjacent to invasive carcinomas, and testing the key molecular profile (KRAS, BRAF, PIK3CA, NRAS, p53, mismatch repair proteins, and SAT-B2) known to be involved in colorectal carcinogenesis, we identified 6 representative, rare and unique cases, in which non-adenomatous lesions were clearly in vicinity and in transition to invasive carcinomas. Furthermore, we identified certain colonic carcinoma-related molecular alterations, and thus further confirmed the neoplastic nature of various non-adenomatous lesions. It was also revealed that non-adenomatous lesions are heterogeneous in both morphology and molecular alterations, and that it is common to have more than one type of lesions be associated with a carcinoma. Moreover, mixed focal adenomatous dysplasia was common, which may be the necessary step in the malignant transformation of the non-adenomatous lesions.

Abstract C58: p53/p16-independent cytoreduction of chemoresistant pancreatic adenocarcinoma by metabolically optimized epigenetic therapy

Abstract Although several reasons for chemorefractoriness of pancreatic ductal adenocarcinoma (PDAC) response have been proposed, a well-established basis is genetic inactivation of the p53/p16 apoptosis axis. Therefore, there is a need for p53/p16-independent PDAC treatments. The key epigenetic regulator DNA methyltransferase 1 (DNMT1) has been scientifically validated as a molecular target for p53-independent cytoreduction of pancreatic cancer, e.g., by activating terminal epithelial programs. Moreover, the pyrimidine nucleoside analog prodrugs decitabine (Dec) or 5-azacytidine (5Aza) are metabolically processed into a nucleotide analog that depletes DNMT1. Motivated by the scientific data, several clinical trials evaluating Dec, 5Aza, or analogs to treat PDAC have been completed. However, results from these trials have been discouraging and contradict encouraging preclinical results. To model the clinical situation, we evaluated Dec and 5Aza efficacy in gemcitabine-resistant PDAC in vitro and in mice. Gemcitabine-resistant PDAC cells were cross-resistant to Dec but not 5Aza. Measurement of the pyrimidine metabolism enzymes needed for prodrug conversion demonstrated the reason: DCK that activates gemcitabine and Dec was suppressed, but UCK2 that activates 5Aza was preserved and upregulated. Also, the catabolic enzyme cytidine deaminase (CDA), which rapidly catabolizes Dec and 5Aza into inactive uridine counterparts, was upregulated. Importantly, these shifts in pyrimidine metabolism that were observed in established gemcitabine-resistant cells were rapidly recapitulated (within hours/days) upon exposure of naïve PDAC cells to Dec or 5Aza, although 5Aza exposure immediately upregulated DCK, whereas Dec exposure immediately upregulated UCK2. Measurements of nucleotide levels by LC-MS/MS indicated that these contrasting shifts were caused by opposite effects of Dec and 5Aza on dCTP levels. These observations suggested potential solutions to overcome resistance: We evaluated alternating Dec with 5Aza, to exploit their mutual priming effects on DCK and UCK2 expression, and the addition of tetrahydrouridine (THU) to inhibit the catabolism of Dec and 5Aza by CDA. This optimized regimen was very efficacious in a murine xenograft model of gemcitabine-resistant PDAC (median vehicle control tumor measurements 972 mm3(range 726-1267.5); median THU-Dec/THU-5Aza 16 mm3 (range 0-87.5); P<0.00001). Terminal epithelial-differentiation of gemcitabine-resistant PDAC cells was confirmed by a significant increase in pancreatic epithelial markers PTF1A, MIST1, and N-cadherin, while apoptosis markers were not induced. In sum, the pyrimidine metabolism network, which senses and regulates nucleotide amounts, automatically dampens Dec (and gemcitabine) and 5Aza activity. Nevertheless, these reactions can be anticipated and exploited instead to extend and deepen DNMT1 depletion and noncytotoxic tumor cytoreduction. Citation Format: Rita Tohme, Xiaorong Gu, Asmaa El Desoky, Caroline Schuerger, Daniel Lindner, Davendra Sohal, Yogen Saunthararajah. p53/p16-independent cytoreduction of chemoresistant pancreatic adenocarcinoma by metabolically optimized epigenetic therapy [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C58.

Is TLE1 Expression Limited to Synovial Sarcoma? Our Experience at Shifa International Hospital, Pakistan.

Introduction: Synovial sarcoma (SS) accounts for 10-15 percent of adult soft tissue sarcomas. Transducin-like enhancer of split 1 (TLE1), a transcriptional repressor, is essential in hematopoiesis, neuronal differentiation, and terminal epithelial differentiation. TLE1 proteins inhibit Wnt signaling and other cell fate determination signals, and so have an established role in repressing differentiation. TLE1 has recently been shown to be a highly sensitive and relatively specific marker of SS.Materials and methods: Study design is retrospective, descriptive. A total of 25 cases of SS and 28 of soft tissue lesions were retrieved from the record. TLE1 (clone 1F5) expression was evaluated and scored as negative (<5% of cells positive), 1+ (5-25% of cells positive), 2+ (26-50% of cells positive), or 3+ (>50 % of cells positive).Result: Twenty-four out of twenty-five (96%) cases of SS showed 3+ TLE1 expression. One (4%) case of poorly differentiated SS showed 2+ positivity. 3+ TLE1 positivity was seen in one (100%) case each of infantile fibrosarcoma and low-grade fibromyxoid sarcoma, while two cases (100%) of schwannoma also showed 3+ positivity. All cases of solitary fibrous tumor) (n=2), clear cell sarcoma of tendons and aponeurosis (n=2), embryonal rhabdomyosarcoma (n=1), and de-differentiated liposarcoma (n=2) showed 2+ positivity. 1+ positivity was seen in alveolar soft part sarcoma (n=2), Ewing's sarcoma (n=4), undifferentiated pleomorphic sarcoma (n=1), myxoid liposarcoma (n=1) and malignant peripheral nerve sheath tumor (n=1). TLE1 was negative in all cases of chordomas (n=2), lipomas (n=2), nodular fasciitis (n=2), malignant perivascular epithelioid cell tumor (n=1) and dermatofibrosarcoma protuberans (n=1).Conclusion: TLE1 may be a reliable immunostain for diagnosing SS, but its expression is not limited to SS. Its expression should be interpreted in the light of morphological features and a panel of antibodies.

Slug and Snail have differential effects in directing colonic epithelial wound healing and partially mediate the restitutive effects of butyrate.

Restitution of wounds in colonic epithelium is essential in the maintenance of health. Microbial products, such as the short-chain fatty acid butyrate, can have positive effects on wound healing. We used an in vitro model of T84 colonic epithelial cells to determine if the Snail genes Slug (SNAI2) and Snail (SNAI1), implemented in keratinocyte monolayer healing, are involved in butyrate-enhanced colonic epithelial wound healing. Using shRNA-mediated Slug/Snail knockdown, we found that knockdown of Slug (Slug-KD), but not Snail (Snail-KD), impairs wound healing in scratch assays with and without butyrate. Slug and Snail had differential effects on T84 monolayer barrier integrity, measured by transepithelial resistance, as Snail-KD impaired the barrier (with or without butyrate), whereas Slug-KD enhanced the barrier, again with or without butyrate. Targeted transcriptional analysis demonstrated differential expression of several tight junction genes, as well as focal adhesion genes. This included altered regulation of Annexin A2 and ITGB1 in Slug-KD, which was reflected in confocal microscopy, showing increased accumulation of B1-integrin protein in Slug-KD cells, which was previously shown to impair wound healing. Transcriptional analysis also indicated altered expression of genes associated with epithelial terminal differentiation, such that Slug-KD cells skewed toward overexpression of secretory cell pathway-associated genes. This included trefoil factors TFF1 and TFF3, which were expressed at lower than control levels in Snail-KD cells. Since TFFs can enhance the barrier in epithelial cells, this points to a potential mechanism of differential modulation by Snail genes. Although Snail genes are crucial in epithelial wound restitution, butyrate responses are mediated by other pathways as well.NEW & NOTEWORTHY Although butyrate can promote colonic mucosal healing, not all of its downstream pathways are understood. We show that the Snail genes Snail and Slug are mediators of butyrate responses. Furthermore, these genes, and Slug in particular, are necessary for efficient restitution of wounds and barriers in T84 epithelial cells even in the absence of butyrate. These effects are achieved in part through effects on regulation of β1 integrin and cellular differentiation state.

Abstract 4316: Disruption and restoration of pioneer transcription factor hub function in bladder cancer

Abstract Urothelial bladder cancer is characterized by the failure to complete transitional epithelial-differentiation. The mechanisms underlying this failure are unknown. To address this gap in knowledge, we analyzed 2000 genes differentially expressed between normal bladder vs. bladder cancer (BLCA) (n=426, TCGA RNA sequencing). Remarkably, genes suppressed in BLCA are found in embryonic stem cells (ESC) in compacted chromatin (decreased H3K4me3, increased CpG methylation), while upregulated genes in BLCA are found in ESCs in open chromatin (H1 ESC Encode).This striking property of genes suppressed in BLCA suggested loss-of-function of pioneer transcription factor (TF) function in BLCA, as pioneer TFs access compacted chromatin to initiate the necessary remodeling for subsequent gene activation. The pioneer master TF hub containing FOXA1, GATA4/6 is highly expressed in normal bladder, and these master TFs were frequently haploinsufficient in BLCA, 32%, 56%, and 24% respectively. To better understand why target genes of FOXA1, the most expressed bladder master TF, are unremodeled and repressed, we pulled down endogenous FOXA1 from UM-UC-6 and -3 BLCA cells and used mass spectrometry and Western blot to analyze interacting coregulators, which execute the biochemical work of chromatin remodeling. These analyses revealed enrichment for corepressors, e.g., DNMT1, in the FOXA1 interactome over coactivators, e.g., ARID1A. Genetic analysis of BLCA show frequent mutation/deletion of coactivators: 43% bi-allelic ARID1A inactivation, while corepressors SMARCA5 and DNMT1 were amplified or unaltered to remain functional. Restoration of ARID1A into ARID1A haploinsufficient BLCA (UC-6) upregulated epithelial-differentiation genes and induced terminal epithelial-differentiation, downregulation of MYC and upregulation of p27 protein. To translate into therapy, we restored coactivator/corepressor balance by pharmacologic corepressor depletion: we used the clinical drug decitabine to deplete DNMT1, to compensate for genetic coactivator inactivation. Decitabine recapitulated the effects of ARID1A introduction, inducing terminal epithelial-differentiation of BLCA cells. This was also seen in vivo in a xenograft model of BLCA (UC-6). Decitabine combined with an inhibitor of its in vivo degradation, tetrahydrouridine, decreased tumor size significantly with vehicle tumor size 917.8 mm3 to treated tumor size 321.0 mm3 (p&amp;lt;0.01). In sum, genetic alterations in BLCA alter composition of a pioneer master TF hub to remove remodeling needed for transitional epithelial-differentiation. Dynamic coactivator/corepressor opposition in gene regulation, however, enables pharmacologic reduction of corepressors to compensate for genetic reduction of coactivators. Citation Format: Caroline Schuerger, Yogen Saunthararajah. Disruption and restoration of pioneer transcription factor hub function in bladder cancer [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 4316.

Logarithmic expansion of LGR5+ cells in human colorectal cancer

Stem cells of the small and large intestine are marked by expression of the Wnt target gene LGR5, a leucine-rich-repeat-containing G protein-coupled receptor. Previous studies reported increased expression of LGR5 in human colorectal cancer (CRC) compared to normal tissue either by immunohistochemistry or in situ hybridization (ISH). However, as these studies were semi-quantitative they did not provide a numerical estimate of the magnitude of this effect. While we confirm that LGR5+ cells are exclusively located at the base of normal human small and large intestinal crypts, representing approximately 6% of total crypt cells, we show this cell population is 10-fold expanded in all grades of CRC, representing as much as 70% of the cells of tumor crypt-like structures. This expansion of the LGR5 compartment coincides with maintenance of crypt-like glandular structure (adenomas, and well and moderately differentiated adenocarcinomas), and is reduced in poorly differentiated CRC, where crypt-like glandular architecture is lost, accompanied by reduced epithelial terminal differentiation. Altogether these results indicate that LGR5+ cell expansion is a hallmark of CRC tumorigenesis occurring during progression to adenoma, supporting CRC as a stem cell disease with implications for CRC therapy.

Abstract 3520: PBRM1 inactivation in renal cell cancer alters master transcription factor hub composition to repress instead of activate epithelial-differentiation

Abstract Clear cell renal cell cancers (RCC) universally inactivate PBRM1 (single allele deletion 100%, bi-allelic deletion/mutation ~40%), however, the mechanisms by which PBRM1 loss contributes to RCC-genesis are unknown. Of ~4000 genes consistently less expressed in RCC than normal renal cortex, &amp;gt;30% were kidney differentiation genes by gene ontology analysis, e.g., 394 (~10%) suppressed genes had specialized renal epithelial functions of cation transport, cell adhesion and excretion (TCGA). Suppression of hundreds of epithelial-differentiation genes suggested disruption to the core transcription factor circuit (protein network hub) activating this program. This master transcription factor hub contains PAX2, PAX8, GATA3 and LHX1, shown by murine knock-outs and human pedigrees with renal defects, and confirmed by our protein interactome studies. Of these master transcription factors, PAX2 and PAX8 expression was preserved in RCC compared to normal kidney, but downstream GATA3 and LHX1 expression was markedly decreased (2 to 4-fold) even though PAX8 localized at these target genes by chromatin immunoprecipitation - this suggested compromised PAX8 transcription activating function. PAX8 protein interactome in RCC cells indicated that PBRM1 is a PAX8 coactivator, and introduction of PBRM1 into PBRM1-haploinsufficient RCC cells (ACHN) by transfection increased activation of GATA3, WT1 and HNF4A, decreased MYC protein, increased p27/CDKN1B protein that mediates cell cycle exits by terminal differentiation, produced morphological changes of epithelial differentiation, and decreased proliferation. Coactivator versus corepressor recruitment by master transcription factors is a dynamic interchange, and the protein interactome studies indicated PBRM1 loss unbalanced stoichiometry of the PAX8-associated protein hub towards corepressors including DNMT1. Depletion of DNMT1 by non-cytotoxic concentrations of decitabine (0.5 µM) produced a remarkable shift from corepressors to coactivators in the PAX8 protein interactome, accompanied by terminal epithelial-differentiation as observed with PBRM1 reintroduction. Cell cycle exits by differentiation do not require p53/p16, and this effect was also produced in vivo in a subcutaneous xenograft model of TP53-mutated refractory/relapsed RCC, especially when decitabine in vivo pharmacology was optimized for non-cytotoxic DNMT1-depletion by avoiding a high Cmax and increasing its half-life and distribution into solid tissue, by administration of low dose in combination with an inhibitor of the enzyme cytidine deaminase that degrades decitabine. PBRM1 loss in RCC thus alters composition of a master transcription factor hub to repress instead of activate terminal epithelial-differentiation, an effect that can be reversed pharmacologically for novel, p53/p16-independent differentiation-restoring therapy. Citation Format: Xiaorong Gu, Francis Enane, Reda Mahfouz, Tomas Radivoyevitch, Bartlomiej Przychodzen, Yvonne Parker, Daniel Lindner, Brian Rini, Yogen Saunthararajah. PBRM1 inactivation in renal cell cancer alters master transcription factor hub composition to repress instead of activate epithelial-differentiation [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 3520. doi:10.1158/1538-7445.AM2017-3520

Transgenic Expression of Human Lysophosphatidic Acid Receptor LPA2 in Mouse Intestinal Epithelial Cells Induces Intestinal Dysplasia.

Lysophosphatidic acid (LPA) acts on LPA2 receptor to mediate multiple pathological effects that are associated with tumorigenesis. The absence of LPA2 attenuates tumor progression in rodent models of colorectal cancer, but whether overexpression of LPA2 alone can lead to malignant transformation in the intestinal tract has not been studied. In this study, we expressed human LPA2 in intestinal epithelial cells (IECs) under control of the villin promoter. Less than 4% of F1-generation mice had germline transmission of transgenic (TG) human LPA2; as such only 3 F1 mice out of 72 genotyped had TG expression. These TG mice appeared anemic with hematochezia and died shortly after birth. TG mice were smaller in size compared with the wild type mouse of the same age and sex. Morphological analysis showed that TG LPA2 colon had hyper-proliferation of IECs resulting in increased colonic crypt depth. Surprisingly, TG small intestine had villus blunting and decreased IEC proliferation and dysplasia. In both intestine and colon, TG expression of LPA2 compromised the terminal epithelial differentiation, consistent with epithelial dysplasia. Furthermore, we showed that epithelial dysplasia was observed in founder mouse intestine, correlating LPA2 overexpression with epithelial dysplasia. The current study demonstrates that overexpression of LPA2 alone can lead to intestinal dysplasia.

Abstract 2044: Characterization of a novel tumor suppressor PRSS8 in colorectal cancer

Abstract PRSS8, a glycosylphosphatidylinositol-anchored epithelial extracellular membrane serine protease prostasin, is expressed in normal epithelia and is involved in terminal epithelial differentiation. Aberrantly expression of PRSS8 was observed in human prostate, breast and gastric cancers, and the reduction might be linked to promoter hypermethylation in vitro. However, the expression of PRSS8 in colorectal cancer and its significance in colorectal carcinogenesis and progression are largely unknown. In this study, the expression levels of PRSS8 were firstly determined in four human colorectal cancer cell lines and in 47 paired human colorectal cancer tissues (cancers and their adjacent non-cancer tissues) by real-time PCR, Western blotting and immunohistochemistry. We found that PRSS8 was significantly reduced in colorectal cancer tissues, compared to adjacent non-cancer tissues, and was in very low levels in the colorectal cancer cell lines. Interestingly, the reduced PRSS8 was associated with histopathological stages and differentiation: PRSS8 was lower in poor differentiated cancers and was higher in well differentiated cancers. In addition, the expression levels of PRSS8 were also determined in esophageal cancer, prostate cancer, breast cancer, liver cancer and lung cancer using tissue microarray (TMA) by immunohistochemical staining. Consistent with those in colorectal cancer, PRSS8 protein levels were reduced in all the five cancers and the reduction was associated with cancer differentiation. Further analysis revealed that the patients with lower expression of PRSS8 in esophageal cancer were likely to have a shorter survival time. Functional studies by gain- and loss-expression of PRSS8 showed that increased PRSS8 expression inhibited colon cancer cell proliferation, promoted apoptosis, attenuated cell migration and epithelial-mesenchymal transition (EMT). Mechanistic study showed tumor suppression of PRSS8 was linked to Wnt/beta-catenin signaling pathways. Moreover, PRSS8 interacted with Sphk1 to target S1P and affect EMT/metastasis. Lastly, PRSS8 inhibited colorectal cancer cell growth in tumor-bearing mice, and purified PRSS8 protein showed therapeutic effects in vitro and nude mice. Taken together, this study has identified PRSS8 as a tumor suppressor in colorectal cancer and acts as a key regulator in colorectal cancer formation, progression and outcomes. Therefore, PRSS8 could be a useful biomarker for monitoring colorectal carcinogenesis and progression, and also could have a therapeutic potential for cancers. Note: This abstract was not presented at the meeting. Citation Format: Yonghua Bao, Kai Li, Qian Wang, Yongchen Guo, Rongfei Han, Zhiguo Chen, Zexin Li, Jianguo Wang, Weixing Zhao, Wenliang Han, Jiaqi Wang, Huijuan Zhang, Pan He, Wancai Yang. Characterization of a novel tumor suppressor PRSS8 in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2044. doi:10.1158/1538-7445.AM2015-2044

Oral-specific ablation of Klf4 disrupts epithelial terminal differentiation and increases premalignant lesions and carcinomas upon chemical carcinogenesis

Squamous cell carcinoma (SSC) of the head and neck is the sixth most common cancer and is rarely diagnosed in early stages. The transcription factor Krϋppel-like factor 4 (Klf4) suppresses cell proliferation and promotes differentiation. Inducible mice carrying an oral-specific ablation of Klf4 (K14-CreER(tam) /Klf4(flox/flox) ) develop mild dysplastic lesions and abnormal differentiation in the tongue. Aiming to analyze whether Klf4 cooperate in oral chemical carcinogenesis,we applied 4-nitroquinoline 1-oxide (4NQO), a tobacco surrogate, to this conditional Klf4 knockout mice. K14-CreER(tam) /Klf4(flox/flox) and control mice were treated with 4NQO for 16 weeks and monitored until week 30. Histopathological samples were used for diagnostic purposes and immunofluorescence detection of epithelial differentiation markers. 4NQO-treated K14-CreER(tam) /Klf4(flox/flox) mice (Klf4KO 4NQO) showed a significant weight loss and developed more severe dysplastic lesions than control mice with 4NQO (P < 0.005). The Klf4KO 4NQO showed a tendency to higher incidence of oral SCC and a marked keratinization pattern in dysplasias, in situ carcinomas and SCC. Also, tongues derived from Klf4KO 4NQO mice exhibited reduced terminal differentiation as judged by cytokeratin 1 staining when compared with 4NQO-treated controls. Klf4 ablation results in more severe dysplastic lesions in oral mucosa, with a tendency to higher incidence of SCC, after chemical carcinogenesis. We show here, in a context similar to the human carcinogenesis, that absence of Klf4 accelerates carcinogenesis and correlates with the absence of cytokeratin 1 expression. These results suggest a potential role for KLF4 as a tumor suppressor gene for the tongue epithelium.

Human papillomavirus type 1 E1^E4 protein is a potent inhibitor of the serine-arginine (SR) protein kinase SRPK1 and inhibits phosphorylation of host SR proteins and of the viral transcription and replication regulator E2.

The serine-arginine-specific protein kinase SRPK1 is a common binding partner of the E1^E4 protein of diverse human papillomavirus types. We show here for the first time that the interaction between HPV1 E1^E4 and SRPK1 leads to potent inhibition of SRPK1 phosphorylation of host serine-arginine (SR) proteins that have critical roles in mRNA metabolism, including pre-mRNA processing, mRNA export, and translation. Furthermore, we show that SRPK1 phosphorylates serine residues of SR/RS dipeptides in the hinge region of the HPV1 E2 protein in in vitro kinase assays and that HPV1 E1^E4 inhibits this phosphorylation. After mutation of the putative phosphoacceptor serine residues, the localization of the E2 protein was altered in primary human keratinocytes; with a significant increase in the cell population showing intense E2 staining of the nucleolus. A similar effect was observed following coexpression of E2 and E1^E4 that is competent for inhibition of SRPK1 activity, suggesting that the nuclear localization of E2 is sensitive to E1^E4-mediated SRPK1 inhibition. Collectively, these data suggest that E1^E4-mediated inhibition of SRPK1 could affect the functions of host SR proteins and those of the virus transcription/replication regulator E2. We speculate that the novel E4 function identified here is involved in the regulation of E2 and SR protein function in posttranscriptional processing of viral transcripts. The HPV life cycle is tightly linked to the epithelial terminal differentiation program, with the virion-producing phase restricted to differentiating cells. While the most abundant HPV protein expressed in this phase is the E4 protein, we do not fully understand the role of this protein. Few E4 interaction partners have been identified, but we had previously shown that E4 proteins from diverse papillomaviruses interact with the serine-arginine-specific protein kinase SRPK1, a kinase important in the replication cycles of a diverse range of DNA and RNA viruses. We show that HPV1 E4 is a potent inhibitor of this host cell kinase. We show that E4 inhibits SRPK1 phosphorylation, not only of cellular SR proteins involved in regulating alternative splicing of RNA but also the viral transcription/replication regulator E2. Our findings reveal a potential E4 function in regulation of viral late gene expression through the inhibition of a host cell kinase.

Melanoma differentiation-associated gene-7/interleukin-24 as a potential prognostic biomarker and second primary malignancy indicator in head and neck squamous cell carcinoma patients.

The significance of melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) expression in head and neck squamous cell carcinoma (HNSCC) remains unclear. This study was designed to investigate and evaluate the clinical significance of MDA-7/IL-24 expression in HNSCC by detecting expression by immunostaining in 131 HNSCC specimens. The function of MDA-7/IL-24 was investigated by real-time polymerase chain reaction (PCR) and Western blot in Ad5.mda-7-infected HNSCC cell lines. Our results showed that MDA-7/IL-24 was mainly expressed in the cytoplasm of HNSCC cells. MDA-7/IL-24 high patients presented with a favorable postoperative prognosis compared with MDA-7/IL-24 low patients, and high expression of MDA-7/IL-24 was significantly correlated with a lower incidence of second primary malignancies (SPMs) in the head and neck regions. In vitro assays showed that high expression of MDA-7/IL-24 could upregulate the expression of the epithelial terminal differentiation markers cytokeratin (KRT) 1, KRT4, KRT13, phosphorylated endoplasmic reticulum stress protein (p)-EIF2a, and the apoptosis-related protein cleaved caspase-3. It also downregulated the epithelial proliferative markers KRT5, KRT14, Integrin β4, and anti-apoptosis protein Bcl-2, which might be partially involved in the underlying mechanisms of Ad.mda-7-mediated HNSCC differentiation and apoptosis. Our results indicate that MDA-7/IL-24 can be a prognostic biomarker and an indicator of second primary malignancies (SPM) in HNSCC.

Tumor Initiating Cells and Chemoresistance: Which Is the Best Strategy to Target Colon Cancer Stem Cells?

There is an emerging body of evidence that chemoresistance and minimal residual disease result from selective resistance of a cell subpopulation from the original tumor that is molecularly and phenotypically distinct. These cells are called “cancer stem cells” (CSCs). In this review, we analyze the potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies for metastatic colon cancer. These include induction of terminal epithelial differentiation of CSCs or targeting some genes expressed only in CSCs and involved in self-renewal and chemoresistance. Ideal targets could be cell regulators that simultaneously control the stemness and the resistance of CSCs. Another important aspect of cancer biology, which can also be harnessed to create novel broad-spectrum anticancer agents, is the Warburg effect, also known as aerobic glycolysis. Actually, little is yet known with regard to the metabolism of CSCs population, leaving an exciting unstudied avenue in the dawn of the emerging field of metabolomics.

Interactions between BdkrB2 and p53 genes in the developing kidney

Gene-environment interactions are implicated in congenital disorders. Accordingly, there is a pressing need to develop animal models of human disease, which are the product of defined gene-environment interactions. Work from our laboratory demonstrates the presence of genetic interactions between the bradykinin B2 receptor (BdkrB2) and the tumor suppressor protein p53 in the developing kidney. Our studies have shown that the Bdkrb2(-/-) embryos exposed to gestational salt stress develop renal dysgenesis. The underlying mechanism is p53 stabilization and mediated apoptosis and repression of the terminal epithelial differentiation program. We also uncovered a novel functional cross-talk between p53 and BdkrB2. Thus, while BdkrB2 is a target for p53-mediated transcriptional activation, BdkrB2 inactivation results in the upregulation of checkpoint kinase 1 (Chk1) levels, thus potentiating phosphorylation of p53 on Ser23 by Chk1, an essential step in the pathway leading to renal dysgenesis in salt-stressed BdkrB2(-/-) mutant mice. Future studies will now focus on defining how this G-protein-coupled receptor is coupled to the activation of p53, a tumor suppressor gene that is mutated in more than 50% of all human cancers.

Enhanced propagation of adult human renal epithelial progenitor cells to improve cell sourcing for tissue-engineered therapeutic devices for renal diseases

Renal cell therapy employing cells derived from adult renal epithelial cell (REC) progenitors promises to reduce the morbidity of patients with renal insufficiency due to acute renal failure and end stage renal disease. To this end, tissue engineered devices addressing the neglected biologic component of renal replacement therapy are being developed. Because human donor tissue is limited, novel enhanced progenitor cell propagation (EP) techniques have been developed and applied to adult human kidney transplant discards from six donors. Changes include more efficient digestion and the amplification of progenitors prior to terminal epithelial differentiation promoted by contact inhibition and the addition of retinoic acid. Differentiated morphology in EP populations was demonstrated by the ability to form polarized epithelium with tight junctions, apical central cilia and expression of brush border membrane enzymes. Evaluation of lipopolysaccharide stimulated interleukin-8 secretion and γ-glutamyl transpeptisade activity in EP derived cells was used to confirm therapeutic equivalence to REC obtained using published techniques, which have previously shown efficacy in large animal models and clinical trials. Yield exceeded 10(16) cells/gram cortex from the only kidney obtained due to an anatomical defect, while the average yield from diseased kidneys ranged from 1.1 × 10(9) to 8.8 × 10(11) cells/gram cortex, representing an increase of more than 10 doublings over standard methods. Application of the EP protocol to REC expansion has solved the problem of cell sourcing as the limiting factor to the manufacture of cell based therapies targeting renal diseases and may provide a method for autologous device fabrication from core kidney biopsies.

Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT)

BackgroundThe glycosylphosphatidylinositol (GPI)-anchored epithelial extracellular membrane serine protease prostasin (PRSS8) is expressed abundantly in normal epithelia and essential for terminal epithelial differentiation, but down-regulated in human prostate, breast, and gastric cancers and invasive cancer cell lines. Prostasin is involved in the extracellular proteolytic modulation of the epidermal growth factor receptor (EGFR) and is an invasion suppressor. The aim of this study was to evaluate prostasin expression states in the transitional cell carcinomas (TCC) of the human bladder and in human TCC cell lines.MethodsNormal human bladder tissues and TCC on a bladder cancer tissue microarray (TMA) were evaluated for prostasin expression by means of immunohistochemistry. A panel of 16 urothelial and TCC cell lines were evaluated for prostasin and E-cadherin expression by western blot and quantitative PCR, and for prostasin gene promoter region CpG methylation by methylation-specific PCR (MSP).ResultsProstasin is expressed in the normal human urothelium and in a normal human urothelial cell line, but is significantly down-regulated in high-grade TCC and lost in 9 (of 15) TCC cell lines. Loss of prostasin expression in the TCC cell lines correlated with loss of or reduced E-cadherin expression, loss of epithelial morphology, and promoter DNA hypermethylation. Prostasin expression could be reactivated by demethylation or inhibition of histone deacetylase. Re-expression of prostasin or a serine protease-inactive variant resulted in transcriptional up-regulation of E-cadherin.ConclusionLoss of prostasin expression in bladder transitional cell carcinomas is associated with epithelial-mesenchymal transition (EMT), and may have functional implications in tumor invasion and resistance to chemotherapy.

TLE1 expression is not specific for synovial sarcoma: a whole section study of 163 soft tissue and bone neoplasms

TLE1, a transcriptional repressor essential in hematopoiesis, neuronal differentiation and terminal epithelial differentiation, has recently been shown in a single tissue microarray study to be a highly sensitive and relatively specific marker of synovial sarcomas. Expression of TLE1 has not, however, been studied in standard sections of soft tissue and bone tumors. We investigated TLE1 expression in a large series of well-characterized mesenchymal tumors, to more fully characterize the range of TLE1 expression. Standard sections of 163 bone and soft tissue tumors were immunostained for TLE1 (sc-9121, 1:100; Santa Cruz Biochemicals) using the Dako Dual Envision+ detection system. Nuclear positivity was scored as negative (<5% of cell positive), 1+ (5–25% of cells positive), 2+ (25–50% of cells positive), and 3+ (>50% of cells positive). Overall, TLE1 was expressed by 18 of 20 (90%) of synovial sarcoma, with 16 cases (89%) showing 2–3+ positivity. However, TLE1 expression was also seen in 53 of 143 (37%) non-synovial sarcoma, with 36 such cases (25%) showing 2–3+ positivity. TLE1 expression was commonly seen in peripheral nerve sheath tumors, including 33% of neurofibromas, 100% of schwannomas, and 30% of malignant peripheral nerve sheath tumors. Among non-neoplastic tissues, nuclear TLE1 expression was variably present in basal keratinocytes, adipocytes, perineurial cells, endothelial cells and mesothelial cells. Our study confirms the excellent sensitivity of TLE1 for synovial sarcoma. However, TLE1 expression is by no means specific for synovial sarcoma, being present in a number of tumors, which enter its differential diagnosis, in particular tumors of peripheral nerve sheath origin. Heterogeneity of TLE1 expression likely explains the differences between the present standard section study and the earlier TMA study. TLE1 may be of value in the differential diagnosis of synovial sarcoma, but should be used only in the context of a panel of antibodies. Morphology, ancillary immunohistochemistry for traditional markers such as cytokeratins and CD34, and molecular confirmation of synovial sarcoma-associated fusion genes should remain the ‘gold standards’ for this diagnosis.

A crucial role of WW45 in developing epithelial tissues in the mouse

The role and molecular mechanisms of a new Hippo signalling pathway are not fully understood in mammals. Here, we generated mice that lack WW45 and revealed a crucial role for WW45 in cell-cycle exit and epithelial terminal differentiation. Many organs in the mutant mouse embryos displayed hyperplasia accompanied by defects in terminal differentiation of epithelial progenitor cells owing to impaired proliferation arrest rather than intrinsic acceleration of proliferation during differentiation. Importantly, the MST1 signalling pathway is specifically activated in differentiating epithelial cells. Moreover, WW45 is required for MST1 activation and translocation to the nucleus for subsequent LATS1/2 activation upon differentiation signal. LATS1/2 phosphorylates YAP, which, in turn, translocates from the nucleus into the cytoplasm, resulting in cell-cycle exit and terminal differentiation of epithelial progenitor cells. Collectively, these data provide compelling evidence that WW45 is a key mediator of MST1 signalling in the coordinate coupling of proliferation arrest with terminal differentiation for proper epithelial tissue development in mammals.