Roles of Tumor Suppressor Signaling on Reprogramming and Stemness Transition in Somatic Cells

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Physiological β-catenin signaling controls self-renewal networks and generation of stem-like cells from nasopharyngeal carcinoma

BACKGROUND: A few reports suggested that low levels of Wnt signaling might drive cell reprogramming, but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally, whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53, which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells. RESULTS: Introduction of increased β-catenin signaling, haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3, resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines, but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties, including enhancement of CD24(+) and CD44(+) populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells, including activation of p53- and RB1-mediated tumor suppressor pathways, up-regulation of Nanog-, Oct4-, Sox2-, and Klf4-mediated pluripotency networks, and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition, TGF-β, Activin, BMPR, FGFR2, and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays, a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9, CD24, CD44, CD90, and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres. CONCLUSIONS: Wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes, tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks

Tumor suppressive role of chromosomes 11, 13, and 14 in esophageal squamous cell carcinoma studied by functional complementation

Despite the abundant evidence for high allelic loss of chromosome 14q in human cancers (Lee et al., 1997; Chang et al., 1995; Mutirangura et al., 1998; Hu et al., 1999; Hu et al., 2000; Dekken et al., 1999), tumor suppressor genes mapped to this chromosome have yet to be identified due to the complexity of the chromosomal alterations reported. To narrow down the search for candidate genes, we performed monochromosome transfer of chromosome 14 into an esophageal squamous cell carcinoma (ESCC) cell line, SLMT-1 S1. Statistically significant suppression of the tumorigenic potential of microcell hybrids (MCHs), containing the transferred chromosome 14, provides functional evidence that tumor suppressive regions on chromosome 14 are essential for ESCC. Tumor segregants (TSs) emerging in the nude mice during the tumorigenicity assay were analyzed by detailed PCR-microsatellite typing to identify non-randomly eliminated critical regions (CRs). A 680 kb CR mapped to 14q32.13 and a ~2.2 Mb CR mapped to 14q32.33 were delineated. Dual color bacterial artificial chromosome fluorescent in situ hybridization (BAC FISH) analysis of MCHs and TSs verified the selective loss of the 14q32.13 region. In contrast, similar transfers of an intact chromosome 11 into SLMT-1 S1 did not significantly suppress tumor formation. These functional complementation studies showing the correlation of tumorigenic potential with critical regions of chromosome 14 validate the importance of the 14q32 region in tumor suppression in ESCC. The present study also paves the path for further identification of novel tumor suppressor genes (TSGs), which are relevant in the molecular pathogenesis of ESCC. Chromosomal regions with a high rate of loss of heterozygosity (LOH) may implicate candidate TSG(s) that is/are involved in the molecular pathogenesis of ESCC. In ESCC, chromosome 13q LOH ranged from 48% - l00%, as independently observed from different groups (Boynton et al., 1991; Huang et al., 1992; Hu et al., 2000; Li et al., 2001; Li et al., 2003; Hu et al., 2003) and 13q genetic aberrations of 22% - 100% revealed by comparative genomic hybridization (CGH) (Pack et al., 1999; Tada et al., 2000; Wei et al., 2002). However, it is still unclear if the loss of genetic materials is a cause or consequence of ESCC, as this indirect evidence only implicates the presence of candidate TSG(s). Direct functional evidence for tumor suppression after transferring genetic materials is still scanty in ESCC. A tumor suppression effect was observed after the transfer of chromosome 13 into SLMT-1 S1. The tumor suppressive effect observed in MCH13-113 suggested TSGs may be located at 13q34. Three critical regions, CR1 and CR2, at 13q12.3, and CR3 at 13q14.11, were delineated during TS deletion analysis. TSGs important for ESCC may be located on 13q12.3 and 13q14.11. The first functional proof from microcell-mediated chromosome transfer (MMCT) that tumor suppressive regions on chromosomes 13 and 14 are essential for ESCC development is provided in the current study

FANCD2 Confers a Malignant Phenotype in Esophageal Squamous Cell Carcinoma by Regulating Cell Cycle Progression

Fanconi anemia patients with germline genetic defects in FANCD2 are highly susceptible to cancers. Esophageal squamous cell carcinoma (ESCC) is a deadly cancer. Little is known about the function of FANCD2 in ESCC. For detailed molecular and mechanistic insights on the functional role of FANCD2 in ESCC, in vivo and in vitro assays and RNA sequencing approaches were used. Utilizing Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology, FANCD2 knockout models were established to examine the functional impact in mouse models for tumor growth and metastasis and in vitro assays for cell growth, cell cycle, and cellular localization. Our RNA sequence analyses were integrated with public datasets. FANCD2 confers a malignant phenotype in ESCC. FANCD2 is significantly upregulated in ESCC tumors, as compared to normal counterparts. Depletion of FANCD2 protein expression significantly suppresses the cancer cell proliferation and tumor colony formation and metastasis potential, as well as cell cycle progression, by involving cyclin-CDK and ATR/ATM signaling. FANCD2 translocates from the nucleus to the cytoplasm during cell cycle progression. We provide evidence of a novel role of FANCD2 in ESCC tumor progression and its potential usefulness as a biomarker for ESCC disease management

Validation analysis for the CTC enumeration pipeline.

<p>(A) CTC counts are significantly higher in cancer patients (n = 56) than those in healthy individuals (n = 21), <i>p</i> = 0.004. (B) A bar chart displaying individual CTC counts for each cancer patient and healthy donor. (C) Receiver operating characteristic (ROC) plot exhibiting the power of CTC counts in differentiating samples from cancer patients and healthy individuals. (D) Univariate and multivariate analyses testing for the correlation of the number of CTCs, dual-positive cells, and total cells with the clinical presentation of cancer (i.e. presence of cancer). (E) Inter-experimental reproducibility of CTC assays. SD: standard deviation CV: coefficient of variation (CV).</p

Sequencing results for the CTC microfluidic outputs from patients.

<p>Sequencing results for the CTC microfluidic outputs from patients.</p

Schematic diagram of the 2-step library preparation.

<p>(A) Libraries were prepared by two PCRs. The primer pair for the first PCR contains 1) target-hybridizing sequences (blue), which bind to and amplify the exon targets; 2) a 10-bp diversifier sequence composed of random nucleotides (orange), which promotes accurate DNA cluster detection during sequencing runs; 3) a portion of P5 and P7 adapter sequences (green). The universal primer pairs for the second PCR add a full P5 and P7 sequencing adapter (green) to the libraries. Primer sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177276#pone.0177276.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177276#pone.0177276.s002" target="_blank">S2</a> Tables. (B) Kaplan-Meier survival curves of cancer patients stratified by existence of mutations in their tumor tissues. Patients were grouped into “No mutation” or “With mutation(s)” for analysis, according to their mutation status of <i>NRAS</i>, <i>ESR1</i>, <i>EGFR</i>, <i>KRAS</i>, <i>BRAF and TP53</i> genes. The log-rank test <i>p</i> values comparing two survival curves are shown in each plot.</p