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Phenotypic plasticity, defined as the ability of individual cells with stable genotypes to exert different phenotypes upon exposure to specific environmental cues, represent the quintessential hallmark of the cancer cell en route from the primary lesion to distant organ sites where metastatic colonization will occur. Phenotypic plasticity is driven by a broad spectrum of epigenetic mechanisms that allow for the reversibility of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT/MET). By taking advantage of the co-existence of epithelial and quasi-mesenchymal cells within immortalized cancer cell lines, we have analyzed the role of EMT-related gene isoforms in the regulation of epithelial mesenchymal plasticity (EMP) in high grade serous ovarian cancer. When compared with colon cancer, a distinct spectrum of downstream targets characterizes quasi-mesenchymal ovarian cancer cells, likely to reflect the different modalities of metastasis formation between these two types of malignancy, i.e. hematogenous in colon and transcoelomic in ovarian cancer. Moreover, upstream RNA-binding proteins differentially expressed between epithelial and quasi-mesenchymal subpopulations of ovarian cancer cells were identified that underlie differential regulation of EMT-related isoforms. In particular, the up- and down-regulation of RBM24 and ESRP1, respectively, represent a main regulator of EMT in ovarian cancer cells. To validate the functional and clinical relevance of our approach, we selected and functionally analyzed the Tropomyosin 1 gene (TPM1), encoding for a protein that specifies the functional characteristics of individual actin filaments in contractile cells, among the ovarian-specific downstream AS targets. The low-molecular weight Tpm1.8/9 isoforms are specifically expressed in patient-derived ascites and promote invasion through activation of EMT and Wnt signaling, together with a broad spectrum of inflammation-related pathways. Moreover, Tpm1.8/9 expression confers resistance to taxane- and platinum-based chemotherapy. Small molecule inhibitors that target the Tpm1 isoforms support targeting Tpm1.8/9 as therapeutic targets for the development of future tailor-made clinical interventions., (© 2024. The Author(s).)

Human embryonal carcinoma (EC) cells comprise the pluripotent stem cells of malignant non-seminomatous germ cell tumors (GCTs) and represent the malignant counterpart of embryonic stem cells (ESCs). WNT/β-catenin signaling has been implicated in regulating adult and embryonic stem cells although its role in EC cells is less investigated. Here, we studied WNT signaling in a panel of representative pluripotent and nullipotent human EC cell lines. We found that EC cell lines show distinct levels of intrinsic WNT signaling and respond differently to ectopic WNT activation. Short-term activation of WNT signaling induced a differentiation-response in the pluripotent EC cells (NT2 and NCCIT) whereas the nullipotent EC cells (TERA1 and 2102Ep) were refractory and maintained high levels of OCT4 and SSEA4 expression. Long-term activation of WNT signaling in NCCIT and, to a lesser extent, TERA1 cells led to (re)gain of OCT4 expression and a switch from SSEA4 to SSEA1 surface antigens ultimately resulting in OCT4 + /SSEA4 - /SSEA1 + profile. Cisplatin treatment indicated that the OCT4 + /SSEA4 - /SSEA1 + NCCIT cells became more resistant to chemotherapy treatment. Our findings are of particular interest for the GCT and ES cell biology and shed light on the role of WNT signaling in human EC cells.

Colorectal cancer (CRC) has long been known for its tight association with chronic inflammation, thought to play a key role in tumor onset and malignant progression through the modulation of cancer stemness. However, the underlying molecular and cellular mechanisms are still largely elusive. Here we show that the IL-6/STAT3 inflammatory signaling axis induces the deacetylation of FRA1 at the Lys-116 residue located within its DNA-binding domain. The HDAC6 deacetylase underlies this key modification leading to the increase of FRA1 transcriptional activity, the subsequent transactivation of NANOG expression, and the acquisition of stem-like cellular features. As validated in a large (n = 123) CRC cohort, IL-6 secretion was invariably accompanied by increased FRA1 deacetylation at K116 and an overall increase in its protein levels, coincident with malignant progression and poor prognosis. Of note, combined treatment with the conventional cytotoxic drug 5-FU together with Tubastatin A, a HDAC6-specific inhibitor, resulted in a significant in vivo synergistic inhibitory effect on tumor growth through suppression of CRC stemness. Our results reveal a novel transcriptional and posttranslational regulatory cross-talk between inflammation and stemness signaling pathways that underlie self-renewal and maintenance of CRC stem cells and promote their malignant behavior. Combinatorial treatment aimed at the core regulatory mechanisms downstream of IL-6 may offer a novel promising approach for CRC treatment.

The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5 + crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5 + CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5 + CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5 + CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5 + CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5 + CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.

The central dogma of molecular biology states that the flow of genetic information moves from DNA to RNA to protein. However, in the last decade this dogma has been challenged by new findings on non-coding RNAs (ncRNAs) such as microRNAs (miRNAs). More recently, long non-coding RNAs (lncRNAs) have attracted much attention due to their large number and biological significance. Many lncRNAs have been identified as mapping to regulatory elements including gene promoters and enhancers, ultraconserved regions and intergenic regions of protein-coding genes. Yet, the biological function and molecular mechanisms of lncRNA in human diseases in general and cancer in particular remain largely unknown. Data from the literature suggest that lncRNA, often via interaction with proteins, functions in specific genomic loci or use their own transcription loci for regulatory activity. In this review, we summarize recent findings supporting the importance of DNA loci in lncRNA function and the underlying molecular mechanisms via cis or trans regulation, and discuss their implications in cancer. In addition, we use the 8q24 genomic locus, a region containing interactive SNPs, DNA regulatory elements and lncRNAs, as an example to illustrate how single-nucleotide polymorphism (SNP) located within lncRNAs may be functionally associated with the individual's susceptibility to cancer.

Lynch syndrome is the most common form of hereditary colorectal cancer (CRC). This review covers the cardinal features of Lynch syndrome with particular emphasis upon its diagnostic criteria, molecular genetics, natural history, genetic counseling, surveillance and management. Considerable attention has been given to the etiologic role of mismatch repair (MMR) genes as well as low penetrance alleles and modifier genes. The American founder mutation, a deletion of exons 1-6 of MSH2, is discussed in some detail, owing to its high frequency in the US (19 000-30 000 carriers). Genetic counseling is essential prior to patients' undergoing DNA testing and again when receiving their test results. Families with a lower incidence of CRC and extracolonic cancers, in the face of being positive for Amsterdam I criteria but who do not have MMR deficiency by tumor testing, are probably not Lynch syndrome, and thereby should preferably be designated as familial CRC of undetermined type. Patients who are either noncompliant or poorly compliant with colonoscopy, and who are MMR mutation positive, may be candidates for prophylactic colectomy, while MMR mutation-positive women who are noncompliant with gynecologic surveillance may be candidates for prophylactic hysterectomy and bilateral salpingo-oophorectomy.

The Smad4(+/E6sad) mouse carries a null mutation in the endogenous Smad4 gene resulting in serrated adenomas and mixed polyposis of the upper gastrointestinal (GI) tract with 100% penetrance. Here, we show by loss of heterozygosity (LOH) analysis and immunohistochemistry (IHC) that, although the majority of the tumors appear at 9 months of age, somatic loss of the wild-type Smad4 allele occurs only at later stages of tumor progression. Hence, haploinsufficiency underlies Smad4-driven tumor initiation in the GI tract. As both the Apc and Smad4 tumor suppressor genes map to mouse chromosome 18, we have bred Smad4(+/E6sad) with the Apc(+/1638N) model to generate two distinct compound heterozygous lines carrying both mutations either in cis (CAS) or in trans (TAS). Strikingly, both models show increased tumor multiplicities when compared with the single mutant littermates, although CAS mice are more severely affected and became moribund at only 5-6 weeks of age. Phenotypic and molecular analyses indicate that Smad4 haploinsufficiency is sufficient to significantly affect tumor initiation and progression both prior to and upon loss of Apc function. Moreover, complete loss of Smad4 strongly enhances Apc-driven tumor formation.

Aggressive fibromatosis (desmoid tumor) is a locally invasive soft tissue neoplasm associated with mutations resulting in beta-catenin-mediated transcriptional activation. This tumor is composed of cells with histological and molecular characteristics common to proliferating mesenchymal cells of dermal wounds. Using immunohistochemistry and RT-PCR, we show that Rhamm, a protein with an important role in wound healing and neoplastic progression, is also expressed at high levels in aggressive fibromatosis. A mouse harboring a targeted deletion in Rhamm was generated, resulting in viable Rhamm-/- animals. Rhamm-/- mice were crossed with Apc/Apc1638N mice, which harbor a targeted mutation in the Apc gene predisposing animals to gastrointestinal and aggressive fibromatosis tumors. Rhamm deficiency significantly decreased the number of aggressive fibromatosis tumors formed, but did not alter the number of gastrointestinal polyps. Cell culture studies show that Rhamm regulates cell proliferation in both fibroblasts and fibromatosis cells under conditions of low density, but not high density. These results suggest that Rhamm regulates proliferation of cells with sparse cell-cell contacts, such as occurs in aggressive fibromatosis; provides the first genetic evidence implicating Rhamm in tumor pathology; and suggest Rhamm blockade as a potential therapeutic target for this otherwise difficult-to-treat neoplasm.

A mouse model with a targeted mutation in the 3' end of the endogenous Brca1 gene, Brca1(1700T), was generated to compare the phenotypic consequences of truncated Brca1 proteins with other mutant Brca1 models reported in the literature to date. Mice heterozygous for the Brca1(1700T) mutation do not show any predisposition to tumorigenesis. Treatment of these mice with ionizing radiation or breeding with Apc, Msh-2 or Tp53 mutant mouse models did not show any change in the tumor phenotype. Like other Brca1 mouse models, the Brca1(1700T) mutation is embryonic lethal in homozygous state. However, homozygous Brca1(1700T) embryos reach the headfold stage but are delayed in their development and fail to turn. Thus, in contrast to Brca1(null) models, the mutant embryos do not undergo growth arrest leading to a developmental block at 6.5 dpc, but continue to proliferate and differentiate until 9.5 dpc. Homozygous embryos die between 9.5-10.5 dpc due to massive apoptosis throughout the embryo. These results indicate that a C-terminal truncating Brca1 mutation removing the last BRCT repeat has a different effect on normal cell function than does the complete absence of Brca1.

Aggressive fibromatosis is a locally invasive soft tissue lesion. Seventy-five per cent of cases harbor a somatic mutation in either the APC or beta-catenin genes, resulting in beta-catenin protein stabilization. Cyclooxygenase-2 (COX-2) is an enzyme involved in prostaglandin synthesis that modulates the formation of colonic neoplasia, especially in cases due to mutations resulting in beta-catenin stabilization. Human aggressive fibromatoses and lesions from the Apc+/Apc1638N mouse (a murine model for Apc-driven fibromatosis) demonstrated elevated COX-2 levels. COX-2 blockade either by the selective agent DFU or by non-selective COX blocking agents results in reduced proliferation in human tumor cell cultures. Breeding mice with Cox-2-/- mice resulted in no difference in number of aggressive fibromatoses formed, but in a smaller tumor size, while there was a decrease in number of GI lesions by 50%. Mice fed various COX blocking agents also showed a decline in tumor size. COX-2 expression was regulated by tcf-dependent transcription in this lesion. COX-2 partially regulates proliferation due to beta-catenin stabilization in aggressive fibromatosis. Although COX blockade alone does not cause tumor regression, this data suggests that it may have a role as an adjuvant therapy to slow tumor growth in this lesion.

Mutation searching was performed in the hMSH2 and hMLH1 genes in 20 Portuguese families representing 124 registered affected individuals. Of the 20, 16 fulfilled the classic 'Amsterdam' criteria for HNPCC, whereas the remaining four families satisfied a modified set of criteria. These criteria required a CRC diagnosed before age 50 years and cancers diagnosed in two other relatives within the HNPCC spectrum. A multi-method approach was performed using the protein truncation test (PTT), single strand conformation polymorphism (SSCP) with two different sets of conditions, heteroduplex analysis (HA) and denaturing gradient gel electrophoresis (DGGE). Putative phenotype-genotype correlations were also explored. Ten different germline mutations were identified. Six of these were found in hMLH1 in seven families and four in hMSH2 in four families. SSCP and DGGE had the highest diagnostic yields with the percentage of variants detected above 67% and together HA and PTT had the lowest. No single technique detected all variants. Trends for the absence of extracolonic manifestations were observed in families carrying hMLH1 germline mutations (four of seven in hMLH1 vs one of four in hMSH2). Most of the families with rectal cancer were associated with hMLH1 (six of seven in hMLH1 vs two of four in hMSH2). A multi-technique approach is necessary to identify a high percentage of germline mutations. Seven novel mutations were found in this Portuguese population.

Familial adenomatous polyposis (FAP) can be considered as a condition of the whole body as extracolonic features derived from all the three embryonic lineages are recorded with varying frequency in addition to the presence of multiple adenomas in the large intestine. Here, we describe two unrelated cases of FAP with unusual extracolonic phenotypes, namely several abnormalities of mesodermal origin strongly resembling Marfan syndrome (MFS) or a Marfan-like habitus. Conventional cytogenetic and FISH analysis did not reveal any gross chromosomal rearrangement on the long arm of chromosome 5 where the APC and FBN2 genes were located. However, in case 2 the FAP-causing mutation in the APC gene was found in the donor splice site of exon 4 and was shown to result in a frameshift and a premature termination codon. We propose that such connective tissue abnormalities may result from germline APC mutations in combination with specific genetic and/or environmental modifying factors.