Mutations In Wnt Pathway Genes Research Articles

Comprehensive characterization of mesothelin expression in colorectal cancer.

245 Background: Mesothelin (MSLN) is highly expressed in several malignancies including colorectal cancer (CRC) compared to normal tissue and is associated with tumor cell proliferation and worse patient survival. MSLN could be a potential target for antigen-specific therapy. Here, we characterized the MSLN expression in CRC and its association with molecular and immune-related markers. Methods: 15,673 CRC samples were tested by NGS (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). Microsatellite-instability (MSI) was tested by fragment analysis, IHC, and NGS. Tumor mutational burden (TMB) totaled somatic mutations per tumor (high>10 mt/MB). Tumors with MSLN-high(H) and MSLN-low(L) expression were classified by top and bottom quartile, respectively. Real world overall survival (OS) was extracted from insurance claims and calculated using Kaplan-Meier estimates for molecularly defined cohorts from tissue collection to last contact. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q<0.05). Results: There was no difference in median age or gender distribution between MSLN-H and MSLN-L CRC tumors. Right-sided tumors had increased median MSLN expression compared to rectal, transverse, and left-sided tumors (6.12 vs 5.29 vs 5.35 vs 4.81 TPM, q<0.05). Median MSLN expression was higher in metastatic compared to primary tumors (1.21-fold, q<0.05). Median MSLN expression was highest in CMS4 (10.17 TPM), but, in MSS CRC, MSLN expression was highest in CMS1 (10.46 TPM, all q<0.05). MSLN-H tumors had higher frequency of KRAS (59.8% vs 36.4% ) and BRAF (13.1% vs 9.2% ) mutations but lower frequency of APC mutations (67.5% vs 77.2%), TMB High (9.8% vs 12%) and dMMR/MSI-H (6.6% vs 8.9%) compared to MSLN-L tumors (q<0.05). Further, MSLN-H tumors had higher MAPK activation score (MPAS) compared to MSLN-L tumors (1.72-fold, q<0.05). Similarly, in the MSS only cohort, MSLN-H tumors had increased frequency of KRAS (62.2% vs 37.9%) BRAF (10.5% vs 5.0%) mutations but decreased APC mutations (70% vs 80.9%) (q<0.05). MSLN-H tumors had higher expression of immune checkpoint genes ( CD274, PDCD1, PDCD1LG2, CTLA4, LAG3, HAVC2 and FOXP3, FC: 1.1-1.8) and T cell inflamed score (3.3-fold) (all q<0.05). MSLN-H was associated with worse OS compared to MSLN-L in all CRC (934 vs 1253 days; HR: 1.21, 95% CI (1.10-1.32), p<0.0001) and MSS CRC (972 vs 1168 days; HR: 1.15, 95% CI (1.06-1.23), p<0.001) but not in MSI-H CRC (p=0.67). Conclusions: Our data suggest a strong association between MSLN expression and increased mutations in the RAS pathway and MAPK activation score, decreased mutations in WNT pathway genes, a differentially modulated immune tumor microenvironment, and poor overall survival. These findings support MSLN as a potential prognostic biomarker candidate in colorectal cancers pending further prospective validation.

Genomic and clinicopathological features of lung adenocarcinomas with micropapillary component

Lung adenocarcinoma (LA) with a micropapillary component (LAMPC) is a histological subtype of lung cancer that has received increasing attention due to its correlation with poor prognosis, and its tendency to recur and metastasize. At present, comprehensive genomic profiles and clinicopathological features for LAMPC remain unclear and require further investigation. From September 2009 to October 2020, a total of 465 LAMPC patients were recruited and divided into four groups according to MPC proportions, and the correlations between varying proportions of MPCs and clinicopathological characteristics were analyzed. Twenty-nine (29) LAMPC patients and 89 LA patients without MPC (non-MPC) that had undergone NGS testing were selected for further study The comprehensively analyze genomic variations and the difference between LAMPC and MPC were determined. In addition, Gene alterations of LAMPC between Chinese and Western populations were also compared using cBioPortal data. A higher proportion of MPCs, associated with higher tumor stage, pleural invasion, and vascular tumor thrombus formation, was determined in LA patients. Compared to non-MPC patients, LAMPC patients were determined to have a lower frequency of single nucleotide variants and a higher frequency of insertion-deletion mutations. Mutations in TP53, CTNNB1, and SMAD4, and ALK rearrangements/fusions were significantly more frequent in LAMPC patients. ERBB2 mutations were only detected in non-MPC patients. Gene mutations in the Wnt pathway were significantly more common in LAMPC patients as compared to non-MPC patients. ALK fusions were more prevalent in younger patients. Patients with KRAS or LBP1B mutations had significantly larger tumor diameters than patients with wild-type KRAS or LBP1B. Patients with KRAS mutations were more likely to develop vascular tumor thrombus. Using the cBioPortal public database, we determined that mutations in EGFR were significantly higher in Chinese patients than in a Memorial Sloan Kettering Cancer Center (MSKCC) Western cohort. ALK fusions were exclusively detected in the Chinese cohort, while mutations in KEAP1 and NOTCH4 were only detected in the MSKCC cohort. Our analysis of signaling pathways revealed that Wnt pathway gene mutations were significantly higher in the Chinese cohort. LA patients with higher proportions of MPCs were determined to have a higher tumor stage, pleural invasion, and vascular tumor thrombosis formation. We comprehensively analyzed the genomic mutation characteristics of LAMPC patients and identified multiple, novel MPC-related gene alterations and pathway changes. Our data provide further understanding of the nature of the LAMPC and potential drug-targeted gene alterations, which may lead to new therapeutic strategies.

Metaplastic Breast Carcinoma: Update on Histopathology and Molecular Alterations.

Metaplastic carcinoma is a rare, triple-negative carcinoma of the breast that exhibits transformation of part or all of its glandular carcinomatous component into a nonglandular, or metaplastic, component. The World Health Organization currently recognizes 5 variants of metaplastic carcinoma based on their histologic appearance. To review the histologic classifications, differential diagnosis, prognosis, and recent laboratory studies of metaplastic breast carcinoma. We reviewed recently published studies that collectively examine metaplastic carcinomas, including results from our own research. Metaplastic breast carcinoma has a broad spectrum of histologic patterns, often leading to a broad differential diagnosis. Diagnosis can typically be rendered by a combination of morphology and immunohistochemical staining for high-molecular-weight cytokeratins and p63. Recent studies elucidate new genes and pathways involved in the pathogenesis of metaplastic carcinoma, including the downregulation of CCN6 and WNT pathway gene mutations, and provide a novel MMTV-Cre;Ccn6fl/fl knockout disease-relevant mouse model to test new therapies.

Acquisition of WNT Pathway Gene Alterations Coincides With the Transition From Precursor Polyps to Traditional Serrated Adenomas.

Colorectal traditional serrated adenomas (TSAs) are often associated with precursor polyps, including hyperplastic polyps and sessile serrated adenoma/polyps. To elucidate the molecular mechanisms involved in the progression from precursor polyps to TSAs, the present study analyzed 15 precursor polyp-associated TSAs harboring WNT pathway gene mutations. Laser microdissection-based sequencing analysis showed that BRAF or KRAS mutations were shared between TSA and precursor polyps in all lesions. In contrast, the statuses of WNT pathway gene mutations were different between the 2 components. In 8 lesions, RNF43, APC, or CTNNB1 mutations, were exclusively present in TSA. RNF43 mutations were shared between the TSA and precursor components in 3 lesions; however, they were heterozygous in the precursor polyps whereas homozygous in the TSA. In 4 lesions with PTPRK-RSPO3 fusions, RNA in situ hybridization demonstrated that overexpression of RSPO3, reflecting PTPRK-RSPO3 fusion transcripts, was restricted to TSA components. Consistent with the results of the genetic and in situ hybridization analyses, nuclear β-catenin accumulation and MYC overexpression were restricted to the TSA component in 13 and 12 lesions, respectively. These findings indicate that the WNT pathway gene alterations are acquired during the progression from the precursor polyps to TSAs and that the activation of the WNT pathway plays a critical role in the development of TSA rather than their progression to high-grade lesions.

Lgr6 is a stem cell marker in mouse skin squamous cell carcinoma.

The G-protein-coupled receptors Lgr4/5/6 are Wnt signalling mediators, but their functions in squamous carcinomas (SCCs) are unclear. Using lineage tracing in Lgr5-EGFP-CreERT2- and Lgr6-EGFP-CreERT2- Rosa26/Tomato reporter mice, we demonstrate that Lgr6, but not Lgr5, acts as an epithelial stem cell marker in vivo in SCCs. We identify, by single molecule in situ hybridisation and cell sorting, rare Lgr6-positive cells in immortalised keratinocytes, and show that their frequency increases in advanced SCCs. Lgr6 expression is enriched in cells with stem cell characteristics, and Lgr6 downregulation in vivo causes increased epidermal proliferation, with expanded lineage tracing from Lgr6+ epidermal stem cells. Surprisingly, Lgr6 germline knockout mice are predisposed to SCC development, by a mechanism that includes compensatory upregulation of Lgr5. These data provide a model for human patients with germline loss of function mutations in WNT pathway genes RSPO1 or LGR4, who show increased susceptibility to squamous tumour development.

WNT Pathway Gene Mutations Are Associated With the Presence of Dysplasia in Colorectal Sessile Serrated Adenoma/Polyps.

Sessile serrated adenoma/polyps (SSA/Ps) are believed to be the major precursor of serrated pathway-derived colorectal carcinomas. To better characterize the process of progression from SSA/Ps to carcinomas, we analyzed 46 SSA/Ps with dysplasia and 45 SSA/Ps without dysplasia using targeted next-generation sequencing and immunohistochemistry. Among the WNT pathway genes analyzed, protein-truncating mutations of RNF43, APC, and ZNRF3 were identified in 23 (50%), 4 (9%), and 3 (7%) SSA/Ps with dysplasia, respectively. In contrast, SSA/Ps without dysplasia rarely had WNT pathway gene mutations, except for 3 lesions with RNF43 mutations (7%). None of the SSA/Ps had CTNNB1 mutations or RSPO fusions. Thus, WNT pathway gene mutations were more common in SSA/Ps with dysplasia than in SSA/Ps without dysplasia (P=3.0×10). Consistently, nuclear β-catenin accumulation and MYC overexpression, indicative of active WNT signaling, were present in most of the SSA/Ps with dysplasia, but were rare in those without dysplasia. BRAF (86%) or KRAS mutations (7%) were identified in the majority of SSA/Ps, regardless of the presence or absence of dysplasia. MLH1 expression was lost in 14 SSA/Ps with dysplasia (30%). The majority of MLH1-deficient SSA/Ps with dysplasia had RNF43 mutations (86%), most of which were frameshift mutations involving mononucleotide repeats. In contrast, MLH1-retained lesions had less frequent RNF43 mutations with no hot spots (34%), and 4 had APC mutations (13%). These results suggest that WNT pathway gene mutations are involved in the development of dysplasia in SSA/Ps and that MLH1-deficient and MLH1-retained SSA/Ps with dysplasia exhibit distinct mutation profiles of WNT pathway genes.

Comprehensive characterization of RSPO fusions in colorectal traditional serrated adenomas.

Traditional serrated adenoma (TSA) is a rare but distinct type of colorectal polyp. Our previous study showed that PTPRK-RSPO3 fusions are frequent and characteristic genetic alterations in TSAs. This study aimed to characterize comprehensively the prevalence and variability of RSPO fusions in colorectal TSAs. We examined RSPO expression and explored novel RSPO fusions in 129 TSAs, including 66 lesions analysed previously for WNT pathway gene mutations. Quantitative polymerase chain reaction (qPCR) analyses identified three and 43 TSAs overexpressing RSPO2 and RSPO3, respectively, whereas the expression of RSPO1 and RSPO4 was marginal or undetectable in all cases. RSPO overexpression was always mutually exclusive with other WNT pathway gene mutations. Known PTPRK-RSPO3 fusions were detected in 37 TSAs, all but one of which overexpressed RSPO3. In addition, rapid amplification of cDNA ends revealed three novel RSPO fusion transcripts, an NRIP1-RSPO2 fusion and two PTPRK-RSPO3 fusion isoforms, in six TSAs. Overall, 43 TSAs had RSPO fusions (33%), whereas four TSAs (3%) overexpressed RSPO in the absence of RSPO fusions. TSAs with RSPO fusions showed several clinicopathological features, including distal localization (P = 0.0063), larger size (P = 0.0055), prominent ectopic crypt foci (P = 8.4 × 10-4 ), association of a high-grade component (P = 1.1 × 10-4 ), and the presence of KRAS mutations (P = 4.5 × 10-5 ). The present study identified RSPO fusion transcripts, including three novel transcripts, in one-third of colorectal TSAs and showed that PTPRK-RSPO3 fusions were the predominant cause of RSPO overexpression in colorectal TSA.

Abstract P1-05-03: The genomic landscape of breast metaplastic carcinoma

Abstract Introduction: Metaplastic breast carcinoma (MBC) is a rare histologic type of triple-negative breast cancer (TNBC), characterized by the presence of cells displaying squamous and/or mesenchymal differentiation. The transcriptomic profiles of MBCs have been reported to vary according to the type of metaplastic elements. The somatic genetic alterations that underpin this breast cancer subtype remain to be fully characterized. Here we sought to define the genomic landscape of MBCs, whether different subtypes of MBC would be driven by distinct constellations of genetic alterations, and to investigate functionally the impact of mutations affecting WNT pathway genes using non-malignant breast epithelial cells. Methods: Thirty-five MBCs were retrieved from the pathology department of the authors' institutions and classified into the MBC histologic subtypes. All but one of the MBCs were of triple-negative phenotype. DNA was extracted from microdissected tumor-normal pairs and subjected to whole-exome sequencing. Somatic genetic alterations were identified using state-of-the-art bioinformatics algorithms. The genomic profiles of MBCs were compared to those of 69 common type TNBCs from The Cancer Genome Atlas. Overall mutation rates were compared using the Mann Whitney U test, and the frequency of mutations in each gene was compared using Fisher's exact test. RNA was extracted from a subset of MBCs and subjected to WNT signaling pathway activation analysis with the RT2 Profiler PCR Array. Triple-negative non-malignant breast epithelial cells (MCF10A and MCF12A) and cancer cell lines were utilized for 2D and 3D functional studies. Results: Whole-exome analysis revealed that MBCs displayed a median of 103 (15-344) somatic mutations, which did not differ from the median number of somatic mutations in common type TNBCs (76, range 14-233). The most frequent recurrently mutated cancer genes included TP53 (69%) and PIK3CA (29%). MBCs more frequently harbored mutations in PI3K pathway genes than common type TNBCs (57% vs 22%, P<0.05), including mutations affecting PIK3CA (29% vs 7%), PIK3R1 (11% vs 0) and PTEN (11% vs 1%). MBCs also more frequently harbored mutations affecting WNT signaling pathway genes (46% vs 26%, P<0.05), including AXIN1 (6% vs 1%), WNT5A (6% vs 0) and APC (3% vs 0). MBC subtype analysis revealed that PIK3CA mutations were only detected in non-chondroid MBCs (53% vs 0), CHERP mutations were only found in chondroid MBCs (25% vs 0), whereas USP5 mutations only found in squamous MBCs (33% vs 0). MBCs with somatic mutations in WNT pathway genes had significantly higher WNT pathway activation than MBCs lacking mutations in these genes (P=0.0244). Consistent with the mesenchymal phenotype frequently exhibited by MBCs, in vitro experiments provided functional evidence that aberrant WNT pathway activation induces an epithelial-to-mesenchymal transition (EMT) phenotype, with downregulation of epithelial markers and upregulation of EMT transcriptional inducers. Conclusions: MBCs are significantly enriched for mutations affecting PI3K and WNT pathways, highlighting the importance of the dysregulation of the WNT pathway in MBC carcinogenesis. Moreover, our findings suggest that specific mutations are significantly associated with distinct histologic subtypes of MBCs. Citation Format: Geyer FC, Ng CK, Piscuoglio S, Wen YH, Wen H-C, Pareja F, Eberle CA, Burke KA, Lim RS, Natrajan R, Mariani O, Brogi E, Norton L, Vincent-Salomon A, Weigelt B, Reis-Filho JS. The genomic landscape of breast metaplastic carcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-03.

Mutation spectrum in the Wnt/β-catenin signaling pathway in gastric fundic gland-associated neoplasms/polyps.

Frequent activation of the Wnt/β-catenin signaling pathway has recently been demonstrated in gastric adenocarcinoma/neoplasia of chief cell predominant type (GA-CCP/GN-CCP) with submucosal involvement. In this study, we examined the activation status of the Wnt/β-catenin signaling pathway in GN-CCP without submucosal involvement, which is referred to as gastric dysplasia-CCP (GD-CCP). We also examined β-catenin expression and the mutation spectrum of PPP2R1A and Wnt pathway genes in 11 cases of GD-CCP, 25 cases of gastric polyps of fundic gland type (GPs-FG), and 21 cases of GPs-FG with dysplasia (GP-FGD). β-catenin nuclear staining was observed in 3 cases of GD-CCP, none of GPs-FG, and 6 cases of GPs-FGD. Mutations in Wnt pathway genes, including PPP2R1A, were observed in 4 cases of GDs-CCP, 10 cases of GPs-FG, and 7 cases of GPs-FGD. Two of these seven GPs-FGD cases showed β-catenin nuclear staining. However, none of the 4 GD-CCP cases with mutations or the 10 GPs-FG cases with mutations showed β-catenin nuclear staining. PPP2R1A mutations were observed in 1 GD-CCP case and 1 GPs-FGD case. Although the mutation spectra of the Wnt pathway genes in GD-CCP and GP-FG differed, based on the absence of β-catenin nuclear staining despite the genetic alterations, GD-CCP is more similar to GP-FG than to GN-CCP, which shows β-catenin nuclear staining and submucosal involvement. Activation of the Wnt/β-catenin signaling by the β-catenin nuclear transition may be required during progression from GD-CCP to GN-CCP. Furthermore, this is the first report describing PPP2R1A mutations in gastric fundic gland-associated neoplasms.

Abstract 1505: Targeted sequencing for the assessment of intratumor heterogeneity

Abstract Adenocarcinoma, the most common subtype of lung cancer, is notorious for eluding early detection. Thus, understanding the early steps of lung tumorigenesis at the genetic level might facilitate the development of more effective strategies for the prevention, early diagnosis, and treatment of lung cancer. Nearly 10% of lungs resected for adenocarcinomas harbored minute discrete foci of cytologically atypical bronchiolalveolar cells that are designated as atypical adenomatous hyperplasia (AAH). It has been postulated that these lesions represent an early stage in glandular neoplasia, which is now viewed as a progression from AAH to adenocarcinoma in situ (AIS) to minimally invasive adenocarcinoma (MIA), although, the biological nature of AAH is poorly understood and very controversial. To delineate tumor clonal heterogeneity as a function of tumor progression in early NSCLC neoplasias, we isolated DNA from different zones of histologic progression within the same lesion and performed targeted next generation sequencing which targets a panel of 134 well-characterized cancer and pharmacogenomics genes. We found that mutational landscape varies significantly between the three groups of patients. The most mutated genes in MIA were EGFR and TP53. 4 out of 5 MIA cases harbored multiple alternations in EGFR catalytic domain and inactivating mutations in TP53 were found in 3 out of 5 patients. Furthermore, mutations in Wnt pathway genes were found in 4 out of 5 cases. Notably, mutations/amplification in Notch family was detected in 3 out of 5 cases (predominantly in the tumor invasive zone). In contrast to MIA, in AIS, only one case out of 5 harbors mutated TP53 or EGFR. Unlike in MIA, only one AIS patient carried mutated APC gene, suggesting that Wnt signaling plays insignificant role in AIS carcinogenesis. On the contrary, we found that AIS carcinogenesis heavily relies on alternations of AKT pathway, since aberrations in KIT, KRAS, HRAS, IGF1R, FGFR3, MET or TSC2 genes were found in 4 out of 5 patients. For AAH cohort we sequenced 25 independent lesions from 6 patients. Sequencing revealed that RAS and AKT/ERK pathways were most frequently affected in these lesions. In 3 cases we were able to track a possible cancer driving mutation from AAH to the primary tumor. This observation provides for the first time a direct link between AAH lesion and primary adenocarcinoma. Most of the patients in all groups carried mutations in genes associated with DNA repair and chromatin remodeling, suggesting that deregulation of the DNA repair machinery is an early onset event of adenocarcinoma tumorigenesis. In all groups our analysis revealed mutations in genes that have been never implicated in lung adenocarcinoma. Our study helps to understand the potential role of heterogeneous clonal events in the progression of early glandular neoplasms including the transition from non-invasive to invasive disease. Citation Format: Eugene G. Izumchenko, Xiaofei Chang, Mariana Brait, William Westra, David Sidransky. Targeted sequencing for the assessment of intratumor heterogeneity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1505. doi:10.1158/1538-7445.AM2014-1505

Alterations in Metabolism and Mitochondrial Respiration Are Associated with Response to Targeted Inhibition of WNT Signaling in Lymphomas

Abstract 778 BackgroundWnt signaling is a highly conserved pathway with diverse roles in proliferation, self-renewal and differentiation in multiple tissue types. Aberrant Wnt signaling and deletions/mutations of Wnt regulatory genes have been found in a variety of malignancies including lymphoma. The factors which govern this pathway and the precise role of Wnt signaling in mediating tumor proliferation are unknown. We previously identified recurrent mutations in Wnt pathway genes including APC and WIF1 in diffuse large B cell lymphoma (DLBCL) using exome sequencing and thus sought to investigate if targeting of Wnt with molecular agents would be a viable treatment approach in NHL. MethodsAlamar blue proliferation assays were used to determine IC50 values for 2 small molecule inhibitors of Wnt signaling: ICG001 (a selective inhibitor of TCF/LEF1 mediated transcription) and salinomycin (an ionophore which has been shown to target Wnt) in a panel of 65 cell lines comprising subtypes of lymphoma (including 7 ABC DLBCL, 16 GCB DLBCL, 10 Burkitt lymphoma, 5 mantle cell lymphoma and 5 primary effusion lymphoma cell lines) and leukemia. Both molecules inhibited proliferation of cell lines in the screening panel at physiologically achievable concentrations (IC50 range for salinomycin 0.11–6.23 μM, IC50 range for ICG001 0.96–28.9 μM). IC50s values for salinomycin and ICG001 correlated well (Pearson r=0.56) suggesting a common mechanism of action between the 2 agents. Furthermore, we found lower IC50 values in primary effusion lymphoma (PEL) cell lines (0.35 μM vs. 1.37 μM for other cell line histologies, p=0.01 for salinomycin and 3.25 μM vs. 6.22 μM, p=0.13 for ICG001), which is a lymphoma subtype associated with aberrant Wnt signaling. We did not note significant differences in cytoxicity among other subsets. Wnt activity was confirmed using TOPFLASH luciferase assays and correlated with sensitivity to ICG001 and salinomycin.We then used gene expression profiles derived from Affymetrix Gene 1.0 arrays from each cell line and a Cox proportional hazards model to identify genes associated with sensitivity or resistance to both agents. A gene list consisting of 367 genes associated with either sensitivity or resistance at p<0.05 for both agents was derived from this analysis. Hierarchical clustering was used to define 2 groups of cell lines based upon these genes and analyzed with gene set enrichment (GSEA). GSEA comparisons of sensitive and resistance groups derived from this method revealed that genes associated with mitochondrial pathways were highly associated with response to both ICG001 and salinomycin (FDR=0.036, p=0.03).Specifically, expression of genes involved in glycolysis and mitochondrial respiration including FBG1, CS, UBQRB5, OXA1L, LDHA and ATP5B was higher in ICG001 and salinomycin-sensitive cell lines. Conversely, we noted higher IC50s in cell lines with higher expression of mitochondrial genes having opposing functions, including PC and CPT1A. This overall suggests that tumor cells utilizing glycolytic and oxidative pathways are sensitive to Wnt inhibition. Remarkably, the GCB DLBCL cell line Pfeiffer also was noted to contain an inactivating mutation in APC and had the lowest noted IC50 among DLBCL cell lines. ConclusionsThe Wnt pathway appears an important oncologic driver in at least a subset of NHLs and we found ICG001 and salinomycin have significant in vitro activity in NHLs. These agents may be most useful in subsets of tumors with high mitochondrial mass and/or oxidative phosphorylation. This work serves as a starting point to characterize the importance of the Wnt pathway in NHL and identifies inhibition of this pathway as a therapeutic opportunity in lymphomas. Disclosures:No relevant conflicts of interest to declare.

This Month in Gastroenterology

This Month in Gastroenterology

Phases of Canonical Wnt Signaling During the Development of Mouse Intestinal Epithelium

Intestinal crypts constitute a niche in which epithelial progenitors respond to Wnt signals, replicate, and prepare to differentiate. Because mutations in Wnt pathway genes lead to intestinal cancer, the role of Wnt signaling in gut epithelial homeostasis is a subject of intense investigation. We studied how Wnt signaling is established during intestine development. We studied spatiotemporal features of Wnt signaling at formative stages in mouse embryos, when villous projections appear and crypt precursors occupy intervillus regions. We used TOP-GAL transgenic and Axin2(LacZ) mice, which report faithfully on canonical Wnt activity, relevant molecular markers, and embryos with aberrant beta-catenin activation. Developing intestines first display evidence for Wnt signaling after appearance of villi. During villus morphogenesis, intervillus cells proliferate actively but lack signs of canonical Wnt signaling. Surprisingly, in late gestation and briefly thereafter, conspicuous Wnt activity is evident in differentiated, postmitotic villus epithelium. Neither Tcf4, a principal transcriptional effector of intestinal Wnt signals, nor candidate Wnt targets CD44 and cyclinD1 are expressed in late fetal villus cells that show high Wnt activity. Instead, those cells express the related factor Tcf3 and a different Wnt target, c-Myc. Premature and deregulated beta-catenin activation causes severe villus dysmorphogenesis in transgenic mice. Relationships among Wnt signaling, epithelial proliferation, and tissue differentiation are reversed in the developing and adult gut. The canonical Wnt pathway has independent, albeit possibly overlapping, functions in early intestinal villi and adult crypts. These observations advance understanding of Wnt functions in intestinal development and disease.

Repressor roles for TCF-4 and Sfrp1 in Wnt signaling in breast cancer

Mutations in Wnt pathway genes are rare in human breast cancer, yet activation of the pathway is evident from the misolocalization of beta-catenin. We searched for relationships in the expression of Wnt pathway genes and found that both secreted frizzled related protein 1 (Sfrp1) and TCF-4 transcripts were all highly downregulated in a common subset of breast cancers relative to normal breast tissue. Sfrp1 has been previously characterized as a Wnt inhibitor, and we found that interfering with its expression in the human mammary epithelial cell line MCF10A activated Wnt signaling. Reduction of TCF-4 levels in breast cancer was surprising as it is a transcription factor that is responsive to Wnt signaling. Therefore, we investigated a possible inhibitory role for TCF-4 in human breast cells as well as further characterizing Sfrp1. We identified CD24 as a Wnt target in MCF10A cells and used its expression a marker of Wnt signaling. Interfering with either Sfrp1 or TCF-4 in this cell line enhanced CD24 expression. Furthermore, removal of TCF/LEF binding sites in a CD24-luciferase reporter resulted in elevated reporter gene expression. Our results indicate that both Sfrp1 and TCF-4 repress Wnt signaling in breast tissue and their downregulation contributes to the activation of Wnt signaling.

Wnt Signals Can Function as Positional Cues in Establishing Cell Polarity

Wnt signaling plays important roles in cell polarization in diverse organisms, and loss of cell polarity is an early event in tumorigenesis caused by mutations in Wnt pathway genes. Despite this, the precise roles of Wnt proteins in cell polarization have remained elusive. In no organism has it been shown that the asymmetric position of a Wnt signal is essential to establishing a cell's polarity. Attempts to test this by ubiquitous expression of Wnt genes have suggested that Wnt signals might act only as permissive factors in cell polarization. Here we find, by using cell manipulations and ectopic gene expression in C. elegans, that the position from which Wnt signals are presented can determine the polarity of both embryonic and postembryonic cells. Furthermore, the position from which a Wnt signal is presented can determine the polarity of Frizzled receptor localization, suggesting that the polarizing effect of Wnt is likely to be direct. These results demonstrate that Wnt proteins can function as positional cues in establishing cell polarity.