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Genomic instability is a hallmark of cancer, and can be caused by oncogene-induced replication stress. Besides driving the evolution of cancer genomes, genomic instability can lead to the activation of inflammatory signaling, involving the cGAS-STING and JAK-STAT pathways. Inflammatory signaling has been associated with pro-tumorigenic features, but has also been associated with favorable response to treatment, including to immune checkpoint inhibitors. To improve our understanding of the relations between genomic instability and to ultimately guide patient selection for treatment, we investigated the cGAS-STING pathway in relation to markers of replication stress and immune cell infiltration in breast cancer. Immunohistochemistry was performed to determine the expression of cGAS-STING signaling components (STING, phospho-TBK1, and phospho-STAT1), replication stress markers (γH2AX and phospho-RPA32), replication stress-related oncogenes (Cyclin E1 and c-Myc) and immune cell markers (CD20, CD4, and CD57) on primary breast cancer samples (n = 380). Clinical data and mRNA expression data from two public breast cancer databases (TCGA and METABRIC) and an immune therapy trial (I-SPY2) were used to investigate the correlation between cGAS-STING pathway activation, genomic instability markers and patient response to immune therapy. We find that phospho-TBK1, and phospho-STAT1 were highly expressed in triple-negative breast cancers (TNBCs). In addition, expression of genomic instability markers γH2AX and pRPA, replication stress-related oncogenes Cyclin E1 and c-Myc, and immune cell markers were all positively correlated with phospho-STAT1 expression (P

Chromosomal instability (CIN) drives cancer cell evolution, metastasis and therapy resistance, and is associated with poor prognosis1. CIN leads to micronuclei that release DNA into the cytoplasm after rupture, which triggers activation of inflammatory signalling mediated by cGAS and STING2,3. These two proteins are considered to be tumour suppressors as they promote apoptosis and immunosurveillance. However, cGAS and STING are rarely inactivated in cancer4, and, although they have been implicated in metastasis5, it is not known why loss-of-function mutations do not arise in primary tumours4. Here we show that inactivation of cGAS-STING signalling selectively impairs the survival of triple-negative breast cancer cells that display CIN. CIN triggers IL-6-STAT3-mediated signalling, which depends on the cGAS-STING pathway and the non-canonical NF-κB pathway. Blockade of IL-6 signalling by tocilizumab, a clinically used drug that targets the IL-6 receptor (IL-6R), selectively impairs the growth of cultured triple-negative breast cancer cells that exhibit CIN. Moreover, outgrowth of chromosomally instable tumours is significantly delayed compared with tumours that do not display CIN. Notably, this targetable vulnerability is conserved across cancer types that express high levels of IL-6 and/or IL-6R in vitro and in vivo. Together, our work demonstrates pro-tumorigenic traits of cGAS-STING signalling and explains why the cGAS-STING pathway is rarely inactivated in primary tumours. Repurposing tocilizumab could be a strategy to treat cancers with CIN that overexpress IL-6R.

Objective Salivary glands of primary SS (pSS) patients characteristically harbour periductal infiltrates, in which lymphoepithelial lesions (LELs) can develop. LELs are composed of hyperplastic ductal epithelium with infiltrating lymphocytes and may assist in the challenging diagnostic process of pSS. As manual identification of LELs remains difficult, we aimed to identify LELs by using an objective digital image analysis (DIA) algorithm that detects intraepithelial lymphocytes. Methods A virtual triple-staining technique developed for this study was used to count intraepithelial lymphocytes in consecutive slides stained for CD3 (T-lymphocytes), high-molecular-weight cytokeratin (hmwCK) (striated ducts) and CD20 (B-lymphocytes) in labial and parotid gland biopsies in a diagnostic cohort of 109 sicca patients. Patients were classified as having pSS or non-SS according to the ACR-EULAR classification criteria. Results T-lymphocytes were detected in almost all analysed ducts of pSS and non-SS sicca patients, whereas intraepithelial B-lymphocytes were present in 59–68% of labial and parotid gland biopsies of pSS patients, against only 2–3% of patients classified as non-SS. Intraepithelial B-lymphocytes were found in almost all striated ducts with hyperplasia (LELs). Remarkably, ∼25% of analysed striated ducts without hyperplasia of pSS patients also contained B-lymphocytes (precursor-LELs). Furthermore, presence of intraepithelial B-lymphocytes was associated with clinical parameters of pSS (i.e. serology). Conclusion The presence of intraepithelial B-lymphocytes in salivary gland biopsies of sicca patients is a clear indicator of pSS and can be used as an objective alternative to LEL scoring. Therefore, identification of B-lymphocyte–containing ducts should be added to the diagnostic histopathological work-up of patients suspected of pSS.

Chromosomal instability (CIN) drives cancer cell evolution, metastasis and therapy resistance, and is associated with poor prognosis

Background: Triple-negative breast cancer (TNBC) has a poor clinical prognosis and is characterized by a lack of druggable targets and a hypoxic tumor microenvironment. Hypoxia-induced glycogen accumulation and utilization are involved in cancer proliferation and therapy resistance, making modulation of glycogen metabolism of therapeutic interest. Therefore, we studied expression of glycogen synthase 1 (GYS1, the key regulator of glycogen synthesis) and glycogen stores in publicly available expression data and human breast tumors including TNBC. Also, we studied downregulation of GYS1 in preclinical breast cancer models, focusing on TNBC. Methods: GYS1 mRNA expression and correlations with survival per clinical subtype were assessed in the METABRIC dataset. A tissue micro-array was constructed from primary tumors of 396 breast cancer patients with long-term follow-up, including normal breast control tissue. Triplicate tissue cores were stained immunohistochemically for GYS1, glycogen and the hypoxic marker carbonic anhydrase 9, and with periodic acid-Schiff staining for glycogen. In four TNBC cell lines and an MDA-MB-231 xenograft model, GYS1 protein expression, glycogen content and cell proliferation in normoxia and hypoxia were evaluated +/- GYS1 knockdown by siRNA or shRNA. Sensitivity of shGYS1 cell lines to drugs targeting mitochondria was tested. Results: In the METABRIC dataset (n = 1904), overexpression of GYS1 mRNA was associated with poor overall survival (HR 1.20 [95% CI 1.05 - 1.38]), which was mainly driven by the TNBC patients (n = 299, HR 1.52 [95% CI 1.09 - 2.14]). Immunohistochemically, most primary breast tumors had elevated GYS1 levels and glycogen content compared to normal breast tissue, with subtype specific analyses ongoing. In all breast cancer cell lines, hypoxia induced GYS1 protein expression and increased glycogen content. Acute siRNA-mediated GYS1 knockdown decreased proliferation of (TN)BC cell lines and MDA-MB-231 spheroids in both hypoxia and normoxia. shGYS1 MDA-MB-231 cells had decreased glycogen levels and shGYS1 MDA-MB-231 xenograft growth was impaired, especially in the first six weeks after inoculation. In control xenografts, immunohistochemical GYS1 expression was most pronounced adjacent to the necrotic tumor core, whereas shGYS1 xenografts lacked GYS1 expression. Finally, shGYS1 MDA-MB-231 cells were more sensitive to inhibitors of mitochondrial protein homeostasis, suggesting a potential synergistic approach to overcome eventual metabolic adaptation. Conclusions: GYS1 is overexpressed in primary breast tumors and high mRNA levels correlate with poor survival in TNBC. GYS1 downregulation impairs TNBC proliferation in vitro and in vivo, highlighting glycogen synthesis as potential novel therapeutic target in TNBC. Citation Format: Ellen C. De Heer, Christos E. Zois, Esther Bridges, Mieke C. Zwager, Tineke van der Sluis, Bert van der Vegt, Carolien P. Schröder, Steven de Jong, Adrian L. Harris, Mathilde Jalving. Glycogen synthesis as potential novel target in triple negative breast cancer: Glycogen synthase 1 expression in human breast cancers and the impact of downregulation on proliferation of preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5813.

Simple SummaryMicroRNAs (miRNAs) are small RNAs that regulate expression of specific target genes. We observed elevated levels of miR-378a-3p in Burkitt lymphoma (BL) and studied its role in the pathogenesis of BL. Inhibition of miR-378a-3p reduced growth of BL cells, confirming its significance in BL. Identification of BL specific target genes of miR-378a-3p revealed four candidates. For two of them, MNT and IRAK4, miR-378a-dependent regulation was confirmed at the protein level. Overexpression of MNT and IRAK4 in BL cell lines resulted in a similar effect as observed upon miR-378a-3p inhibition, suggesting their involvement in the growth regulatory role of miR-378a-3p.MicroRNAs (miRNAs) are small RNA molecules with important gene regulatory roles in normal and pathophysiological cellular processes. Burkitt lymphoma (BL) is an MYC-driven lymphoma of germinal center B (GC-B) cell origin. To gain further knowledge on the role of miRNAs in the pathogenesis of BL, we performed small RNA sequencing in BL cell lines and normal GC-B cells. This revealed 26 miRNAs with significantly different expression levels. For five miRNAs, the differential expression pattern was confirmed in primary BL tissues compared to GC-B cells. MiR-378a-3p was upregulated in BL, and its inhibition reduced the growth of multiple BL cell lines. RNA immunoprecipitation of Argonaute 2 followed by microarray analysis (Ago2-RIP-Chip) upon inhibition and ectopic overexpression of miR-378a-3p revealed 63 and 20 putative miR-378a-3p targets, respectively. Effective targeting by miR-378a-3p was confirmed by luciferase reporter assays for MAX Network Transcriptional Repressor (MNT), Forkhead Box P1 (FOXP1), Interleukin 1 Receptor Associated Kinase 4 (IRAK4), and lncRNA Just Proximal To XIST (JPX), and by Western blot for IRAK4 and MNT. Overexpression of IRAK4 and MNT phenocopied the effect of miR-378a-3p inhibition. In summary, we identified miR-378a-3p as a miRNA with an oncogenic role in BL and identified IRAK4 and MNT as miR-378a-3p target genes that are involved in its growth regulatory role.

The expression of several microRNAs (miRNAs) is known to be changed in Burkitt lymphoma (BL), compared to its normal counterparts. Although for some miRNAs, a role in BL was demonstrated, for most of them, their function is unclear. In this study, we aimed to identify miRNAs that control BL cell growth. Two BL cell lines were infected with lentiviral pools containing either 58 miRNA inhibitors or 44 miRNA overexpression constructs. Eighteen constructs showed significant changes in abundance over time, indicating that they affected BL growth. The screening results were validated by individual green fluorescent protein (GFP) growth competition assays for fifteen of the eighteen constructs. For functional follow-up studies, we focused on miR-26b-5p, whose overexpression inhibited BL cell growth. Argonaute 2 RNA immunoprecipitation (Ago2-IP) in two BL cell lines revealed 47 potential target genes of miR-26b-5p. Overlapping the list of putative targets with genes showing a growth repression phenotype in a genome-wide CRISPR/Cas9 knockout screen, revealed eight genes. The top-5 candidates included EZH2, COPS2, KPNA2, MRPL15, and NOL12. EZH2 is a known target of miR-26b-5p, with oncogenic properties in BL. The relevance of the latter four targets was confirmed using sgRNAs targeting these genes in individual GFP growth competition assays. Luciferase reporter assay confirmed binding of miR-26b-5p to the predicted target site for KPNA2, but not to the other genes. In summary, we identified 18 miRNAs that affected BL cell growth in a loss- or gain-of-function screening. A tumor suppressor role was confirmed for miR-26b-5p, and this effect could at least in part be attributed to KPNA2, a known regulator of OCT4, c-jun, and MYC.

miRNAs are small noncoding RNAs involved in the posttranscriptional regulation of gene expression. Deregulated miRNA levels have been linked to Burkitt lymphoma (BL) pathogenesis. To date, the number of known pathogenesis-related miRNA-target gene interactions is limited. Here, we determined for the first time the miRNA targetomes of primary BL tumors and normal B cells. AG02-RNA immunoprecipitation of two frozen diagnostic BL tissue samples and three CD19(+) B-cell samples isolated from routinely removed tonsils showed distinct miRNA targetomes of BL and normal B cells. In contrast to normal B cells, miRNA target genes in BL were enriched for targets of the oncogenic miR-17 to 92 cluster, and were involved mainly in cell cycle and cell death. Immunohistochemistry on BL and tonsil tissues confirmed altered protein levels for two of six selected miRNA targets, in line with the differential AG02-IP enrichment between BL and normal B cells. A comparison of AG02-IP-enriched genes in primary BL cases with BL cell lines indicated that despite a considerable overlap, the miRNA targetomes of BL cell lines show substantial differences with the targetomes of primary BL tumors. In summary, we identified distinct miRNA targetomes of BL and normal B cells, and showed both the necessity and feasibility of studying miRNA-target gene interactions in primary tumors.

Microsatellite instability (MSI) in tumors results in an accumulation of mutations in (target) genes. Previous studies suggest that the profile of target genes differs according to tumor type. This paper describes the first genome-wide search for target genes for mismatch repair-deficient endometrial cancers. Genes expressed in normal endometrium containing coding repeats were analyzed for mutations in tumors. We identified 44 possible genes of which seven are highly mutated (>15%). Some candidates were also found mutated in colorectal and gastric tumors. The most frequently mutated gene, NRIP1 encoding nuclear receptor-interacting protein 1, was silenced in an endometrial tumor cell line and expression microarray experiments were performed. Silencing of NRIP1 was associated with differences in the expression of several genes in the estrogen-receptor network. Furthermore, an enrichment of genes related to cell cycle (regulation) and replication was observed. We present a new profile of target genes, some of them tissue specific, whereas others seem to play a more general role in MSI tumors. The high-mutation frequency combined with the expression data suggest, for the first time, an involvement of NRIP1 in endometrial cancer development.

Ribosomal Protein L22 (RPL22) encodes a protein that is a component of the 60S subunit of the ribosome. Variants in this gene have recently been linked to cancer development. Mutations in an A8 repeat in exon 2 were found in a recent study in 52% of microsatellite-unstable endometrial tumors. These tumors are particularly prone to mutations in repeats due to mismatch repair deficiency. We screened this coding repeat in our collection of microsatellite-unstable endometrial tumors (EC) and colorectal tumors (CRC). We found 50% mutation frequency for EC and 77% mutation frequency for CRC. These results confirm the previous study on the involvement of RPL22 in EC and, more importantly, reports for the first time such high mutation frequency in this gene in colorectal cancer. Furthermore, considering the high mutation frequency found, our data point toward an important role for RPL22 in microsatellite instability carcinogenesis.

A progressive accumulation of genetic alterations underlies the adenoma-carcinoma sequence of colorectal cancer. This accumulation of mutations is driven by genetic instability, of which there are different types. Microsatellite instability (MSI) is the predominant type present in the tumours of Lynch syndrome patients and in a subset of sporadic tumours. It is generally accepted that MSI can be found in the early stages of tumour progression, such as adenomas; however, the frequencies reported vary widely among studies. Moreover, data on the qualitative differences between adenomas and carcinomas, or between tumours of hereditary and sporadic origin, are scarce. We compared MSI in samples of colorectal adenoma and colorectal carcinoma in order to identify possible differences along the adenoma-carcinoma sequence. We compared germline mismatch repair (MMR) gene mutation carriers and non-carriers, to address possible differences of instability patterns between Lynch syndrome patients and patients with sporadic tumours. We found a comparable relative frequency of mono- and dinucleotide instability in sporadic colorectal adenomas and carcinomas, dinucleotide instability being observed most frequently in these sporadic tumours. In MMR gene truncating mutation carriers, the profile was different: colorectal adenomas showed predominantly mononucleotide instability and in colorectal carcinomas, also more mononucleotide than dinucleotide instability was detected. We conclude that MSI profiles differ between sporadic and Lynch syndrome tumours, and that mononucleotide marker instability precedes dinucleotide marker instability during colorectal tumour development in Lynch syndrome patients. As mononucleotide MSI proves to be highly sensitive for detecting mutation carriers, we propose the use of mononucleotide markers for the identification of possible Lynch syndrome patients.

Non-steroidal anti-inflammatory drugs, e.g. sulindac have been extensively studied for chemoprevention in familial adenomatous polyposis, but not in hereditary non-polyposis colorectal cancer (HNPCC). We evaluated these effects in HNPCC using surrogate end-points for cancer risk. In a randomised double-blind cross-over study, 22 subjects (9 female; age 30-66 years, mean 44), all ascertained or probable mutation carriers for HNPCC, were included. Sulindac 150 mg b.i.d. and placebo were given for 4 weeks each, with 4 weeks in between, with biopsies taken from ascending, transverse and sigmoid colon and rectum by colonoscopy after both periods. Proliferation was determined by Ki-67 staining and apoptosis by staining of cytokeratin 18 cleavage products. Expression of cyclins B1, D3 and E and p21, p27, bax, bcl2 and cox-2 was studied immunohistochemically. Proliferation was higher during sulindac treatment than drug placebo treatment in ascending and transverse colon, but not in sigmoid and rectum. Apoptosis was not affected. Besides an increase in cyclin D3, no differences were found in expression of regulating proteins in the proximal colon. Conclusion: Sulindac induces an increase in epithelial cell proliferation in the proximal colon of subjects with HNPCC. Since colorectal cancer predominantly arises in the proximal colon in HNPCC, these results cast doubts on the potential chemopreventive effects of sulindac in HNPCC. (C) 2007 Elsevier Ltd. All rights reserved.

AimsTumor-specific targeted imaging is rapidly evolving in cancer diagnosis. The folate receptor alpha (FR-alpha) has already been identified as a suitable target for cancer therapy and imaging. FR-alpha is present on similar to 40% of human cancers. FR-beta is known to be expressed on several hematologic malignancies and on activated macrophages, but little is known about FR-beta expression in solid tumors. Additional or simultaneous expression of FR-beta could help extend the indications for folate-based drugs and imaging agents. In this study, the expression pattern of FR-beta is evaluated in ovarian, breast and colorectal cancer.MethodsFR-beta expression was analyzed by semi-quantitative scoring of immunohistochemical staining on tissue microarrays (TMAs) of 339 ovarian cancer patients, 418 breast cancer patients, on 20 slides of colorectal cancer samples and on 25 samples of diverticulitis.ResultsFR-beta expression was seen in 21% of ovarian cancer samples, 9% of breast cancer samples, and 55% of colorectal cancer samples. Expression was weak or moderate. Of the diverticulitis samples, 80% were positive for FR-beta expression in macrophages. FR-beta status neither correlated to known disease-related variables, nor showed association with overall survival and progression free survival in ovarian and breast cancer. In breast cancer, negative axillary status was significantly correlated to FR-beta expression (p=0.022).ConclusionsFR-beta expression was low or absent in the majority of ovarian, breast and colorectal tumor samples. From the present study we conclude that the low FR-beta expression in ovarian and breast tumor tissue indicates limited practical use of this receptor in diagnostic imaging and therapeutic purposes. Due to weak expression, FR-beta is not regarded as a suitable target in colorectal cancer.

Purpose: To determine the frequency of mismatch repair (MMR) gene germline mutations in endometrial cancer patients who were diagnosed at less than 50 years of age; to relate the presence of mutations to family history, histopathologic data, presence of tumor microsatellite instability (MSI), and immunostaining; and to formulate criteria for genetic testing in these patients.Patients and Methods: Endometrial cancer patients (N = 58), who were diagnosed at less than 50 years of age, were included and questioned about their family history. Mutation analysis of the MLH1, MSH2, and MSH6 genes was performed (denaturing gradient gel electrophoresis and sequence analysis to detect small mutations and multiplex ligation-dependent probe amplification to detect large deletions or duplications). For MSI analysis, five consensus markers were used, and immunostaining of the three MMR proteins was performed.Results: In five of 22 patients with a positive first-degree family history for hereditary nonpolyposis colorectal cancer (HNPCC)-related cancers, pathogenic germline mutations were found (one MLH1, three MSH2, and one MSH6). Four mutation carriers belonged to families fulfilling the revised Amsterdam criteria. No mutations were found in the 35 patients without such family history (P = .006). MSI was detected in 20 of 57 cancers, among which four were from mutation carriers. In 23 of 51 cancers, one or more MMR protein was absent; in all five mutation carriers, immunostaining indicated the involved MMR gene.Conclusion: In 23% of the young endometrial cancer patients with at least one first-degree relative with an HNPCC-related cancer, an MMR gene mutation was detected. Therefore, presence of an HNPCC-related cancer in a first-degree relative seems to be an important selection criterion for mutation analysis. Subsequent immunostaining of MMR proteins will point to the gene(s) that should be analyzed. (C) 2003 by American Society of Clinical Oncology.

Patients with a history of sporadic adenomas have increased epithelial cell proliferative activity, an intermediate risk marker for colorectal cancer. Reduction of proliferation by dietary intervention may reflect a decreased colorectal cancer risk. To evaluate whether calcium or resistant starch could reduce proliferative activity throughout the colon, we performed a randomized controlled trial in 111 sporadic adenoma patients. Patients received two placebos, I g of calcium + placebo, or 30 g of amylomaize (19 g of resistant starch) + placebo. After 2 mo, biopsies were collected from the cecum, transverse and sigmoid colon, and rectum during colonoscopy. Epithelial cell proliferation was determined by dividing the number of 5-bromo-2-deoxyuridine-labeled nuclei by the total number of nuclei x 100 (labeling index, LI). LI of luminal, mid, and basal compartments was determined. Twenty-five patients dropped out. In the remaining 86 patients (28 treated with placebo, 30 with calcium + placebo, and 28 with resistant starch + placebo), no difference was observed in total LI, the LI of the three compartments, or the crypt length in the four areas of the colorectum. Colonic epithelial cell proliferative activity throughout the colon of sporadic adenoma patients is not affected by supplementation with 1 g of calcium or 19 g of resistant starch.

Background & Aims: Germline mutations in one of four mismatch repair genes have been found in the majority of families with hereditary nonpolyposis colorectal cancer (HNPCC), but only in a small part of families with atypical HNPCC. The recently cloned EXO1 gene might be involved in the pathogenesis of HNPCC because the EXO1 protein strongly interacts with the MSH2 protein. To determine its role in HNPCC, EXO1 was scanned for germline mutations. Methods: All 14 exons of EXO1 were scanned for mutations in index patients from 33 families with HNPCC fulfilling the Amsterdam criteria and in 225 index patients suspected of HNPCC. Results: Germline variants of EXO1 were detected in 14 patients, including one splice-site mutation in a family with HNPCC and 13 missense mutations in patients with atypical HNPCC. These variants did not occur in more than 200 control individuals. From 13 of these 14 patients, tumors were available for analysis of microsatellite instability and loss of heterozygosity. Six of the tumors showed microsatellite instability. Heterozygosity analysis showed one case without EXO1 allelic loss and 12 tumors with loss of the mutant allele and retention of the normal one. Conclusions: The results indicate a possible association of germline EXO1 variants with HNPCC and atypical HNPCC. GASTROENTEROLOGY 2001;120:1580-1587

Microsatellite instability (MSI) occurs in more than 90% of the tumors of Lynch syndrome patients, and in 15-25% of sporadic colorectal (CRC) and endometrial carcinomas (EC). Previous studies comparing EC and CRC using BAT markers showed that the frequency of unstable markers is lower in EC, and that the size of the mutations is smaller in EC. In the present study, we analyzed the type (insertions/deletions), size, and frequency of mutations occurring at three BAT and three dinucleotide markers in CRC and EC, to elucidate whether it is possible to establish different MSI profiles in carcinomas of different tissue origin. We show that mononucleotide markers nearly always become shorter whereas dinucleotide markers can become shorter or longer, in both CRC and EC. We therefore conclude that the type of mutation is a marker-dependent feature rather than tissue-dependent. However, we observed that the size of the deletions/insertions differs between CRC and EC, with EC having shorter alterations. The frequency of mono- and dinucleotide instability found in both tissues is comparable, with mononucleotide and dinucleotide markers being affected at similar rates. We conclude that it is not possible to define clearly different MSI profiles that could distinguish MSI-high in CRC and EC. We propose that the differences observed might indicate different durations of tumor development and/or differences in tissue turnover between colorectal and endometrial epithelium, rather than reflecting truly different MSI profiles. We therefore suggest that the same MSI tests can be used for both tumor types. (C) 2009 Wiley-Liss, Inc.

The precise role of STAT3 Ser(727) phosphorylation in RET-mediated cell transformation and oncogenesis is not well understood. In this study, we have shown that familial medullary thyroid carcinoma (FMTC) mutants RET(Y791F) and RET(S891A) induced, in addition to Tyr(705) phosphorylation, constitutive STAT3 Ser(727) phosphorylation. Using inhibitors and dominant negative constructs, we have demonstrated that RET(Y791F) and RET(S891A) induce STAT3 Ser(727) phosphorylation via a canonical Ras/ERK1/2 pathway and that integration of the Ras/ERK1/2/ELK-1 and STAT3 pathways was required for up-regulation of the c-fos promoter by FMTC-RET. Moreover, inhibition of ERK1/2 had a more severe effect on cell proliferation and cell phenotype in HEK293 cells expressing RET(S891A) compared with control and RET(WT)-transfected cells. The transforming activity of RET(Y791F) and RET(S891A) in NIH-3T3 cells was also inhibited by U0126, indicating a role of the ERK1/2 pathway in RET-mediated transformation. To investigate the biological significance of Ras/ERK1/2-induced STAT3 Ser(727) phosphorylation for cell proliferation and transformation, N-Ras-transformed NIH-3T3 cells were employed. These cells displayed elevated levels of activated ERK1/2 and Ser(727)-phosphorylated STAT3, which were inhibited by treatment with U0126. Importantly, overexpression of STAT3, in which the Ser(727) was mutated into Ala (STAT3(S727A)), rescued the transformed phenotype of N-Ras-transformed cells. Immunohistochemistry in tumor samples from FMTC patients showed strong nuclear staining of phosphorylated ERK1/2 and Ser(727) STAT3. These data show that FMTC-RET mutants activate a Ras/ERK1/2/STAT3 Ser(727) pathway, which plays an important role in cell mitogenicity and transformation.

Sulindac reduces colorectal cancer risk in genetically susceptible humans and animals. The molecular mechanisms underlying these effects are incompletely understood. Many studies suggest an important role for induction of apoptosis involving the mitochondrial pathway and the death receptor pathway. Alternatively, mechanisms involving the APC-β-catenin-Wnt pathway have been suggested, possibly mediated by p21. We determined the effects of sulindac on apoptosis and expression of death receptor (DR)-4 and DR5, β-catenin, and p21 in normal-appearing colorectal epithelium. Biopsies were obtained before and after sulindac treatment during two chemoprevention studies. Patients (n = 18) with hereditary nonpolyposis colorectal cancer (HNPCC) received 150 mg sulindac bd for 4 weeks in a placebo-controlled crossover design. Patients (n = 6) with familial adenomatous polyposis (FAP) received 150 mg sulindac bd for 6 months. Apoptosis was assessed by M30 staining and expression patterns of DR4, DR5, β-catenin, and p21 were studied immunohistochemically. In HNPCC patients, apoptotic indices were similar following placebo and sulindac. Also in FAP patients, apoptotic indices were not different after sulindac compared with pretreatment values. Expression of DR4 and DR5 was observed in all samples with no consistent differences between placebo/baseline and sulindac. Intensity of membranous β-catenin staining was lower in HNPCC samples following sulindac compared with placebo (P < 0.001). Similar results were obtained in FAP samples (P < 0.01). p21 expressions before and after sulindac treatment were similar in both patient groups. In conclusion, sulindac inhibits β-catenin expression in normal colorectal epithelium from HNPCC and FAP patients without affecting apoptotic indices and DR4, DR5, and p21 expression.

The RET proto-oncogene encodes a receptor tyrosine kinase whose dysfunction plays a crucial role in the development of several neural crest disorders. Distinct activating RET mutations cause multiple endocrine neoplasia type 2A (MEN2A), type 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). Despite clear correlations between the mutations found in these cancer syndromes and their phenotypes, the molecular mechanisms connecting the mutated receptor to the different disease phenotypes are far from completely understood. Luciferase reporter assays in combination with immunoprecipitations, and Western and immunohistochemistry analyses were done in order to characterize the signaling properties of two FMTC-associated RET mutations, Y791F and S891A, respectively, both affecting the tyrosine kinase domain of the receptor. We show that these RET-FMTC mutants are monomeric receptors which are autophosphorylated and activated independently of glial cell line–derived neurotrophic factor. Moreover, we show that the dysfunctional signaling properties of these mutants, when compared with wild-type RET, involve constitutive activation of signal transducers and activators of transcription 3 (STAT3). Furthermore, we show that STAT3 activation is mediated by a signaling pathway involving Src, JAK1, and JAK2, differing from STAT3 activation promoted by RETC634R which was previously found to be independent of Src and JAKs. Three-dimensional modeling of the RET catalytic domain suggested that the structural changes promoted by the respective amino acids substitutions lead to a more accessible substrate and ATP-binding monomeric conformation. Finally, immunohistochemical analysis of FMTC tumor samples support the in vitro data, because nuclear localized, Y705-phosphorylated STAT3, as well as a high degree of RET expression at the plasma membrane was observed.

The predictive value of MLH1 or MSH2 protein expression for the presence of truncating germline mutations was examined in benign and (pre)malignant endometrial samples from 3 patient groups: (I) 10 endometrial cancer patients from hereditary non-polyposis colorectal cancer (HNPCC) families with (n = 6) or without (n = 4) a known germline mutation; (II) 15 women from HNPCC families with (n = 7) or without (n = 8) a known germline mutation, who underwent endometrial sampling for non-malignant reasons; (III) 38 endometrial cancer patients

Introduction: Ovarian cancer (OC) is the second most common and most lethal gynecological cancer. High recurrence rate and poor prognosis underline the need to explore targeted therapies. Potential targets include hormone receptors (androgen receptor [AR], estrogen receptor α [ERα], estrogen receptor β [ERβ], and progesterone receptor [PR]). Clinical studies have shown modest effects of endocrine agents in phase II studies. Nevertheless, the role of hormones and their putative receptors in OC remains largely unknown. To further probe their role, tumor material for hormone receptor analysis was obtained from a group of patients who were treated in a prospective clinical trial. Methods: Between March 2003 and November 2008, 196 epithelial OC patients were included in a prospective multicenter randomized controlled phase II trial and assigned to carboplatin/docetaxel ± celecoxib. The majority (92.5%) of patients had high-stage disease. Histologic material for biomarker analysis was obtained from 121 patients. Histological subtypes were serous (70%), endometrioid (17%), clearcell (9%), and mucinous (4%). Mean PFS and OS were 16 and 32 months. Tissue micro arrays (4 cores/sample) were constructed and stained for AR, ERα, ERβ, and PR and scored semi-quantitatively by two independent observers. A cut-off of >10% positive staining was used to dichotomize results. Receptor expression was correlated to PFS and OS in uni- and multivariate analysis (including age, FIGO stage, grade, histological subtype, and residual disease after surgery). Results: Tumors were positive for AR, ERα, ERβ, and PR in 10%, 31%, 78%, and 21% of the cases. ERα-positivity was significantly correlated with ERβ-positivity (p = 0.021). Both ERα- and ERβ-positivity were significantly correlated with AR-positivity (p Citation Format: Michel van Kruchten, Pauline W. van der Marel, Henriëtte J.G. Arts, Harry Hollema, Linda de Munck, Tineke van der Sluis, Hetty Timmer-Bosscha, Geke A.P. Hospers, An K.L. Reyners. Frequency and prognostic value of hormone receptor expression in epithelial ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4768. doi:10.1158/1538-7445.AM2013-4768

Recently we identified a new variant, S845G, in the MLH3 gene in 7 out of 327 patients suspected of hereditary nonpolyposis colorectal cancer but not fulfilling the Amsterdam criteria and in I out of 188 control subjects. As this variant might play a role in causing sporadic colorectal cancer, we analyzed its prevalence in sporadic colorectal cancer patients. We analyzed a small part of exon I of the MLH3 gene, including the S845G variant, in germline DNA of 467 white sporadic colorectal cancer patients and 497 white controls. The S845G variant was detected in five patients and eight controls; the results thus indicate that this variant does not confer an increased colorectal cancer risk. Another variant (P844L) was clearly a polymorphism. Three other missense variants were rare and the sample size of the study was too small to conclude whether they are pathogenic. In conclusion, no association was observed between two MLH3 variants (P844L and S845G) and colorectal cancer risk. (C) 2004 Elsevier Inc. All rights reserved.