LKB1 deficiency in T cells promotes the development of gastrointestinal polyposis.

Germline mutations in STK11, which encodes the tumor suppressor liver kinase B1 (LKB1), promote Peutz-Jeghers syndrome (PJS), a cancer predisposition syndrome characterized by the development of gastrointestinal (GI) polyps. Here, we report that heterozygous deletion of Stk11 in T cells (LThet mice) is sufficient to promote GI polyposis. Polyps from LThet mice, Stk11+/- mice, and human PJS patients display hallmarks of chronic inflammation, marked by inflammatory immune-cell infiltration, signal transducer and activator of transcription 3 (STAT3) activation, and increased expression of inflammatory factors associated with cancer progression [interleukin 6 (IL-6), IL-11, and CXCL2]. Targeting either T cells, IL-6, or STAT3 signaling reduced polyp growth in Stk11+/- animals. Our results identify LKB1-mediated inflammation as a tissue-extrinsic regulator of intestinal polyposis in PJS, suggesting possible therapeutic approaches by targeting deregulated inflammation in this disease.

Establishing a clinic-based pancreatic cancer and periampullary tumour research registry in Quebec.

BACKGROUND: Enrolling patients in studies of pancreatic ductal adenocarcinoma (pdac) is challenging because of the high fatality of the disease. We hypothesized that a prospective clinic-based study with rapid ascertainment would result in high participation rates. Using that strategy, we established the Quebec Pancreas Cancer Study (qpcs) to investigate the genetics and causes of pdac and other periampullary tumours (pats) that are also rare and underrepresented in research studies. METHODS: Patients diagnosed with pdac or pat were introduced to the study at their initial clinical encounter, with a strategy to enrol participants within 2 weeks of diagnosis. Patient self-referrals and referrals of unaffected individuals with an increased risk of pdac were also accepted. Family histories, epidemiologic and clinical data, and biospecimens were collected. Additional relatives were enrolled in families at increased genetic risk. RESULTS: The first 346 completed referrals led to 306 probands being enrolled, including 190 probands affected with pdac, who represent the population focus of the qpcs. Participation rates were 88.4% for all referrals and 89.2% for pdac referrals. Family history, epidemiologic and clinical data, and biospecimens were ascertained from 91.9%, 54.6%, and 97.5% respectively of patients with pdac. Although demographics and trends in risk factors in our patients were consistent with published statistics for patients with pdac, the qpcs is enriched for families with French-Canadian ancestry (37.4%), a population with recurrent germ-line mutations in hereditary diseases. CONCLUSIONS: Using rapid ascertainment, a pdac and pat research registry with high participation rates can be established. The qpcs is a valuable research resource and its enrichment with patients of French-Canadian ancestry provides a unique opportunity for studies of heredity in these diseases.

Hepatic arterial infusion pump chemotherapy in the management of colorectal liver metastases: expert consensus statement.

Despite significant improvements in systemic therapy for patients with colorectal liver metastases (crlms), response rates in the first-line setting are not optimal, and response rates in the second-line setting remain disappointing. Hepatic arterial infusion pump (haip) chemotherapy has been extensively studied in patients with crlms, but it remains infrequently used. We convened an expert panel to discuss the role of haip in the contemporary management of patients with crlm. Using a consensus process, we developed these statements: haip chemotherapy should be given in combination with systemic chemotherapy.haip chemotherapy should be offered in the context of a multidisciplinary program that includes expertise in hepatobiliary surgery, medical oncology, interventional radiology, nursing, and nuclear medicine.haip chemotherapy in combination with systemic therapy should be considered in patients with unresectable crlms who have progressed on first-line systemic treatment. In addition, haip chemotherapy is acceptable as first-line treatment in patients with unresectable colorectal liver metastases.haip chemotherapy is not recommended in the setting of extrahepatic disease outside the context of a clinical trial.haip chemotherapy in combination with systemic therapy is an option for select patients with resected colorectal liver metastases. These consensus statements provide a framework that clinicians who treat patients with crlm can use when considering treatment with haip.

Characterization of the growth hormone receptor in human dermal fibroblasts and liver during development.

Human tissues express growth hormone receptors (hGHR) by the 3rd mo of gestation. We assessed developmental changes in hGHR function in fibroblasts and liver, testing binding and hormonal response. Fetal cells showed low but reproducible hGH binding. No age-related changes occurred in fibroblasts (9 wk-34 yr). In contrast, there was a fourfold increase in hGH binding in postnatal liver, with a sixfold increase in hGHR mRNA. Both full-length and truncated hGHR mRNAs were detected in all livers. Cross-linking revealed a larger hGH/receptor complex in fetal liver. Fetal hepatocytes produced 10 times more insulin-like growth factor (IGF)-II than IGF-I, and responded to hGH (150 ng/ml) with a significant increase in IGF-II. Fetal hepatocytes secreted three IGF-binding proteins (IGFBPs), including IGFBP1, but not IGFBP3. hGH did not alter fetal hepatocyte IGFBPs but stimulated glucose uptake. Exposure of fibroblasts to hGH decreased hGH binding only in >1-yr postnatal fibroblasts, whereas treatment with dexamethasone (100-400 nM) increased binding only in postnatal cells. Thus, although fetal hepatocytes and fibroblasts possess functional hGHR, these receptors (and/or their signaling pathways) are immature or have adapted to the in utero environment.

Organization and evolution of the human growth hormone receptor gene 5'-flanking region.

Previous studies have identified eight variant human GH receptor (hGHR) messenger RNA (mRNAs; V1-V8), that differ in their 5'-untranslated regions (5'UTRs) but splice into the same site just upstream of the translation start site in exon 2; thus, they encode the same protein. Here we report a novel variant, V9, and describe the mapping of all nine 5'UTR sequences within 40 kb upstream of exon 2. A cluster of three sequences, V2-V9-V3 (termed module A), lies furthest 5', and approximately 16 kb downstream is a second cluster of four exons, V7-V1-V4-V8 (module B). V6 is midway between modules A and B. Module B is about 18 kb upstream of V5, which lies adjacent to exon 2. hGHR expression is under developmental- and tissue-specific regulation, and expression of the variant mRNAs is related to their position within the 5'-flanking region; whereas module A (V2,V9,V3) and V5 variants are widely expressed, module B (V7,V1,V4,V8) and V6 variant mRNAs are detectable only in postnatal liver. Transcriptional start sites for V1 and V9 (representing the two different modules) were identified, showing that postnatal liver-specific expression of V1 is driven from two TATA boxes, whereas the ubiquitous V9 transcript has a single start site and a TATA-less promoter. V9 promoter activity was shown by in vivo and in vitro transfection assays, and an NF-Y binding site was demonstrated by electromobility shift assay. Thus, the regulatory regions of the hGHR gene are complex, and the clustering of seven 5'UTR exons within two modules with distinctly different mRNA expression patterns is the most striking feature.

The baboon: a model for the study of primate growth hormone receptor gene expression during development.

In subprimates, significant onset of growth hormone receptor (GHR) expression occurs only after birth whereas, in the human, GHR mRNA and protein are widely manifest from the first trimester of fetal life. Thus, it is likely that subprimates are not the best models for studying regulation of human GHR gene transcription, especially during early stages in development. Here we have explored the potential of the baboon as a more appropriate model. Baboon GHR cDNAs were cloned from postnatal liver by reverse transcription (RT)-PCR, using human GHR-specific primers. The encoded baboon GHR precursor protein has an identical signal peptide sequence to that of human and rhesus monkey GHRs, and the mature baboon GHR is also 620 amino acids long, with 95% and 98.5% amino acid identity to the human and rhesus monkey receptors respectively. Previous studies in the human have identified eight 5' untranslated region (5' UTR) variants of the GHR mRNA (V1 to V8, numbered according to their relative abundance). We cloned the baboon V1, V3 and V4 homologues by RT-PCR: these variants have a high degree (>92%) of sequence identity with their human counterparts and also diverge at an identical point, 12 nucleotides upstream of the start of translation. The expression pattern of these three GHR mRNA isoforms in baboon liver during development was characterized. Strong expression of baboon V1 and V4 was evident by 49 days of postnatal life (n=5, 49 days and adult (18.6-19.6 kg)); very low levels of V1, but not V4, were observed in younger animals (n=2, 6 and 30 days). In contrast, V3 5' UTR variant mRNA was present in all fetal (n=4, 141-155 days gestation) and postnatal (n=7, 6-19.6 days and adult (18.6 kg)) hepatic specimens examined. Analysis of postnatal kidney and lung (n=2, 19 and 19.6 kg) revealed that V3 transcripts are present in these tissues, but not V1 and V4. Together, these data demonstrate that, as in the human, baboon V1 and V4 expression is developmentally regulated and tissue specific, while the V3 isoform is more widely expressed. Therefore, it is likely that the regulatory regions of the baboon and human GHR genes are well conserved. Our findings suggest that the baboon is an appropriate animal model in which to define the mechanisms regulating GHR gene transcription during primate development.

Fetal- and tumor-specific regulation of growth hormone receptor messenger RNA expression in human liver.

Eight different 5'-untranslated region variants of the human growth hormone receptor (hGHR) mRNA have been identified in adult liver (V1-V8). We have compared the expression of two of these variants (V1 and V3) in several human fetal and postnatal tissues (including liver) as well as in hepatoblastomas (HBs) and hepatocellular carcinomas (HCCs). Using reverse transcription-PCR assays, followed by Southern blotting to confirm the specificity of the amplified fragments, we found that V3 was expressed in all fetal and postnatal liver (n = 13 fetal and 5 postnatal), kidney (n = 4 fetal and 4 postnatal), lung (n = 4 fetal and 2 postnatal), intestine (n = 8 fetal and 4 postnatal), skeletal muscle (n = 1 fetal and 1 postnatal), and adrenal (n = 1 fetal and 1 postnatal) samples. In contrast, V1 was expressed only in postnatal liver. We then screened for V1 and V3 in HBs (n = 17, ages 6-36 months, including 5 with paired normal liver), and HCCs (n = 4, ages 50-75 years, with paired normal liver). V1 was undetectable in 15 of 17 HBs, including all HBs paired with (V1-expressing) normal liver; the absence of V1 did not correlate with patient age, sex, HB subtype, +/- chemotherapy, exon 3-retaining and -deficient hGHR mRNA isoform pattern, or loss of heterozygosity at 11p, 1p, and 1q. The four HCCs showed marked (>20-fold; n = 2) or complete (n = 2) suppression of V1 as compared to paired normal liver. V3 was expressed in all HBs, HCCs, and paired normal livers. Interestingly, V3, but not V1, was detected in two Wilms' tumor and paired normal kidney specimens. Our findings suggest that, in the human, there is tissue-, fetal- and tumor-specific regulation of V1 hGHR mRNA.

Expression of exon 3-retaining and -deleted human growth hormone receptor messenger ribonucleic acid isoforms during development.

Recent investigations have suggested that human GH (hGH) and its receptor may have specific functions during human fetal life. To improve our understanding of the mechanisms of hGH action during gestation, we characterized the ontogenic appearance of hGH receptor messenger ribonucleic acid (mRNA) in multiple human fetal and postnatal tissues. Using RT-PCR assays, followed by Southern hybridization to confirm the specificity of the amplified fragments, we scanned the entire coding region of the hGH receptor mRNA. Transcription of the hGH receptor gene was observed in all fetal tissues studied (liver, kidney, skin, muscle, lung, adrenal, spleen, intestine, central nervous system, pancreas, and placental villi) from the earliest stage that could be examined [7-14.8 weeks fetal age (FA)]. Furthermore, we identified only 2 isoforms of the hGH receptor mRNA-coding region: exon 3 can be retained or deleted. Surprisingly, we found individual-specific, not tissue-specific, expression patterns of these two transcripts when we examined multiple tissues (n = 2-6) from 15 individuals (11.5-33 weeks FA); this individual-specific pattern of expression is maintained in cultured dermal fibroblasts for at least 12 generations (n = 2; 16 and 20 weeks FA). In addition, a cross-sectional study of 78 individuals (9 weeks FA to 43 yr postnatal age) showed that the exon 3-deleted transcript is predominantly expressed in tissues from fetuses of 9-20 weeks FA (P < 0.002). Finally, we showed that the absence of exon 3 from the mRNA is not due to genomic deletion of exon 3 by amplifying exon 3 from genomic DNA of 3 fetuses (13.3-19 weeks FA) expressing only the exon 3-deleted mRNA transcript. We conclude that 1) transcription of the hGH receptor gene occurs in multiple tissues as early as the first trimester of human fetal life; 2) the exon 3-retaining and -deleted transcripts are the only two isoforms of the hGH receptor mRNA-coding region during gestation; and 3) the pattern of expression of these transcripts is individual specific and may be developmentally regulated.