Plasma hPG80 (Circulating Progastrin) as a Novel Prognostic Biomarker for early-stage breast cancer in a breast cancer cohort.

BACKGROUND: Recurrence and metastases are still frequent outcomes after initial tumour control in women diagnosed with breast cancer. Although therapies are selected based on tumour characteristics measured at baseline, prognostic biomarkers can identify those at risk of poor outcomes. Circulating progastrin or hPG80 was found to be associated with survival outcomes in renal and hepatocellular carcinomas and was a plausible prognostic biomarker for breast cancer. METHODS: Women with incident breast cancers from Calgary, Alberta, Canada enrolled in the Breast to Bone (B2B) study between 2010 to 2016 and provided blood samples prior to any treatment initiation. Plasma from these baseline samples were analysed for circulating progastrin or hPG80. Participant characteristics as well as tumour ones were evaluated for their association with hPG80 and survival outcomes (time to recurrence, recurrence - free survival, breast cancer specific survival and overall survival) in Cox proportional hazards regression models. RESULTS: The 464 participants with measurable hPG80 in this study had an average age of 57.03 years (standard deviation of 11.17 years) and were predominantly diagnosed with Stage I (52.2%) and Stage II (40.1%) disease. A total of 50 recurrences and 50 deaths were recorded as of June 2022. In Cox PH regression models adjusted for chemotherapy, radiation therapy, cancer stage and age at diagnosis, log hPG80 (pmol/L) significantly increased the risks for recurrence (Hazard Ratio (HR) = 1.330, 95% Confidence Interval (CI) = (0.995 - 1.777, p = 0.054)), recurrence-free survival (HR = 1.399, 95% CI = (1.106 - 1.770), p = 0.005) and overall survival (HR = 1.385, 95% CI = (1.046 - 1.834), = 0.023) but not for breast cancer specific survival (HR = 1.015, 95% CI = (0.684 - 1.505), p = 0.942). CONCLUSIONS: hPG80 levels measured at diagnosis were significantly associated with the risk of recurrence or death from any cause in women with breast cancer. Since the recurrence rates of breast cancer are still relatively high amongst women diagnosed at an early stage, identifying women at high risk of recurrence at their time of diagnosis is important. hPG80 is a promising new prognostic biomarker that could improve the identification of women at higher risk of poor outcomes.

Essential requirement for ß-arrestin2 in mouse intestinal tumors with elevated Wnt signaling.

ß-Arrestins (Arrb) participate in the regulation of multiple signaling pathways, including Wnt/ß-catenin, the major actor in human colorectal cancer initiation. To better understand the roles of Arrb in intestinal tumorigenesis, a reverse genetic approach (Arrb(-/-)) and in vivo siRNA treatment were used in Apc(Δ14/+) mice. Mice with Arrb2 depletion (knockout and siRNA) developed only 33% of the tumors detected in their Arrb2-WT littermates, whereas Arrb1 depletion remained without significant effect. These remaining tumors grow normally and are essentially Arrb2-independent. Unsupervised hierarchical clustering analysis showed that they clustered with 25% of Apc(Δ14/+);Arrb2(+/+) tumors. Genes overexpressed in this subset reflect a high interaction with the immune system, whereas those overexpressed in Arrb2-dependent tumors are predominantly involved in Wnt signaling, cell adhesion, migration, and extracellular matrix remodeling. The involvement of Arrb2 in intestinal tumor development via the regulation of the Wnt pathway is supported by ex vivo and in vitro experiments using either tumors from Apc(Δ14/+) mice or murine Apc(Min/+) cells. Indeed, Arrb2 siRNAs decreased the expression of Wnt target genes in cells isolated from 12 of 18 tumors from Apc(Δ14/+) mice. In Apc(Min/+) cells, Arrb2 siRNAs completely reversed the increased Wnt activity and colony formation in soft agar induced by Apc siRNA treatment, whereas they did not affect these parameters in basal conditions or in cells expressing constitutively active ß-catenin. We demonstrate that Arrb2 is essential for the initiation and growth of intestinal tumors displaying elevated Wnt pathway activity and identify a previously unsuspected molecular heterogeneity among tumors induced by truncating Apc mutations.

Characterization of a novel PXR isoform with potential dominant-negative properties.

BACKGROUND & AIMS: The nuclear Pregnane X Receptor (PXR, NR1I2) plays a pivotal role in xenobiotic metabolism. Here, we sought to characterize a new PXR isoform (hereafter called small PXR or sPXR) stemming from alternative transcription starting sites downstream of a CpG Island located near exon 3 of the human PXR gene. METHODS: Quantitative RT-PCR, western blot, methylation-specific PCR, luciferase reporter assays, electro-mobility shift assays, and stable sPXR overexpression were used to examine sPXR expression and function in hepatocellular cell lines, healthy human liver (n=99), hepatocellular adenomas (HCA, n=91) and hepatocellular carcinoma samples (HCC, n=213). RESULTS: Liver sPXR mRNA expression varied importantly among individuals and encodes a 37kDa nuclear protein consisting of the ligand-binding domain of PXR that behaves as a dominant-negative of PXR transactivation properties. In vitro methylation of the sPXR upstream promoter abolished its activity, while the demethylation agent 5-aza-2-deoxycytidine increased sPXR mRNA expression in several cell lines. Finally, we observed that sPXR mRNA expression displayed significant differences related to HCA or HCC biology. CONCLUSIONS: This novel PXR isoform, displaying a dominant-negative activity and regulated by DNA methylation, is associated with outcomes of patients with HCC treated by resection, suggesting that it represents a key modulator of PXR.

Cell death and neuronal differentiation of glioblastoma stem-like cells induced by neurogenic transcription factors.

Glioblastoma multiform (GBM) are devastating brain tumors containing a fraction of multipotent stem-like cells which are highly tumorigenic. These cells are resistant to treatments and are likely to be responsible for tumor recurrence. One approach to eliminate GBM stem-like cells would be to force their terminal differentiation. During development, neurons formation is controlled by neurogenic transcription factors such as Ngn1/2 and NeuroD1. We found that in comparison with oligodendrogenic genes, the expression of these neurogenic genes is low or absent in GBM tumors and derived cultures. We thus explored the effect of overexpressing these neurogenic genes in three CD133(+) Sox2(+) GBM stem-like cell cultures and the U87 glioma line. Introduction of Ngn2 in CD133(+) cultures induced massive cell death, proliferation arrest and a drastic reduction of neurosphere formation. Similar effects were observed with NeuroD1. Importantly, Ngn2 effects were accompanied by the downregulation of Olig2, Myc, Shh and upregulation of Dcx and NeuroD1 expression. The few surviving cells adopted a typical neuronal morphology and some of them generated action potentials. These cells appeared to be produced at the expense of GFAP(+) cells which were radically reduced after differentiation with Ngn2. In vivo, Ngn2-expressing cells were unable to form orthotopic tumors. In the U87 glioma line, Ngn2 could not induce neuronal differentiation although proliferation in vitro and tumoral growth in vivo were strongly reduced. By inducing cell death, cell cycle arrest or differentiation, this work supports further exploration of neurogenic proteins to oppose GBM stem-like and non-stem-like cell growth.

Symplekin promotes tumorigenicity by up-regulating claudin-2 expression.

Symplekin is a ubiquitously expressed protein involved in cytoplasmic RNA polyadenylation and transcriptional regulation and is localized at tight junctions (TJs) in epithelial cells. Nuclear symplekin cooperates with the Y-box transcription factor zonula occludens 1-associated nucleic acid-binding protein (ZONAB) to increase the transcription of cell cycle-related genes and also inhibits differentiation of intestinal cells. We detected high levels of nuclear symplekin in 8 of 12 human colorectal cancer (CRC) samples. shRNA-mediated reduction of symplekin expression was sufficient to decrease significantly the anchorage-independent growth and proliferation of HT-29 CRC cells as well as their tumorigenicity when injected into immunodeficient animals. Symplekin down-regulation also was found to alter ion transport through TJs, to promote the localization of ZONAB in the membrane rather than the nucleus, and strongly to enhance cell polarization in a 3D matrix, leading to the formation of spheroids organized around a central lumen. Claudin-2 expression was reduced following symplekin down-regulation, an effect mimicked when ZONAB expression was down-regulated using selective siRNA. Virus-mediated restoration of claudin-2 expression was found to restore nuclear expression of ZONAB in HT29DeltaSym cells and to rescue the phenotypic alterations induced by symplekin down-regulation of cell polarity, paracellular transport, ZONAB localization, cyclin D1 expression, proliferation, and anchorage-independent growth. Finally, siRNA-mediated claudin-2 down-regulation increased the transepithelial resistance and decreased cyclin D1 expression and ZONAB nuclear localization, similar to observations in symplekin-depleted cells. Our results suggest that nuclear overexpression of symplekin promotes tumorigenesis in the human colon and that the regulation of claudin-2 expression is instrumental in this effect.

Corrigendum: Variation of photosynthetic induction in major horticultural crops is mostly driven by differences in stomatal traits.

[This corrects the article DOI: 10.3389/fpls.2022.860229.].

A small dynamic leaf-level model predicting photosynthesis in greenhouse tomatoes.

The conversion of supplemental greenhouse light energy into biomass is not always optimal. Recent trends in global energy prices and discussions on climate change highlight the need to reduce our energy footprint associated with the use of supplemental light in greenhouse crop production. This can be achieved by implementing "smart" lighting regimens which in turn rely on a good understanding of how fluctuating light influences photosynthetic physiology. Here, a simple fit-for-purpose dynamic model is presented. It accurately predicts net leaf photosynthesis under natural fluctuating light. It comprises two ordinary differential equations predicting: 1) the total stomatal conductance to CO2 diffusion and 2) the CO2 concentration inside a leaf. It contains elements of the Farquhar-von Caemmerer-Berry model and the successful incorporation of this model suggests that for tomato (Solanum lycopersicum L.), it is sufficient to assume that Rubisco remains activated despite rapid fluctuations in irradiance. Furthermore, predictions of the net photosynthetic rate under both 400ppm and enriched 800ppm ambient CO2 concentrations indicate a strong correlation between the dynamic rate of photosynthesis and the rate of electron transport. Finally, we are able to indicate whether dynamic photosynthesis is Rubisco or electron transport rate limited.

Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium.

The HMG-box transcription factor Sox9 is expressed in the intestinal epithelium, specifically, in stem/progenitor cells and in Paneth cells. Sox9 expression requires an active beta-catenin-Tcf complex, the transcriptional effector of the Wnt pathway. This pathway is critical for numerous aspects of the intestinal epithelium physiopathology, but processes that specify the cell response to such multipotential signals still remain to be identified. We inactivated the Sox9 gene in the intestinal epithelium to analyze its physiological function. Sox9 inactivation affected differentiation throughout the intestinal epithelium, with a disappearance of Paneth cells and a decrease of the goblet cell lineage. Additionally, the morphology of the colon epithelium was severely altered. We detected general hyperplasia and local crypt dysplasia in the intestine, and Wnt pathway target genes were up-regulated. These results highlight the central position of Sox9 as both a transcriptional target and a regulator of the Wnt pathway in the regulation of intestinal epithelium homeostasis.

Variation of Photosynthetic Induction in Major Horticultural Crops Is Mostly Driven by Differences in Stomatal Traits.

Under natural conditions, irradiance frequently fluctuates, causing net photosynthesis rate (A) to respond slowly and reducing the yields. We quantified the genotypic variation of photosynthetic induction in 19 genotypes among the following six horticultural crops: basil, chrysanthemum, cucumber, lettuce, tomato, and rose. Kinetics of photosynthetic induction and the stomatal opening were measured by exposing shade-adapted leaves (50 µmol m-2 s-1) to a high irradiance (1000 µmol m-2 s-1) until A reached a steady state. Rubisco activation rate was estimated by the kinetics of carboxylation capacity, which was quantified using dynamic A vs. [CO2] curves. Generally, variations in photosynthetic induction kinetics were larger between crops and smaller between cultivars of the same crop. Time until reaching 20-90% of full A induction varied by 40-60% across genotypes, and this was driven by a variation in the stomatal opening rather than Rubisco activation kinetics. Stomatal conductance kinetics were partly determined by differences in the stomatal size and density; species with densely packed, smaller stomata (e.g., cucumber) tended to open their stomata faster, adapting stomatal conductance more rapidly and efficiently than species with larger but fewer stomata (e.g., chrysanthemum). We conclude that manipulating stomatal traits may speed up photosynthetic induction and growth of horticultural crops under natural irradiance fluctuations.

How to improve hospital admission screening for patients at risk of multidrug-resistant organism carriage: a before-and-after interventional study and cost-effectiveness analysis.

BACKGROUND: Infection prevention and control (IPC) is a prioritised task for healthcare workers in emergency department (ED). Here, we examined compliance with admission screening (AS) and additional precautions (AP) measures for patients at risk of infection with multidrug-resistant organisms (MDROs) by using a two-stage, multifaceted educational intervention, also comparing the cost of a developed automated indicator for AS and AP compliance and clinical audits to sustain observed findings. METHODS: In the first stage, staff in the ED of the University Hospitals of Geneva, Switzerland, were briefed on IPC measures (AS and AP). A cross-sectional survey was then conducted to assess barriers to IPC measures. In the second stage, healthcare workers underwent training sessions, and an electronic patient record 'order-set' including AS and AP compliance indicators was designed. We compared the cost-benefit of the audits and the automated indicators for AS and AP compliance. RESULTS: Compliance significantly improved after training, from 36.2% (95% CI 23.6% to 48.8%) to 78.8% (95% CI 67.1% to 90.3%) for AS (n=100, p=0.0050) and from 50.2% (95% CI 45.3% to 55.1%) to 68.5% (95% CI 60.1% to 76.9%) for AP (n=125, p=0.0092). Healthcare workers recognised MDRO screening as an ED task (70.2%), with greater acknowledgment of risk factors at AS considered an ED duty. The monthly cost was higher for clinical audits than the automated indicator, with a reported yearly cost of US$120 203. The initial cost of developing the automated indicator was US$18 290 and its return on investment US$3.44 per US$1 invested. CONCLUSION: Training ED staff increased compliance with IPC measures when accompanied by team discussions for optimal effectiveness. An automated indicator of compliance is cheaper and closer to real-time than a clinical audit.

hPG80 (Circulating Progastrin), a Novel Blood-Based Biomarker for Detection of Poorly Differentiated Neuroendocrine Carcinoma and Well Differentiated Neuroendocrine Tumors.

Current blood-based biomarkers for neuroendocrine neoplasms (NENs) lack both sensitivity and specificity. Human circulating progastrin (hPG80) is a novel biomarker that can be easily measured in plasma by ELISA. This study is the first to examine hPG80 in NENs. Plasma hPG80 was quantified from 95 stage IV NEN patients, using DxPG80 technology (ECS Progastrin, Switzerland) and compared with hPG80 concentrations in two cohorts of healthy donor controls aged 50-80 (n = 252) and 18-25 (n = 137). Median hPG80 in NENs patients was 5.54 pM compared to 1.5 pM for the 50-80 controls and 0.29 pM the 18-25 cohort (p < 0.0001). Subgroup analysis revealed median hPG80 levels significantly higher than for either control cohort in neuroendocrine carcinoma (NEC; n = 25) and neuroendocrine tumors (NET; n = 70) including the small-cell lung cancer (SCLC) sub-cohort (n = 13). Diagnostic accuracy, estimated by AUCs, was high for NENs, as well as both sub-groups (NEC/NET) when compared to the younger and older control groups. Plasma hPG80 in NENs may be a diagnostic blood biomarker for both low- and high-grade NENs; further study is warranted. A prospective multi-center trial is ongoing in NET to evaluate hPG80 as a means of monitoring disease (NCT04750954).

The long road to colorectal cancer therapy: searching for the right signals.

During the last 25 years, improvements in our understanding of signaling processes mediating tumor initiation, growth and dissemination have opened the door towards increasingly efficient, selective, but also complex therapeutic approaches. In parallel, the better characterization of altered signaling pathways in tumor cells, as well as the relationship between these alterations and the sensitivity of tumors to given compounds, have forced us to contemplate the use of biomarkers allowing patient selection and evaluation of treatment efficacy. Such biomarkers should become a corner stone for the success of a more rational and "personalized" clinical approach to colorectal cancer. Here we give an overview of the "signaling pathway-selective" compounds that are currently in use or under clinical development in the setting of colorectal cancer (CRC) and we discuss promising targets, based on our current knowledge of colorectal tumor cell biology, such as Src family, Wnt and Hegdehog signaling.

Assessing the role of initial conditions in the local structural identifiability of large dynamic models.

Structural identifiability is a binary property that determines whether or not unique parameter values can, in principle, be estimated from error-free input-output data. The many papers that have been written on this topic collectively stress the importance of this a priori analysis in the model development process. The story however, often ends with a structurally unidentifiable model. This may leave a model developer with no plan of action on how to address this potential issue. We continue this model exploration journey by identifying one of the possible sources of a model's unidentifiability: problematic initial conditions. It is well-known that certain initial values may result in the loss of local structural identifiability. Nevertheless, literature on this topic has been limited to the analysis of small toy models. Here, we present a systematic approach to detect problematic initial conditions of real-world systems biology models, that are usually not small. A model's identifiability can often be reinstated by changing the value of such problematic initial conditions. This provides modellers an option to resolve the "unidentifiable model" problem. Additionally, a good understanding of which initial values should rather be avoided can be very useful during experimental design. We show how our approach works in practice by applying it to five models. First, two small benchmark models are studied to get the reader acquainted with the method. The first one shows the effect of a zero-valued problematic initial condition. The second one illustrates that the approach also yields correct results in the presence of input signals and that problematic initial conditions need not be zero-values. For the remaining three examples, we set out to identify key initial values which may result in the structural unidentifiability. The third and fourth examples involve a systems biology Epo receptor model and a JAK/STAT model, respectively. In the final Pharmacokinetics model, of which its global structural identifiability has only recently been confirmed, we indicate that there are still sets of initial values for which this property does not hold.

Cohort profile: Actionable Register of Geneva Outpatients and inpatients with SARS-CoV-2 (ARGOS).

PURPOSE: The Actionable Register of Geneva Outpatients and inpatients with SARS-CoV-2 (ARGOS) is an ongoing prospective cohort created by the Geneva Directorate of Health. It consists of an operational database compiling all SARS-CoV-2 test results recorded in the Geneva area since late February 2020. This article aims at presenting this comprehensive cohort, in light of some of the varying public health measures in Geneva, Switzerland, since March 2020. PARTICIPANTS: As of 1 June 2021, the database included 360 525 patients, among which 65 475 had at least one positive test result for SARS-CoV-2. Among all positive patients, 37.6% were contacted only once, 10.6% had one follow-up call, 8.5% had two and 27.7% had three or more follow-up calls. Participation rate among positive patients is 94%. Data collection is ongoing. FINDINGS TO DATE: ARGOS data illustrates the magnitude of COVID-19 pandemic in Geneva, Switzerland, and details a variety of population factors and outcomes. The content of the cohort includes demographic data, comorbidities and risk factors for poor clinical outcome, self-reported COVID-19 symptoms, environmental and socioeconomic factors, prospective and retrospective contact tracing data, travel quarantine data and deaths. The registry has already been used in several publications focusing on symptoms and long COVID-19, infection fatality rate and re-infection. FUTURE PLANS: The data of this large real-world registry provides a valuable resource for various types of research, such as clinical research, epidemiological research or policy assessment as it illustrates the impact of public health policies and overall disease burden of COVID-19.

A novel method to detect hPG80 (human circulating progastrin) in the blood.

hPG80 (human circulating progastrin) is produced and released by cancer cells. We recently reported that hPG80 is detected in the blood of patients with cancers from different origins, suggesting its potential utility for cancer detection. To accurately measure hPG80 in the blood of patients, we developed the DxPG80 test, a sandwich Enzyme-Linked Immunosorbent Assay (ELISA). This test quantifies hPG80 in EDTA plasma samples. The analytical performances of the DxPG80 test were evaluated using standard procedures and guidelines specific to ELISA technology. We showed high specificity for hPG80 with no cross-reactivity with human glycine-extended gastrin (hG17-Gly), human carboxy-amidated gastrin (hG17-NH2) or the CTFP (C-Terminus Flanking Peptide) and no interference with various endogenous or exogenous compounds. The test is linear between 0 and 50 pM hPG80 (native or recombinant). We demonstrated a trueness of measurement, an accuracy and a variability of hPG80 quantification with the DxPG80 test below the 20% relative errors as recommended in the guidelines. The limit of detection of hPG80 and the limit of quantification were calculated as 1 pM and 3.3 pM respectively. In conclusion, these results show the strong analytical performance of the DxPG80 test to measure hPG80 in blood samples.

Prognostic Value of Plasma hPG80 (Circulating Progastrin) in Metastatic Renal Cell Carcinoma.

Precise management of kidney cancer requires the identification of prognostic factors. hPG80 (circulating progastrin) is a tumor promoting peptide present in the blood of patients with various cancers, including renal cell carcinoma (RCC). In this study, we evaluated the prognostic value of plasma hPG80 in 143 prospectively collected patients with metastatic RCC (mRCC). The prognostic impact of hPG80 levels on overall survival (OS) in mRCC patients after controlling for hPG80 levels in non-cancer age matched controls was determined and compared to the International Metastatic Database Consortium (IMDC) risk model (good, intermediate, poor). ROC curves were used to evaluate the diagnostic accuracy of hPG80 using the area under the curve (AUC). Our results showed that plasma hPG80 was detected in 94% of mRCC patients. hPG80 levels displayed high predictive accuracy with an AUC of 0.93 and 0.84 when compared to 18-25 year old controls and 50-80 year old controls, respectively. mRCC patients with high hPG80 levels (>4.5 pM) had significantly lower OS compared to patients with low hPG80 levels (<4.5 pM) (12 versus 31.2 months, respectively; p = 0.0031). Adding hPG80 levels (score of 1 for patients having hPG80 levels > 4.5 pM) to the six variables of the IMDC risk model showed a greater and significant difference in OS between the newly defined good-, intermediate- and poor-risk groups (p = 0.0003 compared to p = 0.0076). Finally, when patients with IMDC intermediate-risk group were further divided into two groups based on hPG80 levels within these subgroups, increased OS were observed in patients with low hPG80 levels (<4.5 pM). In conclusion, our data suggest that hPG80 could be used for prognosticating survival in mRCC alone or integrated to the IMDC score (by adding a variable to the IMDC score or by substratifying the IMDC risk groups), be a prognostic biomarker in mRCC patients.

Pregnane X Receptor (PXR) expression in colorectal cancer cells restricts irinotecan chemosensitivity through enhanced SN-38 glucuronidation.

BACKGROUND: Clinical efficacy of chemotherapy in colorectal cancer is subjected to broad inter-individual variations leading to the inability to predict outcome and toxicity. The topoisomerase I inhibitor irinotecan (CPT-11) is worldwide approved for the treatment of metastatic colorectal cancer and undergoes extensive peripheral and tumoral metabolism. PXR is a xenoreceptor activated by many drugs and environmental compounds regulating the expression of drug metabolism and transport genes in detoxification organs such as liver and gastrointestinal tract. Considering the metabolic pathway of irinotecan and the tissue distribution of Pregnane x Receptor (PXR), we hypothesized that PXR could play a key role in colon cancer cell response to irinotecan. RESULTS: PXR mRNA expression was quantified by RT-quantitative PCR in a panel of 14 colon tumor samples and their matched normal tissues. PXR expression was modulated in human colorectal cancer cells LS174T, SW480 and SW620 by transfection and siRNA strategies. Cellular response to irinotecan and its active metabolic SN38 was assessed by cell viability assays, HPLC metabolic profiles and mRNA quantification of PXR target genes. We showed that PXR was strongly expressed in colon tumor samples and displayed a great variability of expression. Expression of hPXR in human colorectal cancer cells led to a marked chemoresistance to the active metabolite SN38 correlated with PXR expression level. Metabolic profiles of SN38 showed a strong enhancement of SN38 glucuronidation to the inactive SN38G metabolite in PXR-expressing cells, correlated with an increase of UDPglucuronosyl transferases UGT1A1, UGT1A9 and UGT1A10 mRNAs. Inhibition of PXR expression by lentivirus-mediated shRNA, led to SN38 chemoresistance reversion concomitantly to a decrease of UGT1A1 expression and SN38 glucuronidation. Similarly, PXR mRNA expression levels correlated to UGT1A subfamily expression in human colon tumor biopsies. CONCLUSION: Our results demonstrate that tumoral metabolism of SN38 is affected by PXR and point to potential therapeutic significance of PXR quantification in the prediction of irinotecan response. Furthermore, our observations are pharmacologically relevant since many patients suffering from cancer diseases are often exposed to co-medications, food additives or herbal supplements able to activate PXR. A substantial part of the variability observed among patients might be caused by such interactions.

The symplekin/ZONAB complex inhibits intestinal cell differentiation by the repression of AML1/Runx1.

BACKGROUND & AIMS: Symplekin is a ubiquitously expressed protein involved in RNA polyadenylation and transcriptional regulation that localizes at tight junctions in epithelial cells. The association between symplekin and the Y-box transcription factor ZONAB activates proliferation in intestinal and kidney cells. We analyzed symplekin expression in human colonic crypts and investigated its function in differentiation. METHODS: Expression of differentiation markers and transcription factors was assessed in HT29-Cl.16E cells that expressed inducible symplekin short hairpin RNA or were transfected with ZONAB small interfering RNAs. Intestines of AML1(Delta/Delta) mice were stained with alcian blue and analyzed for expression of AML1/Runx1, GAPDH, KLF-4, and Muc-2. Mobility shift and chromatin immunoprecipitation were used to detect AML1 and ZONAB/DbpA binding to promoter regions of the Krüppel-like factor 4 (KLF4) and acute myeloid leukemia-1 (AML1) genes, respectively. RESULTS: The gradient of nuclear symplekin expression decreased from the proliferative toward the differentiated compartment of colonic crypts; symplekin down-regulation promoted the differentiation of HT29-Cl.16E colorectal carcinoma cells into goblet cells. Down-regulation of symplekin or ZONAB/Dbpa induced de novo expression of the transcription factor AML1/Runx1, thereby increasing the expression of KLF4 and promoting goblet cell differentiation. Furthermore, increased AML1 expression was required for the induction of goblet cell differentiation after symplekin down-regulation. KLF4 expression and goblet cell numbers were reduced in the intestines of AML1(Delta/Delta) mice, confirming the role of AML1 as a promoter of intestinal differentiation in vivo. CONCLUSIONS: Symplekin cooperates with ZONAB to negatively regulate intestinal goblet cell differentiation, acting by repression of AML1 and KLF4.

The oncogenic and druggable hPG80 (Progastrin) is overexpressed in multiple cancers and detected in the blood of patients.

BACKGROUND: In colorectal cancer, hPG80 (progastrin) is released from tumor cells, promotes cancer stem cells (CSC) self-renewal and is detected in the blood of patients. Because the gene GAST that encodes hPG80 is a target gene of oncogenic pathways that are activated in many tumor types, we hypothesized that hPG80 could be expressed by tumors from various origins other than colorectal cancers, be a drug target and be detectable in the blood of these patients. METHODS: hPG80 expression was monitored by fluorescent immunohistochemistry and mRNA expression in tumors from various origins. Cancer cell lines were used in sphere forming assay to analyze CSC self-renewal. Blood samples were obtained from 1546 patients with 11 different cancer origins and from two retrospective kinetic studies in patients with peritoneal carcinomatosis or hepatocellular carcinomas. These patients were regularly sampled during treatments and assayed for hPG80. FINDINGS: We showed that hPG80 was present in the 11 tumor types tested. In cell lines originating from these tumor types, hPG80 neutralization decreased significantly CSC self-renewal by 28 to 54%. hPG80 was detected in the blood of patients at significantly higher concentration than in healthy blood donors (median hPG80: 4.88 pM versus 1.05 pM; p < 0.0001) and shown to be correlated to GAST mRNA levels in the matched tumor (i.e., lung cancers, Spearman r = 0.8; p = 0.0023). Furthermore, we showed a strong association between longitudinal hPG80 concentration changes and anti-cancer treatment efficacy in two independent retrospective studies. In the peritoneal carcinomatosis cohort, median hPG80 from inclusion to the post-operative period decreased from 5.36 to 3.00 pM (p < 0.0001, n = 62) and in the hepatocellular carcinoma cohort, median hPG80 from inclusion to remission decreased from 11.54 pM to 1.99 pM (p < 0.0001, n = 63). INTERPRETATION: Because oncogenic hPG80 is expressed in tumor cells from different origins and because circulating hPG80 in the blood is related to the burden/activity of the tumor, it is a promising cancer target for therapy and for disease monitoring. FUNDINGS: ECS-Progastrin.

Reference gene selection for head and neck squamous cell carcinoma gene expression studies.

BACKGROUND: It is no longer adequate to choose reference genes blindly. We present the first study that defines the suitability of 12 reference genes commonly used in cancer studies (ACT, ALAS, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPL27, RPS29, SHAD and TBP) for the normalization of quantitative expression data in the field of head and neck squamous cell carcinoma (HNSCC). RESULTS: Raw expression levels were measured by RT-qPCR in HNSCC and normal matched mucosa of 46 patients. We analyzed the expression stability using geNorm and NormFinder and compared the expression levels between subgroups. In HNSCC and/or normal mucosa, the four best normalization genes were ALAS, GAPDH, RPS18 and SHAD and the most stable combination of two genes was GAPDH-SHAD. We recommend using KALPHA-TBP for the study of T1T2 tumors, RPL27-SHAD for T3T4 tumors, KALPHA-SHAD for N0 tumors, and ALAS-TBP for N+ tumors. ACT, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPS29, SHAD and TBP were slightly misregulated (<1.7-fold) between tumor and normal mucosa but can be used for normalization, depending on the resolution required for the assay. CONCLUSION: In the field of HNSCC, this study will guide researchers in selecting the most appropriate reference genes from among 12 potentially suitable reference genes, depending on the specific setting of their experiments.