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.

Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state.

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides a unique opportunity to derive patient-specific stem cells with potential applications in tissue replacement therapies and without the ethical concerns of human embryonic stem cells (hESCs). However, cellular senescence, which contributes to aging and restricted longevity, has been described as a barrier to the derivation of iPSCs. Here we demonstrate, using an optimized protocol, that cellular senescence is not a limit to reprogramming and that age-related cellular physiology is reversible. Thus, we show that our iPSCs generated from senescent and centenarian cells have reset telomere size, gene expression profiles, oxidative stress, and mitochondrial metabolism, and are indistinguishable from hESCs. Finally, we show that senescent and centenarian-derived pluripotent stem cells are able to redifferentiate into fully rejuvenated cells. These results provide new insights into iPSC technology and pave the way for regenerative medicine for aged patients.

Identification of a pharmacologically tractable Fra-1/ADORA2B axis promoting breast cancer metastasis.

Metastasis confronts clinicians with two major challenges: estimating the patient's risk of metastasis and identifying therapeutic targets. Because they are key signal integrators connecting cellular processes to clinical outcome, we aimed to identify transcriptional nodes regulating cancer cell metastasis. Using rodent xenograft models that we previously developed, we identified the transcription factor Fos-related antigen-1 (Fra-1) as a key coordinator of metastasis. Because Fra-1 often is overexpressed in human metastatic breast cancers and has been shown to control their invasive potential in vitro, we aimed to assess the implication and prognostic significance of the Fra-1-dependent genetic program in breast cancer metastasis and to identify potential Fra-1-dependent therapeutic targets. In several in vivo assays in mice, we demonstrate that stable RNAi depletion of Fra-1 from human breast cancer cells strongly suppresses their ability to metastasize. These results support a clinically important role for Fra-1 and the genetic program it controls. We show that a Fra-1-dependent gene-expression signature accurately predicts recurrence of breast cancer. Furthermore, a synthetic lethal drug screen revealed that antagonists of the adenosine receptor A2B (ADORA2B) are preferentially toxic to breast tumor cells expressing Fra-1. Both RNAi silencing and pharmacologic blockade of ADORA2B inhibited filopodia formation and invasive activity of breast cancer cells and correspondingly reduced tumor outgrowth in the lungs. These data show that Fra-1 activity is causally involved in and is a prognostic indicator of breast cancer metastasis. They suggest that Fra-1 activity predicts responsiveness to inhibition of pharmacologically tractable targets, such as ADORA2B, which may be used for clinical interference of metastatic breast cancer.

Cellular senescence in vivo: a barrier to tumorigenesis.

Cellular senescence is characterized by a largely irreversible cell cycle arrest that can be triggered by many types of intrinsic and extrinsic stress. These include telomere malfunction, oncogene activation and tumor suppressor gene inactivation. Ultimately, such events culminate in the activation of a tumor suppressor gene network. Since the first description of Oncogene-Induced cellular Senescence (OIS) little over a decade ago, many subsequent studies have confirmed that OIS prevents cells from undergoing oncogenic transformation in vitro. However, it has long been debated whether any in vivo correlates exist. It is only since recent years that evidence has been accumulating indicating that OIS in vivo does correspond to a major protective mechanism against cancer. In this review, we highlight some of the recent developments.

Mesenchymal stem cell features of Ewing tumors.

The cellular origin of Ewing tumor (ET), a tumor of bone or soft tissues characterized by specific fusions between EWS and ETS genes, is highly debated. Through gene expression analysis comparing ETs with a variety of normal tissues, we show that the profiles of different EWS-FLI1-silenced Ewing cell lines converge toward that of mesenchymal stem cells (MSC). Moreover, upon EWS-FLI1 silencing, two different Ewing cell lines can differentiate along the adipogenic lineage when incubated in appropriate differentiation cocktails. In addition, Ewing cells can also differentiate along the osteogenic lineage upon long-term inhibition of EWS-FLI1. These in silico and experimental data strongly suggest that the inhibition of EWS-FLI1 may allow Ewing cells to recover the phenotype of their MSC progenitor.

[Cellular senescence and the myth of Janus]. / La sénescence cellulaire - Un nouveau mythe de Janus?

Cellular senescence is, essentially, a permanent proliferation arrest induced by various cellular stresses or inappropriate stimuli. This arrest, which is associated with dramatic changes in cell morphology, metabolism and gene expression, involves a complex signalling network aiming at stable inactivation of CDKs, major cell cycle regulators. Notably, several tumour suppressors, such as p53, pRb or p16(Ink4a), play key roles both in the initiation of the senescence program and in its maintenance, which often involves epigenetic changes. While having widely recognized roles in tumour suppression and wound healing, senescence, like the roman god Janus, recently revealed another darker face. Mostly due to altered secretion phenotype favouring inflammation, senescent cells strongly influence surrounding tissue contributing to the development of age-related pathologies, including cancer.

Plasma hPG80 (Circulating Progastrin) as a Novel Prognostic Biomarker for Hepatocellular Carcinoma.

Alpha-fetoprotein (AFP) is the most widely used biomarker for hepatocellular carcinoma (HCC) prognosis. However, AFP is not useful in establishing a prognosis for patients with a tumor in the early stages. hPG80 (circulating progastrin) is a tumor promoting peptide present in the blood of patients with various cancers, including HCC. In this study, we evaluated the prognostic value of plasma hPG80 in patients with HCC, alone or in combination with AFP. A total of 168 HCC patients were tested prospectively for hPG80 and analyzed retrospectively. The prognostic impact of hPG80 and AFP levels on patient survival was assessed using Kaplan-Meier curves and log-rank tests. hPG80 was detected in 84% of HCC patients. There was no correlation between hPG80 and AFP levels in the training and validation cohorts. Both cohorts showed higher sensitivity of hPG80 compared to AFP, especially at early stages. Patients with high hPG80 (hPG80+) levels (optimal cutoff value 4.5 pM) had significantly lower median overall survival (OS) compared to patients with low hPG80 (hPG80-) levels (12.4 months versus not reached respectively, p < 0.0001). Further stratification by combining hPG80 and AFP levels (cutoff 100 ng/mL) improved prognosis in particular for those patients with low AFP level (hPG80-/AFP+ and hPG80-/AFP-, 13.4 months versus not reached respectively, p < 0.0001 and hPG80+/AFP+ and hPG80+/AFP-, 5.7 versus 26 months respectively, p < 0.0001). This was corroborated when analyses were performed using the BCLC staging especially at early stages. Our findings show that hPG80 could serve as a new prognostic biomarker in HCC. Used in combination with AFP, it improves the stratification of the patients in good and poor prognosis, especially for those patients with negative AFP and early-stage HCC.

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).

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.

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.

EWS/FLI-1 silencing and gene profiling of Ewing cells reveal downstream oncogenic pathways and a crucial role for repression of insulin-like growth factor binding protein 3.

Ewing tumors are characterized by abnormal transcription factors resulting from the oncogenic fusion of EWS with members of the ETS family, most commonly FLI-1. RNA interference targeted to the junction between EWS and FLI-1 sequences was used to inactivate the EWS/FLI-1 fusion gene in Ewing cells and to explore the resulting phenotype and alteration of the gene expression profile. Loss of expression of EWS/FLI-1 resulted in the complete arrest of growth and was associated with a dramatic increase in the number of apoptotic cells. Gene profiling of Ewing cells in which the EWS/FLI-1 fusion gene had been inactivated identified downstream targets which could be grouped in two major functional clusters related to extracellular matrix structure or remodeling and regulation of signal transduction pathways. Among these targets, the insulin-like growth factor binding protein 3 gene (IGFBP-3), a major regulator of insulin-like growth factor 1 (IGF-1) proliferation and survival signaling, was strongly induced upon treating Ewing cells with EWS/FLI-1-specific small interfering RNAs. We show that EWS/FLI-1 can bind the IGFBP-3 promoter in vitro and in vivo and can repress its activity. Moreover, IGFBP-3 silencing can partially rescue the apoptotic phenotype caused by EWS/FLI-1 inactivation. Finally, IGFBP-3-induced Ewing cell apoptosis relies on both IGF-1-dependent and -independent pathways. These findings therefore identify the repression of IGFBP-3 as a key event in the development of Ewing's sarcoma.

Targeting the Wnt Pathway and Cancer Stem Cells with Anti-progastrin Humanized Antibodies as a Potential Treatment for K-RAS-Mutated Colorectal Cancer.

Purpose: Patients with metastatic colorectal cancer suffer from disease relapse mainly due to cancer stem cells (CSC). Interestingly, they have an increased level of blood progastrin, a tumor-promoting peptide essential for the self-renewal of colon CSCs, which is also a direct ß-catenin/TCF4 target gene. In this study, we aimed to develop a novel targeted therapy to neutralize secreted progastrin to inhibit Wnt signaling, CSCs, and reduce relapses.Experimental Design: Antibodies (monoclonal and humanized) directed against progastrin were produced and selected for target specificity and affinity. After validation of their effectiveness on survival of colorectal cancer cell lines harboring B-RAF or K-RAS mutations, their efficacy was assessed in vitro and in vivo, alone or concomitantly with chemotherapy, on CSC self-renewal capacity, tumor recurrence, and Wnt signaling.Results: We show that anti-progastrin antibodies decrease self-renewal of CSCs both in vitro and in vivo, either alone or in combination with chemotherapy. Furthermore, migration and invasion of colorectal cancer cells are diminished; chemosensitivity is prolonged in SW620 and HT29 cells and posttreatment relapse is significantly delayed in T84 cells, xenografted nude mice. Finally, we show that the Wnt signaling activity in vitro is decreased, and, in transgenic mice developing Wnt-driven intestinal neoplasia, the tumor burden is alleviated, with an amplification of cell differentiation in the remaining tumors.Conclusions: Altogether, these data show that humanized anti-progastrin antibodies might represent a potential new treatment for K-RAS-mutated colorectal patients, for which there is a crucial unmet medical need. Clin Cancer Res; 23(17); 5267-80. ©2017 AACR.

Absence of major defects in non-homologous DNA end joining in human breast cancer cell lines.

Structural abnormalities of chromosomes, including translocations and deletions, are extremely frequent in human cancer cells and particularly in breast cancer cells. One hypothesis to account for these alterations is a deficiency in the repair of DNA double-strand breaks (DSB). This repair process relies on two distinct pathways, homologous recombination (HR) and non-homologous DNA end joining (NHEJ). To investigate this latter pathway, we have studied the ability of cell-free extracts from a variety of human cells to rejoin different types of DSBs. The end joining activity of eleven sporadic breast cancer cell lines (BCCLs) was compared with that of control cells including primary human fibroblasts and cells harbouring a limited number of chromosome abnormalities. In vitro rejoining activity was not detected in extracts from MO59J DNA-PKcs-deficient cells and was strongly inhibited by wortmannin in control extracts. In contrast, most sporadic BCCLs and BRCA1 or BRCA2 deficient cells demonstrated similar efficiencies and accuracies of in vitro NHEJ than control cells. Only two BCCLs, SKBR3 and MDA-MB-453 exhibited decreased in vitro NHEJ. This study therefore indicates that a major defect in the NHEJ pathway is unlikely to account for the high number of chromosomes abnormalities observed in sporadic and hereditary BCCLs.

p53 and p16(INK4A) independent induction of senescence by chromatin-dependent alteration of S-phase progression.

Senescence is triggered by various cellular stresses that result in genomic lesions and DNA damage response activation. However, the role of chromatin and DNA replication in senescence induction remains elusive. Here we show that downregulation of p300 histone acetyltransferase activity induces senescence by a mechanism that is independent of the activation of p53, p21(CIP1) and p16(INK4A). This inhibition leads to a global H3, H4 hypoacetylation, initiating senescence-associated heterochromatic foci formation during S phase, together with a global decrease in replication fork velocity, and alteration of DNA replication timing. This replicative stress occurs without DNA damage and checkpoint activation, but results in a robust G2/M cell cycle arrest, within only one cell cycle. These results provide new insights into the control of S-phase progression by p300, and identify an unexpected chromatin-dependent alternative mechanism for senescence induction, which could possibly be exploited to treat cancer by senescence induction without generating further DNA damage.

SUMOylation of DRIL1 directs its transcriptional activity towards leukocyte lineage-specific genes.

DRIL1 is an ARID family transcription factor that can immortalize primary mouse fibroblasts, bypass RAS(V12)-induced cellular senescence and collaborate with RAS(V12) or MYC in mediating oncogenic transformation. It also activates immunoglobulin heavy chain transcription and engages in heterodimer formation with E2F to stimulate E2F-dependent transcription. Little, however, is known about the regulation of DRIL1 activity. Recently, DRIL1 was found to interact with the SUMO-conjugating enzyme Ubc9, but the functional relevance of this association has not been assessed. Here, we show that DRIL1 is sumoylated both in vitro and in vivo at lysine 398. Moreover, we provide evidence that PIASy functions as a specific SUMO E3-ligase for DRIL1 and promotes its sumoylation both in vitro and in vivo. Furthermore, consistent with the subnuclear localization of PIASy in the Matrix-Associated Region (MAR), SUMO-modified DRIL1 species are found exclusively in the MAR fraction. This post-translational modification interferes neither with the subcellular localization nor the DNA-binding activity of the protein. In contrast, DRIL1 sumoylation impairs its interaction with E2F1 in vitro and modifies its transcriptional activity in vivo, driving transcription of subset of genes regulating leukocyte fate. Taken together, these results identify sumoylation as a novel post-translational modification of DRIL1 that represents an important mechanism for targeting and modulating DRIL1 transcriptional activity.