Melissa M. Johnson

Regulation of liver regeneration and hepatocarcinogenesis by suppressor of cytokine signaling 3

Suppressor of cytokine signaling 3 (SOCS3) down-regulates several signaling pathways in multiple cell types, and previous data suggest that SOCS3 may shut off cytokine activation at the early stages of liver regeneration (Campbell, J.S., L. Prichard, F. Schaper, J. Schmitz, A. Stephenson-Famy, M.E. Rosenfeld, G.M. Argast, P.C. Heinrich, and N. Fausto. 2001.J. Clin. Invest. 107:1285–1292). We developed Socs3 hepatocyte-specific knockout (Socs3 h-KO) mice to directly study the role of SOCS3 during liver regeneration after a two-thirds partial hepatectomy (PH). Socs3 h-KO mice demonstrate marked enhancement of DNA replication and liver weight restoration after PH in comparison with littermate controls. Without SOCS3, signal transducer and activator of transcription 3 (STAT3) phosphorylation is prolonged, and activation of the mitogenic extracellular signal-regulated kinase 1/2 (ERK1/2) is enhanced after PH. In vitro, we show that SOCS3 deficiency enhances hepatocyte proliferation in association with enhanced STAT3 and ERK activation after epidermal growth factor or interleukin 6 stimulation. Microarray analyses show that SOCS3 modulates a distinct set of genes, which fall into diverse physiological categories, after PH. Using a model of chemical-induced carcinogenesis, we found that Socs3 h-KO mice develop hepatocellular carcinoma at an accelerated rate. By acting on cytokines and multiple proliferative pathways, SOCS3 modulates both physiological and neoplastic proliferative processes in the liver and may act as a tumor suppressor.

Increased plasma levels of the APC-interacting protein MAPRE1, LRG1, and IGFBP2 preceding a diagnosis of colorectal cancer in women.

Longitudinal blood collections from cohort studies provide the means to search for proteins associated with disease before clinical diagnosis. We investigated plasma samples from the Womens Health Initiative (WHI) cohort to determine quantitative differences in plasma proteins between subjects subsequently diagnosed with colorectal cancer (CRC) and matched controls that remained cancer-free during the period of follow-up. Proteomic analysis of WHI samples collected before diagnosis of CRC resulted in the identification of six proteins with significantly (P < 0.05) elevated concentrations in cases compared with controls. Proteomic analysis of two CRC cell lines showed that five of the six proteins were produced by cancer cells. Microtubule-associated protein RP/EB family member 1 (MAPRE1), insulin-like growth factor–binding protein 2 (IGFBP2), leucine-rich alpha-2-glycoprotein (LRG1), and carcinoembryonic antigen (CEA) were individually assayed by enzyme linked immunosorbent assay (ELISA) in 58 pairs of newly diagnosed CRC samples and controls and yielded significant elevations (P < 0.05) among cases relative to controls. A combination of these four markers resulted in a receiver operating characteristics curve with an area under the curve value of 0.841 and 57% sensitivity at 95% specificity. This combination rule was tested in an independent set of WHI samples collected within 7 months before diagnosis from cases and matched controls resulting in 41% sensitivity at 95% specificity. A panel consisting of CEA, MAPRE1, IGFBP2, and LRG1 has predictive value in prediagnostic CRC plasmas. Cancer Prev Res; 5(4); 655–64. ©2012 AACR.

Postmenopausal estrogen and progestin effects on the serum proteome

BackgroundWomens Health Initiative randomized trials of postmenopausal hormone therapy reported intervention effects on several clinical outcomes, with some important differences between estrogen alone and estrogen plus progestin. The biologic mechanisms underlying these effects, and these differences, have yet to be fully elucidated.MethodsBaseline serum samples were compared with samples drawn 1 year later for 50 women assigned to active hormone therapy in both the estrogen-plus-progestin and estrogen-alone randomized trials, by applying an in-depth proteomic discovery platform to serum pools from 10 women per pool.ResultsIn total, 378 proteins were quantified in two or more of the 10 pooled serum comparisons, by using strict identification criteria. Of these, 169 (44.7%) showed evidence (nominal P < 0.05) of change in concentration between baseline and 1 year for one or both of estrogen-plus-progestin and estrogen-alone groups. Quantitative changes were highly correlated between the two hormone-therapy preparations. A total of 98 proteins had false discovery rates < 0.05 for change with estrogen plus progestin, compared with 94 for estrogen alone. Of these, 84 had false discovery rates <0.05 for both preparations. The observed changes included multiple proteins relevant to coagulation, inflammation, immune response, metabolism, cell adhesion, growth factors, and osteogenesis. Evidence of differential changes also was noted between the hormone preparations, with the strongest evidence in growth factor and inflammation pathways.ConclusionsSerum proteomic analyses yielded a large number of proteins similarly affected by estrogen plus progestin and by estrogen alone and identified some proteins and pathways that appear to be differentially affected between the two hormone preparations; this may explain their distinct clinical effects.

Autoantibody Signatures Involving Glycolysis and Splicesome Proteins Precede a Diagnosis of Breast Cancer among Postmenopausal Women

We assessed the autoantibody repertoire of a mouse model engineered to develop breast cancer and the repertoire of autoantibodies in human plasmas collected at a preclinical time point and at the time of clinical diagnosis of breast cancer. In seeking to identify common pathways, networks, and protein families associated with the humoral response, we elucidated the dynamic nature of tumor antigens and autoantibody interactions. Lysate proteins from an immortalized cell line from a MMTV-neu mouse model and from MCF7 human breast cancers were spotted onto nitrocellulose microarrays and hybridized with mouse and human plasma samples, respectively. Immunoglobulin-based plasma immunoreactivity against glycolysis and spliceosome proteins was a predominant feature observed both in tumor-bearing mice and in prediagnostic human samples. Interestingly, autoantibody reactivity was more pronounced further away than closer to diagnosis. We provide evidence for dynamic changes in autoantibody reactivity with tumor development and progression that may depend, in part, on the extent of antigen-antibody interactions.

Detection of Elevated Plasma Levels of Epidermal Growth Factor Receptor Before Breast Cancer Diagnosis among Hormone Therapy Users

Applying advanced proteomic technologies to prospectively collected specimens from large studies is one means of identifying preclinical changes in plasma proteins that are potentially relevant to the early detection of diseases such as breast cancer. We conducted 14 independent quantitative proteomics experiments comparing pooled plasma samples collected from 420 estrogen receptor-positive (ER(+)) breast cancer patients ≤17 months before their diagnosis and matched controls. Based on the more than 3.4 million tandem mass spectra collected in the discovery set, 503 proteins were quantified, of which 57 differentiated cases from controls with a P value of <0.1. Seven of these proteins, for which quantitative ELISA assays were available, were assessed in an independent validation set. Of these candidates, epidermal growth factor receptor (EGFR) was validated as a predictor of breast cancer risk in an independent set of preclinical plasma samples for women overall [odds ratio (OR), 1.44; P = 0.0008] and particularly for current users of estrogen plus progestin (E + P) menopausal hormone therapy (OR, 2.49; P = 0.0001). Among current E + P users, the EGFR sensitivity for breast cancer risk was 31% with 90% specificity. Whereas the sensitivity and specificity of EGFR are insufficient for a clinically useful early detection biomarker, this study suggests that proteins that are elevated preclinically in women who go on to develop breast cancer can be discovered and validated using current proteomic technologies. Further studies are warranted to examine the role of EGFR and to discover and validate other proteins that could potentially be used for early detection of breast cancer.

Polymorphic markers in the 5α‐reductase type II gene and the incidence of prostate cancer

In the prostate, the enzyme encoded by the SRD5A2 gene (5α‐reductase) converts testosterone to dihydrotestosterone, a potent androgen that has been hypothesized to play a role in the genesis of prostate cancer. Several polymorphisms have been identified in the SRD5A2 gene, including a valine‐to‐leucine substitution (V89L) at codon 89, a variable number of TA dinucleotide repeats and a missense substitution at codon 49 resulting in an amino acid substitution of alanine with threonine (A49T). To investigate the influence of these polymorphisms on prostate cancer risk, we conducted a case‐control study nested within the Beta‐Carotene and Retinol Efficacy Trial. Genotypes were determined by PCR‐based capillary electrophoresis using genomic DNA isolated from 300 cases and 300 controls matched on the basis of race, age at enrollment (within 5 years), enrollment study center and year of randomization. There was no association between V89L genotypes and prostate cancer risk. The age‐ and race‐adjusted odds ratio (OR) associated with the VL and LL genotypes were 1.06 (95% confidence interval (CI) = 0.75‐1.49) and 0.99 (95% CI = 0.57‐1.73), respectively, as compared to the VV genotype. The age‐ and race‐adjusted odds ratio for men having 1 TA(9) or TA(18) allele was 0.98 (95% CI = 0.64‐1.48) when compared to men without TA repeats. The corresponding odds ratio for men without the TA(0) alleles was 0.68 (95% CI = 0.21‐2.19). The age‐ and race‐adjusted odds ratio associated with having at least 1 T allele at codon 49 was 1.11 (95% CI = 0.58‐2.11), as compared to the AA genotype. Our results do not support the hypothesis that the V89L and A49T polymorphisms in the SRD5A2 gene are related to the risk of prostate cancer, but are compatible with the suggestion from earlier studies that men who are homozygous for the TA(9) or (18) alleles and men who have the TA(9)/TA(18) genotype are at a modestly reduced risk.

Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice: evidence for stromal induction of hepatocellular carcinoma.

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet‐derived growth factor C (PDGF‐C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF‐CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast‐like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell‐derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte‐derived PDGF‐C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF‐CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF‐C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.

A Novel Soluble Form of Tim-3 Associated with Severe Graft-versus-Host Disease

The T cell Ig and mucin domain 3 (Tim-3) receptor has been implicated as a negative regulator of adaptive immune responses. We have utilized a proteomic strategy to identify novel proteins associated with graft versus host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). Mass spectrometry analysis of plasma from subjects with mid-gut and upper-gut GVHD compared with those without GVHD identified increased levels of a protein identified with high confidence as Tim-3. A follow-up validation study using an immunoassay to measure Tim-3 levels in individual plasma samples from 127 patients demonstrated significantly higher plasma Tim-3 concentrations in patients with the more severe mid-gut GVHD, compared with those with upper-gut GVHD (P = .005), patients without GVHD (P = .002), and normal controls (P < .0001). Surface expression of Tim-3 was increased on CD8(+) T cells from patients with grade 2 to 4 acute GVHD (P = .01). Mass spectrometry-based profiling of plasma from multiple subjects diagnosed with common diseases provided evidence for restricted release of soluble Tim-3 in the context of GVHD. These findings have mechanistic implications for the development of novel strategies for targeting the Tim-3 immune regulatory pathway as an approach to improving control of GVHD.

An Autoimmune Response Signature Associated with the Development of Triple-Negative Breast Cancer Reflects Disease Pathogenesis

The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple-negative breast cancer (TNBC) in the before diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Womens Health Initiative cohort, collected before clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in estrogen receptor(+) breast cancer. Importantly, autoantibodies directed against networks involving BRCA1, TP53, and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC before diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected before occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig-bound proteins yielding a predominance of cytokeratins, including several associated with a mesenchymal/basal phenotype among cases compared with controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC.

Proteomic risk markers for coronary heart disease and stroke: validation and mediation of randomized trial hormone therapy effects on these diseases

BackgroundWe previously reported mass spectrometry-based proteomic discovery research to identify novel plasma proteins related to the risk of coronary heart disease (CHD) and stroke, and to identify proteins with concentrations affected by the use of postmenopausal hormone therapy. Here we report CHD and stroke risk validation studies for highly ranked proteins, and consider the extent to which protein concentration changes relate to disease risk or provide an explanation for hormone therapy effects on these outcomes.MethodsFive proteins potentially associated with CHD (beta-2 microglobulin (B2M), alpha-1-acid glycoprotein 1 (ORM1), thrombospondin-1(THBS1), complement factor D pre-protein (CFD), and insulin-like growth factor binding protein 1 (IGFBP1)) and five potentially associated with stroke (B2M, IGFBP2, IGFBP4, IGFBP6, and hemopexin (HPX)) had high discovery phase significance level ranking and an available ELISA assay, and were included in case-control validation studies within the Women’s Health Initiative (WHI) hormone therapy trials. Protein concentrations, at baseline and 1 year following randomization, were assessed for 358 CHD cases and 362 stroke cases, along with corresponding disease-free controls. Disease association, and mediation of estrogen-alone and estrogen plus progestin effects on CHD and stroke risk, were assessed using logistic regression.ResultsB2M, THBS1, and CFD were confirmed (P <0.05) as novel CHD risk markers, and B2M, IGFBP2, and IGFBP4 were confirmed as novel stroke disease risk markers, while the assay for HPX proved to be unreliable. The change from baseline to 1 year in B2M was associated (P <0.05) with subsequent stroke risk, and trended similarly with subsequent CHD risk. Change from baseline to 1 year in IGFBP1 was also associated with CHD risk, and this change provided evidence of hormone therapy effect mediation.ConclusionsPlasma B2M is confirmed to be an informative risk marker for both CHD and stroke. The B2M increase experienced by women during the first year of hormone therapy trial participation conveys cardiovascular disease risk. The increase in IGFBP1 similarly conveys CHD risk, and the magnitude of the IGFBP1 increase following hormone therapy may be a mediator of hormone therapy effects. Plasma THBS1 and CFD are confirmed as CHD risk markers, and plasma IGFBP4 and IGFBP2 are confirmed as stroke risk markers.Clinical trials registrationClinicalTrials.gov identifier: NCT00000611