Squamous Cell Carcinoma Antigen 1 Research Articles

Prospective Analysis of Squamous Cell Carcinoma Antigen-1 and -2 for Diagnosing Sinonasal Inverted Papilloma.

Background: The goal of this research was to confirm whether preoperative serum squamous cell carcinoma antigen (SCCA)-1 and -2 levels are useful diagnostic markers for sinonasal inverted papilloma (IP) in a prospective study. Methods: Participants were 102 patients who underwent consecutive endoscopic sinus surgery: 18 with IP, two with other types of papilloma, 77 with chronic rhinosinusitis, four with sinonasal cancer, and one with hemangioma. SCCA-1 and SCCA-2 were measured preoperatively by an automatic chemiluminescence immunoassay and an enzyme-linked immunosorbent assay, respectively. Results: SCCA-1 and SCCA-2 values were significantly correlated (r = 0.603, p < 0.001). Receiver operating characteristic analysis for differentiating papilloma (IP and other types of papilloma) from other diseases yielded an area under the curve of 0.860, with a Youden index of 1.75. Combined with SCCA-2 analysis, the detection system had a sensitivity and specificity of 0.65 and 0.98, respectively. While our study did not find a strong link between SCCA levels and skin or lung diseases, smoking status may influence SCCA levels in IP patients (p = 0.035). We recommend a cutoff value of 1.8 ng/mL for SCCA-1 in IP diagnosis. Conclusions: SCCA-1 and SCCA-2 when combined with imaging and pathology hold promise for enhancing the preoperative detection of IP, which would be a valuable contribution to clinical practice.

Bile detection of squamous cell carcinoma antigen (SCCA) in extrahepatic cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a rare biliary tract tumor with poor prognosis that often is challenging to diagnose and the majority of patients present with advanced stage. Squamous cell carcinoma antigen 1 (SCCA1) overexpression has been found in different tumors associated with poor prognosis and chemoresistance. To assess the presence and possible prognostic role of SCCA1/2 isoforms in bile and serum of patients with CCA. Forty seven surgical patients (36 with CCA and 11 with benign diseases) were prospectively included in the study. Serum and bile specimens were collected at the time of surgery and free and IgM-complexed SCCA was quantified by ELISA (Xeptagen, srl). Free or IgM linked SCCA was rarely found in serum, while SCCA was detectable in bile samples of patients with CCA, especially in those with extrahepatic form (43% vs 17%, p=0.008), but not in controls. Despite similar tumor stage, these positive patients presented a trend toward a higher percentage of portal invasion (27% vs 15%) and of tumor recurrence than negative cases (62% vs 40%), although the difference was not statistically significant. These preliminary results indicate that bile testing for SCCA is a specific marker of extrahepatic CCA, with potential prognostic value.

Squamous cell carcinoma antigen 1 is associated to poor prognosis in esophageal cancer through immune surveillance impairment and reduced chemosensitivity.

Squamous cell carcinoma antigen‐1 (SCCA1) overexpression is associated with poor prognosis and chemoresistance in several tumor types, however, the underlying mechanisms remain elusive. Here, we report SCCA1 in relation to the immune and peritumoral adipose tissue microenvironment in early and advanced esophageal adenocarcinoma (EAC). In our series of patients with EAC, free SCCA1 serum levels were associated with significantly worse overall survival, and SCCA1‐IgM serum levels showed a trend to a worse overall survival. Serum SCCA1 and intratumoral SCCA1 were inversely correlated with immune activation markers. In agreement with these findings, SCCA1 induced the expression of the immune checkpoint molecule programmed death ligand‐1 on monocytes and a direct correlation of these 2 molecules was observed in sequential tumor sections. Furthermore, SCCA1 mRNA expression within the tumor was inversely correlated with stem cell marker expression both within the tumor and in the peritumoral adipose tissue. In vitro, in EAC cell lines treated with different chemotherapeutic drugs, cell viability was significantly modified by SCCA1 presence, as cells overexpressing SCCA1 were significantly more resistant to cell death. In conclusion, poor prognosis in EAC overexpressing SCCA1 is due to reduced tumor chemosensitivity as well as intratumoral immunity impairment, likely induced by this molecule.

Clinical significance of progastrin-releasing peptide, neuron-specific enolase, chromogranin a, and squamous cell cancer antigen in pulmonary neuroendocrine tumors

Background/aim It is not always easy to diagnose pulmonary neuroendocrine tumors (PNETs). The aim of the present study is to make a differential diagnosis by studying the same markers in patients with non-small-cell lung carcinoma (NSCLC), patients with benign lung disease (chronic obstructive pulmonary disease and pneumonia), and healthy volunteers to determine the roles of these markers in pulmonary neuroendocrine tumor diagnosis and to identify their power. Materials and methods A total of 100 participants including 23 PNET patients and 28 NSCLC patients who were pathologically diagnosed but not yet treated, 25 participants with benign disease, and 24 healthy volunteers were included in this cross-sectional study.Results No significant difference was found between the chromogranin A (CgA) and squamous cell carcinoma antigen 1 (SCCA1) values among the groups (PNET, NSCLC, benign, healthy volunteers), but the difference in progesterone-releasing peptide (ProGRP), neuron-specific enolase (NSE), and adjusted NSE was statistically significant (P values were respectively ProGRP, P = 0.006; NSE, P = 0.015; NSE adjusted, P = 0.09). In a comparison of the PNET and NSCLC groups, having a ProGRP value higher than 84.6 pg/mL revealed PNET with 60.9% sensitivity and 89.3% specificity (P = 0.001). Conclusion The ProGRP value is the only indicator that distinguishes the PNET group from the other 3 groups.

Diagnostic specificity and sensitivity of PIVKAII, GP3, CSTB, SCCA1 and HGF for the diagnosis of hepatocellular carcinoma in patients with alcoholic liver cirrhosis.

Introduction Despite some new treatment possibilities, the improvement in survival rate for hepatocellular carcinoma (HCC) patients is still poor due to late diagnosis. The aim of this study was to investigate the diagnostic sensitivity and specificity of protein induced by vitamin K absence or antagonist-II (PIVKAII), Glypican-3 (GP3), Cystatin B (CSTB), squamous cell carcinoma antigen 1 (SCCA1) and hepatocyte growth factor (HGF) as potential tumour markers for HCC in patients with alcoholic liver cirrhosis (ALC) using imaging techniques (MSCT and MRI) as reference standards. Patients and methods Eighty-three participants were included: 20 healthy volunteers, 31 patients with ALC and 32 patients with HCC. Peripheral blood sampling was performed for each participant, and serum concentrations of PIVKAII, GP3, CSTB, SCCA1 and HGF were determined using commercial ELISA kits. Results Only serum concentrations of PIVKAII were significantly higher in HCC patients as compared with ALC and healthy controls (cut-off: 2.06 µg/L; AUC: 0.903), whereas individual diagnostic performance of other individual compounds was inadequate. The 'best' combination of tumour markers in our study includes all tested markers with AUC of 0.967. Conclusion While novel diagnostic tumour markers are urgently needed, the examined potential tumour markers, with the exception of PIVKAII seem to be inadequate for diagnosing HCC in ALC. Furthermore, probably the future is in finding the best optimal combination of tumour markers for diagnosing HCC based on cost-effectiveness.

Abstract 4548: High throughput siRNA screens uncover a high rate of USP8 and ESCRT pathway dependency in squamous carcinoma cell lines

Abstract USP8 and the ESCRT pathway (endosomal sorting complex required for transport) are required for cellular homeostasis through key functions in shuttling ubiquitinated proteins towards lysosomal degradation, and through regulating the maturation of autophagosomal structures. In large-scale siRNA screens, the vast majority of cancer cell lines do not demonstrate dependency on USP8 or ESCRT pathway components for viability. However, we identified three cell lines (HCC70, HCC1954 and PFSK1) that are acutely dependent on USP8 and other ESCRT components (HGS, TSG101) for viability. Using a transcriptome-wide association approach, we found that these three USP8-dependent cell lines also had in common the expression of SerpinB3, also known as SCCA1 (Squamous Cell Carcinoma Antigen 1). After selecting additional Squamous Cell Carcinoma cell lines for further validation of USP8-dependency by siRNA screening, we found that Squamous Carcinoma cell lines, irrespective of their SerpinB3 expression status, were far more likely to be dependent on USP8 for viability (23%; N = 22) than cancer cell lines of non-Squamous epithelial origin (4.5%; N = 67). To unravel possible molecular mechanisms underpinning the requirement of USP8 for the viability of USP8-dependent Squamous Carcinoma cell lines (including SCABER, BICR22, CALU1 and EBC1), we conducted additional transcriptome association studies and genetic interaction screens to uncover genes that are synthetic lethal with USP8 knock-down. Citation Format: Wendy Zhong, Elissa Cosgrove, Elissa Swearingen, Mike Ollmann, Jayee Banerjee, Vivienne Watson, Peter Jaeckel, Mariana Pfreimer, Silvia Materna-Reichelt, Holger Beckmann, Paul Kassner, Astrid Ruefli-Brasse, Olivier Nolan-Stevaux. High throughput siRNA screens uncover a high rate of USP8 and ESCRT pathway dependency in squamous carcinoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4548.

Overexpression of Squamous Cell Carcinoma Antigen 1 Is Associated with the Onset and Progression of Human Hepatocellular Carcinoma

Previous studies indicated Squamous Cell Carcinoma Antigen 1 (SCCA1) may be involved in tumorigenesis and progress of various human malignancies by inhibiting cell apoptosis and promoting cell proliferative activity. The aim of the study was to further investigate SCCA1 expression in different extent of liver diseases and evaluate the clinical significance and prognostic value in HCC. Eighty nine patient-matched tumors and peritumoral surgical specimens and 56 liver biopsies specimens from 23 patients with chronic hepatitis B (CHB), 19 with dysplastic nodule (DN), and 14 with HCC were enrolled. An additional four normal liver (NL) samples were used as controls. SCCA1 expression in liver tissue was measured by immunochemistry. Another 28 HCC specimens and paired non-tumor tissues were used for SCCA1 detection by Western blot. The prognostic value of SCCA1 expression in HCC was evaluated by the Cox proportional hazards regression model analysis. Western blot analysis showed SCCA1 positive rate in HCC was higher than the matched adjacent noncancerous tissues (p <0.001). Immunohistochemistry revealed that SCCA1-positive rate increased gradually from NL, CHB, PNT to DN and HCC (p <0.05). Clinicopathological analysis showed that SCCA1 expression was positively associated with tumor differentiation (p = 0.043) and patients' Child-Pugh score (p = 0.021). The SCCA1-poistive group showed better overall survival than the negative group (p = 0.029). Importantly, SCCA1 expression was an independent prognostic factor for the overall survival of HCC patients (hazard ratio = 3.757, p <0.001). SCCA1 expression pattern may relate to the progression of chronic liver diseases. Furthermore, our study supports a potential association of negative SCCA1 expression with poor outcome in HCC.

Abstract B54: Squamous cell carcinoma antigen 1 (SCCA1) promotes mammary tumorigenesis through the induction of misfolded protein stress

Abstract SCCA1, a member of the serine/cysteine protease inhibitor family, is upregulated in several cancers such as the cervix, lung, head and neck, liver, skin, and breast, and has been used as a diagnostic/prognostic marker. However, the biological function of SCCA1 in tumorigenesis remains largely unclear. Here we show that ectopic expression of SCCA1 in the non-tumorigenic mammary epithelial cell line MCF10A leads to spindle shaped cell morphology, increased cell migration, resistance to anoikis, foci formation in soft agar, as well as orthotopic tumor formation in immune-compromised mice. Gene expression analysis revealed that SCCA1 induces changes that are associated with epithelial-mesenchymal transition (EMT). Mechanistically, SCCA1 interferes with cellular protein homeostasis leading to a heightened unfolded protein response including the nuclear translocation of the transcriptionally active form of ATF6α. This increased ATF6α signaling results in NF-κB activation and increased pro-inflammatory response including an IL-6 autocrine signaling loop that drives EMT and transformation. These findings indicate a previously unknown role of SCCA1 in promoting tumorigenesis by inducing misfolded protein stress, NF-κB activation, and pro-tumorigenic cytokine signaling. Citation Format: Namratha Sheshadri, Erica Ullman, Joseph Catanzaro, Ji-An Pan, Emily Chen, Wei-Xing Zong. Squamous cell carcinoma antigen 1 (SCCA1) promotes mammary tumorigenesis through the induction of misfolded protein stress. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B54.

Proteomic analysis of human cervicovaginal fluid collected before preterm premature rupture of the fetal membranes

A significant obstetric complication facing contemporary materno-fetal medicine is preterm premature rupture of the fetal membranes (preterm PROM), which occurs in 30% of all preterm births. The objective of this study was to identify differentially expressed proteins in the cervicovaginal fluid of asymptomatic women before the clinical manifestation of preterm PROM. The preterm PROM group comprised of women with samples collected 6-23 days before PROM, who subsequently delivered preterm (n=5). Women who spontaneously delivered at term served as gestation-matched controls (n=10). Two-dimensional difference in-gel electrophoresis was used to distinguish differential expression between the pooled groups and fold changes were subsequently confirmed by two-dimensional PAGE of individual samples. Spots of interest were identified by mass spectrometry. Proteins that were significantly reduced with impending preterm PROM included the following: thioredoxin (2.7-fold), interleukin 1 receptor antagonist (1.7-fold), fatty acid-binding protein 5 (2.1-fold), cystatin A (dimer; 1.9-fold), monocyte/neutrophil elastase inhibitor (1.6-fold), squamous cell carcinoma antigen-1 (2.1-fold) and γ-glutamyl cyclotransferase (3.0-fold). By contrast, annexin A3 (3.7-fold) and vitamin D binding protein (3.9-fold) were significantly increased with impending preterm PROM. Western blot analysis was also performed on an independent cohort of preterm PROM and control samples to validate these candidate biomarkers. These proteins have known biological functions in oxidative balance, anti-inflammatory activity, metabolism or protease inhibition that may facilitate membrane rupture.

SERPINB3 in the Chicken Model of Ovarian Cancer: A Prognostic Factor for Platinum Resistance and Survival in Patients with Epithelial Ovarian Cancer

Serine protease inhibitors (SERPINs) appear to be ubiquitously expressed in a variety of species and play important roles in pivotal physiological processes such as angiogenesis, immune responses, blood coagulation and fibronolysis. Of these, squamous cell carcinoma antigen 1 (SCCA1), also known as a SERPINB3, was first identified in squamous cell carcinoma tissue from the cervix of women. However, there is little known about the SERPINB3 expression in human epithelial ovarian cancer (EOC). Therefore, in the present study, we investigated the functional role of SERPINB3 gene in human EOC using chickens, the most relevant animal model. In 136 chickens, EOC was found in 10 (7.4%). SERPINB3 mRNA was induced in cancerous, but not normal ovaries of chickens (P<0.01), and it was abundant only in the glandular epithelium of cancerous ovaries of chickens. Further, several microRNAs, specifically miR-101, miR-1668 and miR-1681 were discovered to influence SERPINB3 expression via its 3′-UTR which suggests that post-transcriptional regulation influences SERPINB3 expression in chickens. SERPINB3 protein was localized predominantly to the glandular epithelium in cancerous ovaries of chickens, and it was abundant in the nucleus of both chicken and human ovarian cancer cell lines. In 109 human patients with EOC, 15 (13.8%), 66 (60.6%) and 28 (25.7%) patients showed weak, moderate and strong expression of SERPINB3 protein, respectively. Strong expression of SERPINB3 protein was a prognostic factor for platinum resistance (adjusted OR; odds ratio, 5.94; 95% Confidence Limits, 1.21–29.15), and for poor progression-free survival (PFS; adjusted HR; hazard ratio, 2.07; 95% CI; confidence interval, 1.03–4.41). Therefore, SERPINB3 may play an important role in ovarian carcinogenesis and be a novel biomarker for predicting platinum resistance and a poor prognosis for survival in patients with EOC.

IgM-Linked SerpinB3 and SerpinB4 in Sera of Patients with Chronic Liver Disease

BackgroundEpidemiological studies indicate that a growing number of cirrhotic patients will develop hepatocellular carcinoma (HCC) in the next decade. Recent findings have demonstrated that Squamous cell carcinoma antigen 1 (SCCA1) and 2 (SCCA2) isoforms, now classified as serpinB3 and serpinB4, are over-expressed in HCC, but not in normal liver. As reported, high levels of circulating SCCA-IgM immunocomplexes in patients with cirrhosis are significantly associated with HCC development.AimTo ascertain whether IgM-linked SCCA isoforms circulate in patients with chronic liver disease, compared to total SCCA-IgM levels.Methodology and Findings79 patients with chronic liver disease were studied, including 17 patients with chronic hepatitis, 36 patients with cirrhosis and 26 with HCC. 28 blood donors were used as control. Monoclonal antibodies against serpinB3 and serpinB4 were used as catcher antibodies to set up specific ELISA assays, while total SCCA-IgM immunocomplexes were detected by commercially available ELISA assay. Overall, the results revealed a better diagnostic sensitivity of total SCCA-IgM assay, compared to both serpinB3 and serpinB4 IgM-linked assays. SerpinB4-IgM median values obtained with SCC103 antibody were moderately higher in patients with cirrhosis than in those with HCC, median values: 0.168 (IQR 0.140–0.427) vs. 0.140 (IQR 0.140–0.278), (p = 0.177). A trend toward decreasing serpinB4-IgM/serpinB3-IgM median ratio was observed in patients with advanced liver disease, being 1.08 in patients with HCC, 1.10 in patients with cirrhosis and 1.40 in patients with chronic hepatitis (p = 0.079).ConclusionsIgM-linked SCCA isoforms in serum of patients with chronic liver diseases were quantified for the first time. Although the number of patients was limited, this preliminary study reveals that the relative balance of the two serpin isoforms is altered in HCC and it is characterized by a lower serpinB4-IgM/serpinB3-IgM ratio, determined by lower serpinB4 levels.

Ferritin light chain and squamous cell carcinoma antigen 1 are coreceptors for cellular attachment and entry of hepatitis B virus

Overexpression of squamous cell carcinoma antigen 1 (SCCA1) in hepatitis G2 (HepG2) and Chinese hamster ovary cells can increase hepatitis B virus (HBV) binding capacity by interacting with the preS1 domain of the HBV surface antigen. However, the magnitude of increase in binding capacity was higher by several orders in the former, indicating the existence of additional factor(s) produced by HepG2 cells, which facilitates HBV attachment. Ferritin light chain (FTL) was identified as the sole high hit candidate by screening human liver cDNA library using a bacterial two-hybrid system with either preS or SCCA1 as the bait. Subsequent in vitro protein–protein interaction assays confirmed the binding activity of FTL to both preS and SCCA1, as well as the formation of triple complex preS-FTL-SCCA1, and narrowed down the binding sites on FTL. In vitro overexpression of FTL could further enhance HBV attachment in both HepG2 and Chinese hamster ovary cells, which were already overexpressing SCCA1. Importantly, in vivo co-expression of human FTL and SCCA1 in mouse liver by means of tailvein hydrodynamic injection increased serum levels of HBV surface antigen transiently 24 hours post challenge with HBV-positive human sera, and a large amount of HBV core antigen-positive hepatocytes around blood vessels could be identified by immunohistochemical staining 48 hours post challenge. The data strongly suggest that FTL and SCCA1 may serve as coreceptors in HBV cellular attachment and virus entry into hepatocytes.

Tissue specific expression and estrogen regulation of SERPINB3 in the chicken oviduct

Serine protease inhibitors (SERPINs) comprise the largest superfamily of protease inhibitors and appear to be ubiquitously expressed in a variety of species. Of these, squamous cell carcinoma antigen 1 (SCCA1), also known as a SERPINB3, was first identified in squamous cell carcinoma tissue from the cervix of women. However, there is little known about the expression and hormonal regulation of SERPINB3 in chickens. Therefore, the avian SERPINB3 gene was compared with those of other species with respect to structure, phylogenetic evolution and tissue- and cell-specific expression in hens. Chicken SERPINB3 has moderate homology to mammalian SERPINB3 proteins (36–47%). Of particular note, SERPINB3 mRNA was most abundant in the chicken oviduct and cell-specific expression was in glandular (GE) and luminal (LE) epithelial cells of the oviduct of laying hens. Treatment of young chicks with DES (diethylstilbestrol, a synthetic nonsteroidal estrogen) induced SERPINB3 mRNA and protein in GE and LE, but not in other cell types of the oviduct. Western blot analyses determined that immunoreactive SERPINB3 protein was also increased by DES in LE and GE of the oviduct of chicks. Collectively, these results indicate that SERPINB3 is an estrogen-induced gene expressed only in LE and GE of the chicken oviduct and implicate SERPINB3 in regulation of oviduct development and egg formation.

Inhibition of Staphylococcus aureus cysteine proteases by human serpin potentially limits staphylococcal virulence

Bacterial proteases are considered virulence factors and it is presumed that by abrogating their activity, host endogenous protease inhibitors play a role in host defense against invading pathogens. Here we present data showing that Staphylococcus aureus cysteine proteases (staphopains) are efficiently inhibited by Squamous Cell Carcinoma Antigen 1 (SCCA1), an epithelial-derived serpin. The high association rate constant (k(ass)) for inhibitory complex formation (1.9×10(4) m/s and 5.8×10(4) m/s for staphopain A and staphopain B interaction with SCCA1, respectively), strongly suggests that SCCA1 can regulate staphopain activity in vivo at epithelial surfaces infected/colonized by S. aureus. The mechanism of staphopain inhibition by SCCA1 is apparently the same for serpin interaction with target serine proteases whereby the formation of a covalent complex result in cleavage of the inhibitory reactive site peptide bond and associated release of the C-terminal serpin fragment. Interestingly, the SCCA1 reactive site closely resembles a motif in the reactive site loop of native S. aureus-derived inhibitors of the staphopains (staphostatins). Given that S. aureus is a major pathogen of epithelial surfaces, we suggest that SCCA1 functions to temper the virulence of this bacterium by inhibiting the staphopains.

Up-regulation of serpin SCCA1 is associated with epidermal barrier disruption

Parakeratosis, the persistent presence of nuclei in the stratum corneum (SC) is associated with serious disruption of skin barrier function. Squamous cell carcinoma antigen 1 (SCCA1) is strongly up-regulated in inflamed and parakeratotic skin. To find a biochemical marker for the SC barrier disruption, especially the disruption associated with parakeratosis. An ELISA assay system was established to quantify SCCA1 in the extract of tape-stripped cornified cells. Transepidermal water loss (TEWL) and other skin parameters were measured and compared with the amount of SCCA1. Localization of SCCA1 was investigated immunohistochemically in various skin diseases with parakeratosis. Nuclei and SCCA1 on the skin surface were detected by staining of corniocytes collected on an adhesive-coated slide glass. SCCA1 showed strong up-regulation in lesional skin with psoriasis (466-fold), hayfever skin caused by Japanese ceder pollen (232-fold) and sun-exposed skin of healthy individuals (90-fold) compared to their normal sun-protected skin. The increased levels of SCCA1 were well correlated with increased values of TEWL and the number of parakeratotic cells in the SC. Furthermore, subjects with high levels of SCCA1 in the epidermis were more susceptible to barrier disruption by external stimuli, and this was accompanied with a further increase of SCCA1. We confirmed that localization of SCCA1 was limited to parakeratotic areas by using the skin surface staining technique. Immunohistochemical study also demonstrated that SCCA1 was always present at high levels in parakeratotic epidermis. All of our findings indicate that SCCA1 plays an important role in the induction of epidermal barrier disruption. SCCA1 may be a critical determinant of barrier function in the epidermis.

Role of squamous cell carcinoma antigen-1 on liver cells after partial hepatectomy in transgenic mice

Squamous Cell Carcinoma Antigen-1 (SCCA1) overexpression has been observed in tumours of epithelial origin and in hepatocellular carcinoma. Previous data indicate that this serpin inhibits apoptosis, while its proliferative activity was only recently proposed. The aim of this study was to evaluate the effect of SCCA1 on liver cells in a transgenic mouse model after partial hepatectomy. Twenty-one C57BL/6J mice (11 transgenic for human SCCA1, 10 wild-type) underwent partial hepatectomy and were sacrified after one week. Apoptosis and proliferation markers were determined in the liver at sacrifice, while a cytokine panel was measured in serum. Transgenic mice showed a relative liver weight significantly higher than wild-type mice at sacrifice (mean +/- SD, 5.38+/-0.50% vs 4.84+/-0.29%, p=0.0221), while no difference (p=0.2403) was observed in two untreated control groups (6 transgenic, 6 wild-type mice). Active caspase-3 was significantly lower in transgenic mice than in wild-type mice (p=0.0047). The transgenic mouse group showed overall higher proliferative activity at sacrifice, compared to wild-type mice, with increased proliferation parameters. Cytokine analysis revealed a remarkable and opposite sex-dependent behaviour of interleukin (IL)-6 after hepatectomy. At variance with wild-type mice, a significant IL-6 increase was documented only in transgenic females (p=0.0313), even more relevant than that observed in wild-type males. In conclusion, transgenic mice expressing SCCA1 showed higher liver regenerative potential compared to wild-type mice, supporting the dual role of this serpin as an anti-apoptotic and pro-proliferative stimulus for liver cells in vivo.

Crystal structure of SCCA1 and insight about the interaction with JNK1

Squamous cell carcinoma antigen 1 (SCCA1), which belongs to serine proteinase inhibitor (serpin) superfamily, inhibits papain-like cysteine proteinase. Recently, it has been reported that SCCA1 acts not only as a proteinase inhibitor but also as an inhibitor of UV-induced apoptosis via suppression of the activity of c-Jun NH 2-terminal kinase (JNK1). The present study determined the crystal structure of SCCA1, suggesting that the reactive center loop (RCL) of SCCA1, a recognition site of proteinase, is very flexible and located away form the main-body of SCCA1. We show that the inhibitory effect of SCCA1 on the kinase activity of JNK1 is lost when the RCL was truncated. Furthermore, we found that a mutant protein created by replacing one amino acid in RCL maintain the suppressive activity to JNK1, whereas the inhibitory effect to proteinase is obviously decreased.

Expression of squamous cell carcinoma antigen‐1 in liver enhances the uptake of hepatitis B virus envelope‐derived bio‐nanocapsules in transgenic rats

We previously developed the bio-nanocapsule, which consists of hepatitis B virus envelope L proteins. The bio-nanocapsule can be used to deliver genes and drugs specifically to the human liver-derived tissues in xenograft models, presumably by utilizing the human liver-specific mechanism of hepatitis B virus infection. The hepatitis B virus tropism is highly restricted to humans and higher primates. Thus, to evaluate the in vivo therapeutic effects of forthcoming bio-nanocapsule-based medicines, it will be crucial to develop an animal model whose liver is susceptible to both bio-nanocapsule and hepatitis B virus. In the present study, we aimed to establish a bio-nanocapsule-susceptible animal model using transgenic rats expressing squamous cell carcinoma antigen-1 (SCCA1), which has been proposed to be a receptor for hepatitis B virus, interacting with the hepatitis B virus envelope protein and enhancing the cellular uptake of hepatitis B virus. We show that the recombinant SCCA1 protein interacts directly with bio-nanocapsule and inhibits its attachment to the cultured human liver-derived cells. Furthermore, we have established a transgenic rat that specifically expresses SCCA1 in the liver and also demonstrate that the amount of bio-nanocapsule accumulated in the liver is significantly increased by the SCCA1 expression. Histological analysis suggests that bio-nanocapsule is preferentially incorporated into the SCCA1-expressing hepatocytes but not into macrophages, such as Küppfer cells, nor into endothelial cells. Therefore, this animal model is expected to be useful for the development of bio-nanocapsule-based medicines.

Squamous cell carcinoma antigen-1 (SERPINB3) polymorphism in chronic liver disease

The serpin squamous cell carcinoma antigen (SCCA, SERPINB3) has been found over-expressed in primary liver cancer and at lower extent in cirrhosis and chronic hepatitis. A novel SCCA-1 variant (SCCA-PD), presenting a single mutation in the reactive centre (Gly351Ala), has been recently identified (rs3180227). To explore SCCA-1 polymorphism in patients with HCV infection as single etiologic factor and different extent of liver disease. One hundred and fourty-eight patients with chronic HCV infection (45 chronic hepatitis, 53 cirrhosis, 50 HCC) and 50 controls were evaluated. SCCA-1 polymorphism was studied by restriction fragment length polymorphism and confirmed randomly by direct sequencing. Circulating SCCA-IgM complex was determined by ELISA. SCCA-PD was detected with higher frequency in cirrhotic patients (45.3%, odds ratio=2.62; 95%CI 1.13-6.10, p=0.038) than in patients with chronic hepatitis or in controls (24.4% and 24%, respectively). Intermediate figures were found in hepatocarcinoma (36.0%). SCCA-IgM in serum was lower in patients carrying SCCA-PD than in wild type patients and the difference was statistically significant in cirrhotic patients (mean+/-S.D.=117.45+/-54.45 U/ml vs. 268.52+/-341.27 U/ml, p=0.026). The newly identified SCCA-PD variant was more frequently found in liver cirrhosis, suggesting that patients carrying this polymorphism are more prone to develop progressive liver fibrosis.

AFP, PIVKAII, GP3, SCCA-1 and follisatin as surveillance biomarkers for hepatocellular cancer in non-alcoholic and alcoholic fatty liver disease

BackgroundThe incidence and mortality of hepatocellular cancer (HCC) complicating alcoholic and non-alcoholic fatty liver diseases (ALD and NAFLD) is rising in western societies. Despite knowing the at risk populations for HCC development, the lack of sensitive and specific means of surveillance hampers disease detection at curable stages. The most widely used serum HCC marker is alpha-fetoprotein (AFP), while PIVKA-II, glypican-3 (GP3) and Squamous Cell Carcinoma Antigen -1 (SCCA-1) have been proposed as new biomarkers. Assessment of these HCC biomarkers has largely been performed in patients with viral hepatitis. We conducted a cross sectional study assessing the value of these serum proteins, as well a novel candidate biomarker -follistatin – in patients with HCC arising on a background of ALD or NAFLD.MethodsPre-treatment serum samples from 50 patients with HCC arising on a background of ALD (n = 31) or NAFLD (n = 19) were assessed by specific ELISA assay for PIVKAII, Glypican-3, SCCA-1 and Follistatin. Results were compared and contrasted with a control patient group with biopsy proven steatohepatitis-related cirrhosis (n = 41). The diagnostic accuracy of each of the candidate biomarkers was evaluated using receiver operating characteristic (ROC) curve analysis, reporting the area under the curve (AUC) and its 95% confidence interval (CI). Performance was compared to that of the established biomarker, AFP.ResultsSerum levels of all proteins were assessed by specific ELISA assays. GP3, SCCA-1 and follistatin had no HCC surveillance benefit in these patients. AFP and PIVKAII were superior to the other markers, particularly in combination.ConclusionWe conclude that while novel means of surveillance are urgently required, the combination of AFP and PIVKAII for HCC is an improvement on AFP alone in ALD/NAFLD patients. Furthermore, our data in this homogenous subset of patients- particularly that confirming no role for SCCA-1 – suggests that the choice of optimal biomarkers for HCC surveillance may be determined by the aetiology of underlying chronic liver disease.