SPARC Expression Research Articles

Study on the mechanism of OSM participating in myocardial fibrosis by inhibiting TGFβ-induced EndMT of cardiac microvascular endothelial cells through SPARC/SMAD signaling.

Cardiac fibrosis constitutes a crucial element in the progression of diverse chronic cardiac conditions. Notably, a significant correlation has been observed between the endothelial-to-mesenchymal transition (EndMT) and the emergence of cardiac fibrosis. To investigate mechanisms, we employed immunofluorescence for α-SMA and CD31 analysis, Western blotting for CD34, vimentin, and SPARC overexpression. CCK8, wound healing, and transwell assay-assessed cell viability, invasion, and migration. SPARC overexpression plasmid was constructed and validated by Western blotting. Fibrosis levels were quantified via Masson staining, and collagen 1 and 3 expressions were measured using ELISA assays. Notably, in TGF-β-induced H5V cells, the downregulation of CD31 and CD34 expression, along with the upregulation of α-SMA and vimentin, suggests the induction of EndMT in cardiac fibrosis. Interestingly, OSM treatment mitigated EndMT progression, cell invasion, migration, and the expression of p-SMAD2, p-SMAD3, and SPARC in TGF-β-treated H5V cells. Further analysis revealed that OSM alleviated TGFβ-induced EndMT, invasion, and migration of cardiac microvascular endothelial cells by suppressing SPARC/SMAD signaling. Moreover, OSM therapy notably mitigated myocardial tissue fibrosis, along with a reduction in the expression of collagen 1, collagen 3, α-SMA, and CD34, while augmenting CD31 and vimentin expression in ISO-induced myocardial tissue. Additionally, OSM exhibited the ability to suppress myocardial tissue fibrosis and the expression of EndMT markers as well as SPARC/SMAD signals in ISO-induced myocardial tissue. Our comprehensive analysis unveiled that OSM contributes significantly to myocardial fibrosis modulation by inhibiting TGFβ-mediated EndMT in myocardial microvascular endothelial cells via SPARC/SMAD signaling.

Exploring new dimensions of immune cell biology in Anopheles gambiae through genetic immunophenotyping.

Mosquito immune cells, or hemocytes, are integral components of the innate immune responses that define vector competence. However, the lack of genetic resources has limited their characterization and our understanding of their functional roles in immune signaling. To overcome these challenges, we engineered transgenic Anopheles gambiae that express fluorescent proteins under the control of candidate hemocyte promoters. Following the characterization of five transgenic constructs through gene expression and microscopy-based approaches, we examine mosquito immune cell populations by leveraging advanced spectral imaging flow cytometry. Our results comprehensively map the composition of mosquito hemocytes, classifying them into twelve distinct populations based on size, granularity, ploidy, phagocytic capacity, and the expression of PPO6, SPARC, and LRIM15 genetic markers. Together, our novel use of morphological properties and genetic markers provides increased resolution into our understanding of mosquito hemocytes, highlighting the complexity and plasticity of these immune cell populations, while providing the foundation for deeper investigations into their roles in immunity and pathogen transmission.

CAF-Associated Genes in Breast Cancer for Novel Therapeutic Strategies.

Breast cancer (BC) is the most common cancer in women, and therapeutic strategies for it are based on the molecular subtypes of luminal BC, HER2 BC, and triple-negative BC (TNBC) because each subtype harbors different unique genetic aberrations. Recently, features of the tumor microenvironment (TME), especially cancer-associated fibroblasts (CAFs), have been demonstrated to play a critical role in BC progression, and we would like to understand the molecular features of BC CAFs for novel therapeutic strategies. In a recent study, 115 CAF-associated genes (CAFGs) were identified in a public database of microdissection and microarray data (GSE35602) from 13 colorectal cancer (CRC) tumors. Using a public database (GSE10797) of 28 BC tumors, a similar analysis was performed. In BC, 59 genes from the 115 CAFGs identified in CRC (CRC CAFGs) were also closely associated with a CAFs marker, SPARC (R = 0.6 or beyond), and POSTN was of particular interest as one of the BC CAFGs with the highest expression levels and a close association with SPARC expression (R = 0.94) in the cancer stroma of BC tumors. In BC stroma, POSTN was followed in expression levels by DKK3, MMP2, PDPN, and ACTA2. Unexpectedly, FAP and VIM were not as highly associated with SPARC expression in the cancer stroma of BC tumors and exhibited low expression. These findings suggested that ACTA2 might be the most relevant conventional CAFs marker in BC, and ACTA2 was actually correlated in expression with many CRC CAFGs, such as SPARC. Surprisingly, the SE ratio values of the BC CAFGs were much lower (average SE = 3.8) than those of the CRC CAFGs (SE = 10 or beyond). We summarized the current understanding of BC CAFs from the literature. Finally, in triple-negative BC (TNBC) (n = 5), SPARC expression uniquely showed a close association with COL11A1 and TAGLN expression, representing a myofibroblast (myCAFs) marker in the cancer stroma of the BC tumors, suggesting that myCAFs may be molecularly characterized by TNBC in contrast to other BC phenotypes. In summary, CAFs could have unique molecular characteristics in BC, and such TME uniqueness could be therapeutically targeted in BC.

Astrocyte modulation of synaptic plasticity mediated by activity-dependent Sonic hedgehog signaling.

The influence of neural activity on astrocytes and their reciprocal interactions with neurons has emerged as an important modulator of synapse function. Astrocytes exhibit activity-dependent changes in gene expression, yet the molecular mechanisms by which they accomplish this have remained largely unknown. The molecular signaling pathway, Sonic hedgehog (Shh), mediates neuron-astrocyte communication and regulates the organization of cortical synapses. Here, we demonstrate that neural activity stimulates Shh signaling in cortical astrocytes and upregulates expression of Hevin and SPARC, astrocyte derived molecules that modify synapses. Whisker stimulation and chemogenetic activation both increase Shh activity in deep layers of the somatosensory cortex, where neuron-astrocyte Shh signaling is predominantly found. Experience-dependent Hevin and SPARC require intact Shh signaling and selective loss of pathway activity in astrocytes occludes experience-dependent structural plasticity. Taken together, these data identify Shh signaling as an activity-dependent, neuronal derived cue that stimulates astrocyte interactions with synapses and promotes synaptic plasticity.

EXPRESSION OF SPP1 AND SPARC GENES IN TUMOR TISSUE OF PATIENTS WITH BREAST CANCER.

Breast cancer (BCa) is one of the most common oncological diseases in women in Ukraine and worldwide, which determines the need to search for new diagnostic and prognostic markers. In this aspect, the study of multicellular proteins, in particular osteopontin (OPN) and osteonectin (ON), in BCа tissue is relevant. The aim of the work was to investigate the expression of SPP1 and SPARC at the mRNA and protein levels in BCa tissue and to assess their relationship with the main clinicopathological BCa characteristics and the survival rates of patients. The work was based on the analysis of the results of the examination and treatment of 60 patients with stage II-III BCa and 15 patients with breast fibroadenomas. SPP1 and SPARC mRNA levels were determined by real-time PCR. The study of the expression of protein products of the SPP1 and SPARC genes was carried out by the immunohistochemical method. We have established that the BCa tissue was characterized by 3.5 (p < 0.05) and 7.4 (p < 0.05) lower levels of SPP1 and SPARC mRNA, respectively, compared to the tissue of benign neoplasms, while OPN and ON expression levels were 1.6 (p < 0.05) and 5.6 (p < 0.05) times higher, respectively, compared to fibroadenoma tissue. The analysis of the relationship between the expression of SPP1 and SPARC at the protein and mRNA levels in BCa tissue and the main clinicopathological BCa characteristics revealed its dependence on the presence of metastases in regional lymph nodes, differentiation grade, and the molecular BCa subtype. Also, high expression levels of SPP1 and OPN were associated with worse patient survival rates. The obtained results indicate the perspective of using SPP1 and SPARC expression indices in BCa tissue to assess the aggressiveness of the cancer course and optimize the tactics of treating patients.

Biocompatibility and Cytotoxicity of Pulp-Capping Materials on DPSCs, With Marker mRNA Expressions

ObjectivesThe present study aimed to (i) investigate biocompatibility and cytotoxicity of pulp capping materials on viability of hDPSCs, (ii) determine angiogenic, odontogenic, and osteogenic marker mRNA expressions, and (iii) observe changes in surface morphology of the hDPSCs using SEM. MethodsImpacted third molars were used to isolate the hDPSCs, which were treated with extract release fluids of the pulp capping materials (Harvard BioCal–Cap, NeoPutty MTA, TheraCal LC, and Dycal). Effects of the capping materials on cell viability were assessed using MTS assay and the apoptotic/necrotic cell ratios and ROS levels from a flow cytometry. Marker expressions (ALP, OCN, Col1A, SPARC (ON) and VEGF) were determined by RT-qPCR. Changes in surface morphology of the hDPSCs were visualized by SEM. ResultsThe MTS assay results at days 1, 3, 5 and 7 indicated that Harvard BioCal–Cap, NeoPutty MTA and TheraCal LC did not adversely affect cell viability when compared to the control group. According to the MTS assay results at day 14, no significant difference was found among Dycal, Harvard BioCal–Cap, NeoPutty MTA and TheraCal LC affecting the cell viability. Dycal was the only capping material that increases the ROS level. High levels of VEGF expression were observed in Harvard BioCal-Cap, TheraCal LC, and NeoPutty MTA. NeoPutty MTA and Dycal up-regulated OCN expression, whereas TheraCal LC up-regulated Col1A and SPARC expression. Only Dycal increased ALP expression. The hDPSCs were visualized in characteristic spindle morphology by SEM due to TheraCal LC and Harvard BioCal–Cap. ConclusionsNeoPutty MTA and Harvard BioCal–Cap showed suitable biocompatibility values, and in particular, these pulp capping materials were observed to support the angiogenic marker. Clinical SignificanceSince it was thought that cytotoxicity and biocompatibility values would contribute significantly to future studies, a new pulp capping material, NeoPutty MTA and Harvard BioCal-Cap, were used in this study.

Inhibiting immune escape in lung adenocarcinoma: the role of SPARC in suppressing CD276 function

SPARC is an acidic, cysteine-rich, calcium-binding member of the non-collagen glycoproteins originating in bone. Although implicated in the development of several cancers, the functions and mechanisms of SPARC remain unclear. The aim of this study was to investigate whether smooth muscle cell (SMC)-associated SPARC acts an important tumour suppressor in lung adenocarcinoma (LUAD). SPARC inhibits immune evasion in LUAD by suppressing CD276 functionality. We downloaded RNA-sequencing data from patients with LUAD in The Cancer Genome Atlas and identified a set of genes showing SMC-specific expression in these samples. We then screened for differentially expressed genes (DEGs). Enrichment analysis using the Gene Expression Omnibus identified key genes, while immune pathway analysis explored the expression of immune checkpoints involved in LUAD immune regulation. We further validated the differential expression of SPARC and CD276 using immunohistochemistry. Among the upregulated DEGs, SPARC exhibited high enrichment in SMCs, whereas 13 immune checkpoints, such as LAIR1 and CTLA4, were excluded. The infiltration level of the CD276 immune checkpoint was lower in the high-risk group than in the low-risk group. While our results suggest a correlation between SPARC, CD276 and LUAD, additional studies are needed to validate these findings and elucidate the underlying mechanisms before definitive conclusions can be drawn regarding their utility in clinical practice.

TDO2 promotes bladder cancer progression via AhR-mediated SPARC/FILIP1L signaling

The enzyme tryptophan 2,3-dioxygenase (TDO2) has been implicated in the dysregulation across a variety of human cancers. Despite this association, the implications of TDO2 in the progression of bladder cancer have eluded thorough understanding. In this study, we demonstrate that TDO2 expression is notably elevated in bladder cancer tissues and serves as an unfavorable prognostic factor for overall survival. Through a series of biological functional assays, we have determined that TDO2 essentially enhances cell proliferation, metastatic potential, and imparts a decreased sensitivity to the chemotherapeutic agent cisplatin. Our mechanistic investigations reveal that TDO2 augments aryl hydrocarbon receptor (AhR) signaling pathways and subsequently upregulates the expression of SPARC and FILIP1L. Importantly, we have identified a positive correlation between TDO2 levels and the basal/squamous subtype of bladder cancer, and we provide evidence to suggest that TDO2 expression is modulated by the tumor suppressors RB1 and TP53. From a therapeutic perspective, we demonstrate that the targeted inhibition of TDO2 with the molecular inhibitor 680C91 markedly attenuates tumor growth and metastasis while concurrently enhancing the efficacy of cisplatin. These findings open a new therapeutic avenue for the management of bladder cancer.

Abstract 3419: Demographic, health history, and lifestyle factors in association with biomarkers of colorectal cancer prognosis: A pilot study

Abstract Background/Objectives: Multiple demographic, health history, and lifestyle factors have been associated with prognosis of colorectal cancer (CRC), but the mechanisms underlying these associations remain poorly understood. Knowledge of these mechanisms could reveal new strategies to improve outcomes among CRC patients. The primary objective of this project was to explore the association of these factors, which were assessed pre-diagnostically, with expression of two biomarkers in CRC tumors, SPARC and PD-L1, for which lower and higher levels of expression, respectively, have been previously associated with poorer CRC prognosis. Methods: Participants were drawn from the British Columbia Generations Project (BCGP). At the time of recruitment, they completed a detailed questionnaire that ascertained demographic factors (e.g., biological sex and household income), health history (e.g., personal history of CRC screening), and lifestyle factors (daily fruit and vegetable consumption and alcohol consumption). Formalin-fixed paraffin-embedded blocks (FFPE) with adequate volumes of tumor were obtained for 49 incident CRC cases diagnosed within the BCGP. Cores were extracted from the blocks to create tumor tissue microarrays (TMAs). Slides created from thin sections of the TMAs were stained with SPARC and PD-L1 antibodies and then imaged and analyzed to calculate H-scores as measures of expression in both epithelial and non-epithelial tissues. Linear regression analyses were conducted to evaluate associations between the various factors and ln-transformed H-scores. Results: Compared to non-smokers, smokers, on average, had 47% lower SPARC H-scores (p=0.05) in the epithelial tissues of their CRC tumors. Individuals with incomes higher than $74,999/year had 33% higher SPARC H-scores (p=0.04) in their CRC tumor non-epithelial tissues than those who earned less than $74 999/year. Females had 2.8-fold greater PD-L1 H-scores (p=0.005) in their CRC tumor epithelial tissues than males. Compared to those without a history of CRC screening, those with a history of CRC screening had 2.2 and 2.0-fold greater PD-L1 H-scores in their epithelial and non-epithelial CRC tumor tissues, respectively. Conclusion: Larger-scale studies with prognostic data are needed to confirm our findings, but our results suggest that differences in the expression of SPARC and PDL-1 may contribute to the previously observed impacts of some demographic, healthy history, and lifestyle factors on CRC prognosis. Citation Format: Umaimah Zanif, Isabella Tai, Stephen Yip, Sindy Babinszky, Katy Milne, Peter Watson, Rachel Murphy, Parveen Bhatti. Demographic, health history, and lifestyle factors in association with biomarkers of colorectal cancer prognosis: A pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3419.

Screening of differentially expressed genes in gastric cancer based on GEO database and function and pathway enrichment analysis

To explore the core genes related to the diagnosis and prognosis of gastric cancer (GC) based on Gene Expression Omnibus (GEO) database and screen the molecular targets involved in the occurrence and development of GC. GC microarray data GSE118916, GSE54129 and GSE79973 were downloaded from GEO database, and the differentially expressed genes (DEGs) were screened. Enrichment analysis of the signaling pathways and molecular functions were preformed and protein-protein interaction networks (PPI) were constructed to identify the hub genes, whose expression levels and diagnostic and prognostic values were verifies based on gastric adenocarcinoma data from TCGA. The expression levels of these core genes were also detected in different GC cell lines using qRT- PCR. Seventy-seven DEGs were identified, which encodes proteins located mainly in the extracellular matrix and basement membrane with activities of oxidoreductase and extracellular matrix receptor and ligand, involving the biological processes of digestion and hormone metabolism and the signaling pathways in retinol metabolism and gastric acid secretion. Nine hub genes were obtained, among which SPARC, TIMP1, THBS2, COL6A3 and THY1 were significantly up- regulated and TFF1, GKN1, TFF2 and PGC were significantly down-regulated in GC. The abnormal expressions of SPARC, TIMP1, THBS2, COL6A3, TFF2 and THY1 were significantly correlated with the survival time of GC patients. ROC curve analysis showed that aberrant expression of TIMP1 SPARC, THY1 and THBS2 had high diagnostic value for GC. High expressions of SPARC, TIMP1, THBS2 and COL6A3 were detected in GC tissues. In the GC cell lines, qRT- PCR revealed different expression patterns of these hub genes, but their expressions were largely consistent with those found in bioinformatics analyses. SPARC, TIMP1, THBS2 and other DEGs are probably involved in GC occurrence and progression and may serve as potential candidate molecular markers for early diagnosis and prognostic evaluation of GC.

A Comprehensive Study of Meningioma Biomineralization: Morphological, Crystallographic, and Immunohistochemical Aspects.

Biomineralization of brain tissues occurs both in normal and pathological conditions. Dura mater biomineralization is widespread and occurs in 1-72% of cases, depending on the patient's age and research method. The amount of biomineral deposits under the conditions of tumor growth in the meninges only increases, reaching 100% in the case of psammomatous meningiomas. Since calcifications are often found in the meninges, the problem of differential diagnosis with calcified meningiomas arises. A total of 30 samples of meningiomas with signs of biomineralization-dense structure, characteristic crunch, psammoma bodies (group I) and 30 samples of meningiomas without any signs of biomineralization were examined as controls (group II). To detect pathological biomineralization, the meningioma tissue was studied using the methods of macroscopic description, histology, histochemistry, and immunohistochemistry, scanning electron microscopy with microanalysis, and transmission electron microscopy. A significantly higher level of caspase3 and features of the expression of osteoblastic markers (a lower level of OPG expression and a higher level of the presence of RANKL in group I, the absence of fluctuations in the expression of SPARC) may indicate a dystrophic type of development of biomineral deposits in meningiomas.

Immunohistochemical expression of SPARC in odontogenic keratocysts: a comparative study with other odontogenic cysts

BackgroundSecreted protein acidic and rich in cysteine (SPARC) has been shown to modulate aggressive behavior in several benign and malignant tumors. Little is known about SPARC expression in odontogenic keratocyst (OKC), an odontogenic cyst with an aggressive nature. To the best of our knowledge, only one study has been investigated the expression of this protein in OKCs. This study aimed to characterize SPARC expression in OKCs. Additionally, to determine whether SPARC is associated with aggressive behavior in OKCs, SPARC expression in OKCs was compared with radicular cysts (RCs), dentigerous cysts (DCs) and calcifying odontogenic cysts (COCs). These odontogenic cysts showed no or less aggressive behavior.MethodsSPARC expression was evaluated in 38 OKCs, 39 RCs, 35 DCs and 14 COCs using immunohistochemistry. The percentages of positive cells and the intensities of immunostaining in the epithelial lining and the cystic wall were evaluated and scored.ResultsGenerally, OKCs showed similar staining patterns to RCs, DCs and COCs. In the epithelial lining, SPARC was not detected, except for ghost cells in all COCs. In the cystic wall, the majority of positive cells were fibroblasts. Compared between 4 groups of odontogenic cysts, SPARC expression in OKCs was significantly higher than those of RCs (P < 0.001), DCs (P < 0.001) and COCs (P = 0.001).ConclusionsA significant increase of SPARC expression in OKCs compared with RCs, DCs and COCs suggests that SPARC may play a role in the aggressive behavior of OKCs.

Osteonectin (SPARC) prognostic value in prostate cancer

BackgroundProstate cancer (PCa) is common malignancy among men worldwide. To date only few molecular markers are available to predict its course and outcome. SPARC is considered to be promising prognostic marker of PCa due to its involvement in various cancer processes. Materials and methodsstudy was conducted on PCa surgical primary tumor samples, obtained from 84 patients. Level of SPARC mRNA expression was estimated using RT-qPCR. To identify SPARC protein (osteonectin) in prostate tissue, immunohistochemical analysis was conducted. Bioinformatical analysis was performed on UALCAN and TNMplot resources. Resultsbioinformatical analysis demonstrated that SPARC mRNA levels are decreased in PCa samples, in comparison to normal tissue. In patients with lymph node metastases its levels are 1.26 times higher; p = 4.66E-02, than in N0 category. Ex vivo study demonstrated that SPARC expression was elevated on both mRNA and protein levels in PCa patients with lymph node metastases (by 2.34 and 1.91, respectively, p < 0.05). We established higher levels of SPARC mRNA and protein in PCa patients with T3 tumors, as well as high Gleason score. Estimation of survival rates demonstrated that PCa patients with a high level of SPARC mRNA and protein have decreased overall 2-year survival. ConclusionsSPARC protein was overexpressed on mRNA and protein levels in patients with presence of lymph node metastases and higher Gleason score of tumors. Also, both mRNA and protein upregulation were associated with worse survival rates. The current study has therefore provided further evidence that SPARC is indeed linked to the prognosis and aggressiveness of human PCa.

Peritumoral SPARC expression induced by exosomes from nasopharyngeal carcinoma infected Epstein-Barr virus: A poor prognostic marker.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) cells have high metastatic potential. Recent research has revealed that the interaction of between tumor cells and the surrounding stroma plays an important role in tumor invasion and metastasis. In this study, we showed the prognostic value of expression of SPARC, an extracellular matrix protein with multiple cellular functions, in normal adjacent tissues (NAT) surrounding NPC. In the immunohistochemical analysis of 51 NPC biopsy specimens, SPARC expression levels were significantly elevated in the NAT of EBER (EBV-encoded small RNA)-positive NPC compared to that in the NAT of EBER-negative NPC. Moreover, increased SPARC expression in NAT was associated with a worsening of overall survival. The enrichment analysis of RNA-seq of publicly available NPC and NAT surrounding NPC data showed that high SPARC expression in NPC was associated with epithelial mesenchymal transition promotion, and there was a dynamic change in the gene expression profile associated with interference of cellular proliferation in NAT, including SPARC expression. Furthermore, EBV-positive NPC cells induce SPARC expression in normal nasopharyngeal cells via exosomes. Induction of SPARC in cancer-surrounding NAT cells reduced intercellular adhesion in normal nasopharyngeal structures and promoted cell competition between cancer cells and normal epithelial cells. These results suggest that epithelial cells loosen their own binding with the extracellular matrix as well as stromal cells, facilitating the invasion of tumor cells into the adjacent stroma by activating cell competition. Our findings reveal a new mechanism by which EBV creates a pro-metastatic microenvironment by upregulating SPARC expression in NPC.

Expression of secretory calcium-binding phosphoprotein (scpp) genes in medaka during the formation and replacement of pharyngeal teeth

BackgroundAnalyses of tooth families and tooth-forming units in medaka with regard to tooth replacement cycles and the localization of odontogenic stem cell niches in the pharyngeal dentition clearly indicate that continuous tooth replacement is maintained. The secretory calcium-binding phosphoprotein (scpp) gene cluster is involved in the formation of mineralized tissues, such as dental and bone tissues, and the genes encoding multiple SCPPs are conserved in fish, amphibians, reptiles, and mammals. In the present study, we examined the expression patterns of several scpp genes in the pharyngeal teeth of medaka to elucidate their roles during tooth formation and replacement.MethodsHimedaka (Japanese medaka, Oryzias latipes) of both sexes (body length: 28 to 33 mm) were used in this study. Real-time quantitative reverse transcription-polymerase chain reaction (PCR) (qPCR) data were evaluated using one-way analysis of variance for multi-group comparisons, and the significance of differences was determined by Tukey’s comparison test. The expression of scpp genes was examined using in situ hybridization (ISH) with a digoxigenin-labeled, single-stranded antisense probe.ResultsqPCR results showed that several scpp genes were strongly expressed in pharyngeal tissues. ISH analysis revealed specific expression of scpp1, scpp5, and sparc in tooth germ, and scpp5 was continually expressed in the odontoblasts of teeth attached to pedicles, but not in the osteoblasts of pedicles. In addition, many scpp genes were expressed in inner dental epithelium (ide), but not in odontoblasts, and scpp2 consistently showed epithelial-specific expression in the functional teeth. Taken together, these data indicate that specific expression of scpp2 and scpp5 may play a critical role in pharyngeal tooth formation in medaka.ConclusionWe characterized changes in the expression patterns of scpp genes in medaka during the formation and replacement of pharyngeal teeth.

Cell-type-specific alternative polyadenylation promotes oncogenic gene expression in non-small cell lung cancer progression

Disrupted alternative polyadenylation (APA) is frequently involved in tumorigenesis and cancer progression by regulating the gene expression of oncogenes and tumor suppressors. However, limited knowledge of tumor-type- and cell-type-specific APA events may lead to novel APA events and their functions being overlooked. Here, we compared APA events across different cell types in non-small cell lung cancer (NSCLC) and normal tissues and identified functionally related APA events in NSCLC. We found several cell-specific 3'-UTR alterations that regulate gene expression changes showed prognostic value in NSCLC. We further investigated the function of APA-mediated 3'-UTR shortening through loss of microRNA (miRNA)-binding sites, and we identified and experimentally validated several oncogene-miRNA-tumor suppressor axes. According to our analyses, we found SPARC as an APA-regulated oncogene in cancer-associated fibroblasts in NSCLC. Knockdown of SPARC attenuates lung cancer cell invasion and metastasis. Moreover, we found high SPARC expression associated with resistance to several drugs except cisplatin. NSCLC patients with high SPARC expression could benefit more compared to low-SPARC-expression patients with cisplatin treatment. Overall, our comprehensive analysis of cell-specific APA events shed light on the regulatory mechanism of cell-specificoncogenes and provided opportunities for combination of APA-regulated therapeutic target and cell-specific therapy development.

Abstract P1134: RNA Elongation Mediates Rapid Induction Of Gene Expression During Endothelial-to-mesenchymal Transition

Endothelial-to-mesenchymal transition (EndMT) is triggered by stress stimuli and characterized by the conversion of endothelial cells (EC) into mesenchymal cells. Stress stimuli such as hypoxia or inflammation depend on rapid gene expression mediated by RNA elongation. RNA elongation is controlled by the super elongation complex (SEC) through transient pausing of RNA Polymerase II (RNAPII). We aimed to analyze the regulatory role of RNA elongation on rapid gene expression during the onset of EndMT. In an in vitro EndMT model, SEC scaffolds AFF1 and AFF4 were upregulated compared to controls. SEC inhibition using chemical compounds reduced the protein levels of AFF1 and AFF4. EndMT was reduced by SEC inhibition, whereas overexpression of AFF4 increased EndMT as measured by mesenchymal marker gene expression. Rapid gene expression depends on the release of paused RNAPII at promoter sites. ChIP-sequencing revealed a reduction of RNAPII occupancy at promoter sites in EndMT compared to controls, suggesting the release of RNAPII during EndMT. Transcriptome-wide RNA elongation rates were increased as early as 10 min after induction of EndMT, as determined by analysis of nascent 4sU-labeled RNA. SEC inhibition reduced global RNA elongation rates, suggesting a regulatory contribution of RNA elongation in EndMT. Among the genes that were immediately upregulated after the induction of EndMT was the SPARC mRNA. RNA initiation and elongation of SPARC mRNA were increased 10 min after EndMT induction, which was reduced by SEC inhibition. At 72 h after EndMT induction, inhibition of SEC decreased SPARC mRNA, protein concentration and SPARC delivery to the supernatant. Silencing of SPARC reduced the angiogenic potential of EC and enhanced EndMT induction, which was attenuated by treatment with recombinant SPARC. Treatment with recombinant SPARC also improved endothelial impedance under EndMT conditions. Spatial transcriptomics revealed increased SPARC expression in brachiocephalic artery plaques from LDLR-/- mice. In conclusion, we observed increased RNA elongation rates associated with reduced RNAPII pausing during EndMT that mediate rapid gene expression of target genes such as SPARC. Our results suggest that RNA elongation plays a regulatory role in EndMT.

SPARC Is Highly Expressed in Young Skin and Promotes Extracellular Matrix Integrity in Fibroblasts via the TGF-β Signaling Pathway.

The matricellular secreted protein acidic and rich in cysteine (SPARC; also known as osteonectin), is involved in the regulation of extracellular matrix (ECM) synthesis, cell-ECM interactions, and bone mineralization. We found decreased SPARC expression in aged skin. Incubating foreskin fibroblasts with recombinant human SPARC led to increased type I collagen production and decreased matrix metalloproteinase-1 (MMP-1) secretion at the protein and mRNA levels. In a three-dimensional culture of foreskin fibroblasts mimicking the dermis, SPARC significantly increased the synthesis of type I collagen and decreased its degradation. In addition, SPARC also induced receptor-regulated SMAD (R-SMAD) phosphorylation. An inhibitor of transforming growth factor-beta (TGF-β) receptor type 1 reversed the SPARC-induced increase in type I collagen and decrease in MMP-1, and decreased SPARC-induced R-SMAD phosphorylation. Transcriptome analysis revealed that SPARC modulated expression of genes involved in ECM synthesis and regulation in fibroblasts. RT-qPCR confirmed that a subset of differentially expressed genes is induced by SPARC. These results indicated that SPARC enhanced ECM integrity by activating the TGF-β signaling pathway in fibroblasts. We inferred that the decline in SPARC expression in aged skin contributes to process of skin aging by negatively affecting ECM integrity in fibroblasts.

FERMT2 upregulation in CAFs enhances EMT of OSCC and M2 macrophage polarization.

FERMT2 upregulation was associated with malignant tumor behaviors, including epithelial-to-mesenchymal (EMT). This study aimed to characterize the expression profile of FERMT2 in oral squamous cell carcinoma (OSCC) and to explore its involvement in the tumor microenvironment sculptured by oral cancer-associated fibroblasts (OCAFs). Previous bulk-seq (TCGA-HNSC) and single-cell RNA-seq data sets were retrieved for bioinformatic analysis. Human OSCC lines SCC15 and CAL27, primary normal oral fibroblasts (NOFs), OCAFs, and THP-1 cells were used for intro studies. FERMT2 expression was significantly higher in CAFs compared with OSCC tumor cells and normal fibroblasts. Higher FERMT2 expression might independently predict unfavorable disease-specific survival (DSS) in patients with OSCC. Knockdown of FERMT2 suppressed the expression and secretion of IGFBP7, SPARC, TIMP3, COL4A1, and IGFBP4 in OCAFs. OCAFs with FERMT2 knockdown had significantly weakened capability to induce the invasion of OSCC cells and the expression of mesenchymal markers. FERMT2 knockdown impaired the inducing effect of OCAFs on the migration of M0 macrophages and the expression of M2 macrophage markers. FERMT2 could modulate the production and secretion of IGFBP7, SPARC, COL4A1, and IGFBP4 in OCAFs, thereby inducing the EMT of OSCC and M2 macrophage polarization.

SPARC accelerates biliary tract cancer progression through CTGF-mediated tumor-stroma interactions: SPARC as a prognostic marker of survival after neoadjuvant therapy.

In biliary tract cancer (BTC), malignancy is strongest at the invasion front. To improve the BTC prognosis, the invasion front should be controlled. We evaluated tumor-stroma crosstalk at the tumor center and at the invasion front of BTC lesions. We investigated the expression of SPARC, a marker of cancer-associated fibroblasts, and determined its ability to predict BTC prognosis after neoadjuvant chemoradiotherapy (NAC-RT). We performed immunohistochemistry to evaluate SPARC expression in resected specimens from patients that underwent BTC surgery. We established highly invasive (HI) clones in two BTC cell lines (NOZ, CCLP1), and performed mRNA microarrays to compare gene expression in parental and HI cells. Among 92 specimens, stromal SPARC expression was higher at the invasion front than at the lesion center (p = 0.014). Among 50 specimens from patients treated with surgery alone, high stromal SPARC expression at the invasion front was associated with a poor prognosis (recurrence-free survival: p = 0.033; overall survival: p = 0.017). Coculturing fibroblasts with NOZ-HI cells upregulated fibroblast SPARC expression. mRNA microarrays showed that connective tissue growth factor (CTGF) was upregulated in NOZ-HI and CCLP1-HI cells. A CTGF knockdown suppressed cell invasion in NOZ-HI cells. Exogeneous CTGF upregulated SPARC expression in fibroblasts. SPARC expression at the invasion front was significantly lower after NAC-RT, compared to surgery alone (p = 0.003). CTGF was associated with tumor-stroma crosstalk in BTC. CTGF activated stromal SPARC expression, which promoted tumor progression, particularly at the invasion front. SPARC expression at the invasion front after NAC-RT may serve as a prognosis predictor.