T Antigen Research Articles

4-[(5-Methyl-1H-pyrazol-3-yl)amino]-2H-phenyl-1-phthalazinone Inhibits MCPyV T Antigen Expression in Merkel Cell Carcinoma Independent of Aurora Kinase A.

Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV), and MCPyV-positive tumor cells depend on expression of the virus-encoded T antigens (TA). Here, we identify 4-[(5-methyl-1H-pyrazol-3-yl)amino]-2H-phenyl-1-phthalazinone (PHT)-a reported inhibitor of Aurora kinase A-as a compound inhibiting growth of MCC cells by repressing noncoding control region (NCCR)-controlled TA transcription. Surprisingly, we find that TA repression is not caused by inhibition of Aurora kinase A. However, we demonstrate that β-catenin-a transcription factor repressed by active glycogen synthase kinase 3 (GSK3)-is activated by PHT, suggesting that PHT bears a hitherto unreported inhibitory activity against GSK3, a kinase known to function in promoting TA transcription. Indeed, applying an in vitro kinase assay, we demonstrate that PHT directly targets GSK3. Finally, we demonstrate that PHT exhibits in vivo antitumor activity in an MCC xenograft mouse model, suggesting a potential use in future therapeutic settings for MCC.

Regulation of Virus Replication by BK Polyomavirus Small T Antigen.

Polyomavirus small T antigen (tAg) plays important roles in regulating viral replication, the innate immune response, apoptosis, and transformation for SV40, Merkel cell polyomavirus (MCPyV), murine polyomavirus (MuPyV), and JC polyomavirus (JCPyV). However, the function of BK polyomavirus (BKPyV) tAg has been much less studied. Here, we constructed mutant viruses that do not express tAg, and we showed that, in contrast with other polyomaviruses, BKPyV tAg inhibits large T antigen (TAg) gene expression and viral DNA replication. However, this occurs only in an archetype viral background. We also observed that the transduction of cells with a lentivirus-expressing BKPyV tAg kills the cells. We further discovered that BKPyV tAg interacts not only with PP2A A and C subunits, as has been demonstrated for other polyomavirus tAg proteins, but also with PP2A B''' subunit members. Knocking down either of two B''' subunits, namely STRN or STRN3, mimics the phenotype of the tAg mutant virus. However, a virus containing a point mutation in the PP2A binding domain of tAg only partially affected virus TAg expression and DNA replication. These results indicate that BKPyV tAg downregulates viral gene expression and DNA replication and that this occurs in part through interactions with PP2A. IMPORTANCE BK polyomavirus is a virus that establishes a lifelong infection of the majority of people. The infection usually does not cause any clinical symptoms, but, in transplant recipients whose immune systems have been suppressed, unchecked virus replication can cause severe disease. In this study, we show that a viral protein called small T antigen is one of the ways that the virus can persist without high levels of replication. Understanding which factors control viral replication enhances our knowledge of the virus life cycle and could lead to potential interventions for these patients.

Immortalization of mouse primary astrocytes

In cell culture studies, immortalized primary cells have become a useful tool to investigate the molecular and cellular functions of different types of cells. Several immortalization agents, such as human telomerase reverse transcriptase (hTERT) and Simian Virus 40 (SV40) T antigens, are commonly used for primary cell immortalization. Astrocytes, as the most abundant glial cell type in the central nervous system, are promising therapeutical targets for many neuronal disorders, such as Alzheimer’s disease and Parkinson’s disease. Immortalized primary astrocytes can provide useful information for astrocytes biology, astrocytes-neuron interactions, glial interactions and astrocytes-associated neuronal diseases. In this study, we successfully purified primary astrocytes with immuno-panning method and examined the astrocyte functions after immortalization through both hTERT and SV40 Large-T antigens. As expected, both immortalized astrocytes presented unlimited lifespan and highly expressed multiple astrocyte-specific markers. However, SV40 Large-T antigen, but not hTERT, immortalized astrocytes displayed fast ATP-induced calcium wave in culture. Hence, SV40 Large-T antigen could be a better choice for primary astrocyte immortalization, which closely mimics the cell biology of primary astrocytes in culture. In summary, the purification and immortalization of primary astrocytes presented in this study can be used for studying astrocyte biology under physiological and pathological conditions.

Establishment and characterization of immortalized human vocal fold fibroblast cell lines.

Vocal fold scarring is abnormal scar tissue in the lamina propria layer of the vocal fold. To facilitate investigation of vocal fold scarring, we established and characterized immortalized human vocal fold fibroblast (iHVFF) cell lines. Human vocal fold fibroblasts were immortalized by introducing Simian virus 40 large T antigen (SV40TAg) by transfection. Successfully transfected fibroblasts were sorted using flow cytometry. Immunofluorescence cytochemistry and western blot were applied to analyze the expression of fibronectin, vimentin, alpha-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP). Cell proliferation rate was measured by CCK-8 assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA expression level. The iHVFFs continued to proliferate for more than 30 generations and appeared spindle-shaped. The expression of Vimentin and α-SMA were detected in both iHVFFs and primary fibroblasts, and enhanced expression of FAP was observed in iHVFFs. Furthermore, iHVFFs exhibited an increased proliferative capability compared with the primary fibroblasts. RT-qPCR results suggested that collagen type III alpha 1 chain (COL3A1), interleukin-6, cyclooxygenase 2 (COX2), hyaluronan synthase 2 (HAS2), hepatocyte growth factor (HGF) in the iHVFFs significantly increased, whereas transforming growth factor-β1 (TGF-β1), elastin and matrix metallopeptidase-1 (MMP-1) expression significantly downregulated. No differences in mRNA expression of α-SMA, fibronectin and collagen type I alpha 2 chain (COL1A2) were noted between iHVFFs and primary fibroblasts. iHVFFs can be used as a novel tool cell for future researches on the mechanisms of pathogenesis and treatment of vocal fold scarring.

Generation of human immortalized chondrocytes from osteoarthritic and healthy cartilage

After a few passages of in vitro culture, primary human articular chondrocytes undergo senescence and loss of their phenotype. Most of the available chondrocyte cell lines have been obtained from cartilage tissues different from diarthrodial joints, and their utility for osteoarthritis (OA) research is reduced. Thus, the goal of this research was the development of immortalized chondrocyte cell lines proceeded from the articular cartilage of patients with and without OA. Using telomerase reverse transcriptase (hTERT) and SV40 large T antigen (SV40LT), we transduced primary OA articular chondrocytes. Proliferative capacity, degree of senescence, and chondrocyte surface antigen expression in transduced chondrocytes were evaluated. In addition, the capacity of transduced chondrocytes to synthesize a tissue similar to cartilage and to respond to interleukin (IL)-1β was assessed. Coexpression of both transgenes (SV40 and hTERT) were observed in the nuclei of transduced chondrocytes. Generated chondrocyte cell lines showed a high proliferation capacity and less than 2% of senescent cells. These cell lines were able to form 3D aggregates analogous to those generated by primary articular chondrocytes, but were unsuccessful in synthesizing cartilage-like tissue when seeded on type I collagen sponges. However, generated chondrocyte cell lines maintained the potential to respond to IL-1β stimulation. Through SV40LT and hTERT transduction, we successfully immortalized chondrocytes. These immortalized chondrocytes were able to overcome senescence in vitro, but were incapable of synthesizing cartilage-like tissue under the experimental conditions. Nonetheless, these chondrocyte cell lines could be advantageous for OA investigation since, similarly to primary articular chondrocytes, they showed capacity to upregulate inflammatory mediators in response to the IL-1β cytokine.Cite this article: Bone Joint Res2023;12(1):46-57.

Genome-Wide Analysis on Transcriptome and Methylome in Prevention of Mammary Tumor Induced by Early Life Combined Botanicals.

Breast cancer (BC) is the most common malignancy and the second leading cause of cancer death among women in the United States. The consumption of natural dietary components such as broccoli sprouts (BSp) and green tea polyphenols (GTPs) has demonstrated exciting potential in reducing the risk of BC through the regulation of epigenetic mechanisms. However, little is known about their impacts on reversing epigenomic aberrations that are centrally involved in the initiation and progression of BC. Previously, we have determined the efficacy of combined BSp and GTPs treatment on the inhibition of the growth of a mammary tumor in a transgenic Her2/neu mouse model. We sought to extend our previous study to identify universal biomarkers that represent common mechanistic changes among different mouse models in response to this dietary regime by including a new transgenic mouse model, C3(1)-SV40 TAg (SV40). As a result, we identified novel target genes that were differentially expressed and methylated in response to dietary botanicals when administered singly (BSp and GTPs) and in combination (BSp + GTPs) in both mouse models. We discovered more differentially expressed and methylated genes in the combination treatment group compared to the singly administered groups. Subsequently, several biological pathways related to epigenetic regulations were identified in response to the combination treatment. Furthermore, when compared to the BSp and GTPs treatment alone, the combinatorial treatment showed a more significant impact on the regulation of the epigenetic modifier activities involved in DNA methylation and histone modifications. Our study provides key insights about the impact of the combined administration of BSp and GTPs on BC using a multi-omics analysis, suggesting a combinatorial approach is more efficacious in preventing and inhibiting BC by impacting key tumor-related genes at transcriptomic and methylomic levels. Our findings could be further extrapolated as a comprehensive source for understanding the epigenetic modifications that are associated with the effects of these dietary botanicals on BC prevention.

Modeling lung diseases using reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2)

BackgroundA healthy alveolar epithelium is critical to the gas exchange function of the lungs. As the major cell type of alveolar epithelium, alveolar type 2 (AT2) cells play a critical role in maintaining pulmonary homeostasis by serving as alveolar progenitors during lung injury, inflammation, and repair. Dysregulation of AT2 cells may lead to the development of acute and chronic lung diseases and cancer. The lack of clinically relevant AT2 cell models hampers our ability to understand pulmonary diseases. Here, we sought to establish reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2) and investigate their potential in forming alveolar organoids to model pulmonary diseases.MethodsPrimary mouse pulmonary alveolar cells (mPACs) were isolated and immortalized with a retroviral expression of SV40 Large T antigen (LTA). Cell proliferation and survival was assessed by crystal violet staining and WST-1 assays. Marker gene expression was assessed by qPCR, Western blotting, and/or immunostaining. Alveolar organoids were generated by using matrigel. Ad-TGF-β1 was used to transiently express TGF-β1. Stable silencing β-catenin or overexpression of mutant KRAS and TP53 was accomplished by using retroviral vectors. Subcutaneous cell implantations were carried out in athymic nude mice. The retrieved tissue masses were subjected to H & E histologic evaluation.ResultsWe immortalized primary mPACs with SV40 LTA to yield the imPACs that were non-tumorigenic and maintained long-term proliferative activity that was reversible by FLP-mediated removal of SV40 LTA. The EpCAM+ AT2-enriched subpopulation (i.e., imPAC2) was sorted out from the imPACs, and was shown to express AT2 markers and form alveolar organoids. Functionally, silencing β-catenin decreased the expression of AT2 markers in imPAC2 cells, while TGF-β1 induced fibrosis-like response by regulating the expression of epithelial-mesenchymal transition markers in the imPAC2 cells. Lastly, concurrent expression of oncogenic KRAS and mutant TP53 rendered the imPAC2 cells a tumor-like phenotype and activated lung cancer-associated pathways. Collectively, our results suggest that the imPAC2 cells may faithfully represent AT2 populations that can be further explored to model pulmonary diseases.

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria.

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria's distribution, fusion, and fission events without staining the cells.

Abstract 1376: Dysfunctional and tolerant CD8 T cell persistence in premalignant lesions

Abstract T cells have the potential to carry out potent anti-tumor responses. Most studies examining T cell-cancer interactions have been directed toward late-stage tumors, but much less is known about how T cells respond to premalignant lesions. Using a clinically relevant liver cancer mouse model (Albumin-Stop-Large T Antigen; AST) in which the SV40 Large T Antigen (TAG) serves as oncogene and tumor neoantigen, we previously demonstrated that TAG-specific CD8 T cells (TCR-TAG) rapidly become dysfunctional (unable to produce cytokines and cytolytic molecules) after entering premalignant liver lesions, although they persist long-term. We hypothesized that peripheral tolerance mechanisms, which prevent autoimmunity against self-antigens, drive T cell dysfunction in premalignant lesions. To test this, we compared CD8 T cell (TCR-GAG) responses to a liver self-antigen (GAG) to TCR-TAG responses to the TAG neoantigen in premalignant hepatocytes. We found that liver-reactive TCR-GAG were unable to produce cytokines, similar to neoantigen-specific TCR-TAG; however, in contrast, they failed to persist. To test whether oncogenic transformation of hepatocytes promoted T cell persistence, we crossed Alb-GAG and AST mice to generate a model in which GAG and TAG was expressed in premalignant hepatocytes (ASTxAlb-GAG). We found that both TCR-GAG and TCR-TAG transferred into ASTxAlb-GAG mice bearing premalignant lesions driven by the TAG oncogene rapidly lost the ability to produce cytokines; however, only TCR-TAG persisted while TCR-GAG quickly disappeared. To understand why the TCR-GAG failed to persist, we assessed proliferation and cell cycling after T cells encountered their cognate antigen. While both TCR-GAG and TCR-TAG were rapidly activated and began proliferating, TCR-GAG exited cell cycle and began decreasing in number after four cell divisions, while TCR-TAG continued to divide and increase in number. Antigen amount and localization may regulate T cell proliferation and persistence. We found much higher levels of TAG-encoding versus GAG-encoding mRNA in the liver, suggesting that higher antigen expression may be required for T cell persistence. T cell responses can be directed against both shared tumor self-antigens and neoantigens. By dissecting the determinants of CD8 T cell function and persistence in response to self and non-self-antigens, these studies could inform strategies to boost anti-tumor CD8 T cell responses in cancer patients. Citation Format: Jessica J. Roetman, Megan M. Erwin, Minna K. Apostolova, Mary Philip. Dysfunctional and tolerant CD8 T cell persistence in premalignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1376.

Abstract 1336: Characterization of a novel transplantable model of obesity-driven basal-like breast cancer

Abstract Introduction: Breast cancer (BC) is a leading cause of cancer-related death in women, with the poorest prognosis found in the basal-like subtype. Although obesity is an established risk factor for basal-like BC, existing mouse models to study the interaction of basal-like BC and diet-induced obesity (DIO) are limited. The C3(1)/SV40 large T-antigen (TAg) transgenic mouse is an established, human-relevant basal-like mammary cancer model developed on an obesity-resistant FVB genetic background. The goal of this study was to create and characterize a transplantable C3TAg model on an obesity-sensitive C57BL/6J (B6) genetic background. Methods: Male C3TAg transgenic FVB mice were backcrossed with female wild-type B6 mice for 10 generations. Spontaneous mammary tumors from these female B6:C3TAg mice were dissociated and subcloned, generating 4 distinct cell lines differing in their expression of metabolic genes. A pilot orthotopic transplant study using the 4 candidate cell lines (at 3 cell concentrations of each line) in 96 B6 mice identified cell line 2.51 as an attractive model for further evaluation based on in vivo growth characteristics and initial genomic analyses. To assess the impact of DIO in this model, 61 female B6 mice were randomized to receive either control (n=30) or DIO (n=31) diet regimens for 24 weeks, then were orthotopically injected into the 4th mammary fat pad with cell line 2.51 (2.5x105 cells/mouse). The mice continued on their respective diets, were monitored for tumor development, and were euthanized 4 weeks post-injection. Tumors were collected, weighed and flash frozen for genomic, immunologic and biochemical analyses. Results: All 4 cell lines tested in the pilot study generated basal-like mammary tumors when orthotopically transplanted. Tumor gene expression profiling (via Affymetrix microarray) revealed inflammation and stem-like gene expression profiles discordant between cell lines tested, with tumors induced by cell line 2.51 showing striking enrichment in immune signatures relative to the other cell lines. DIO, relative to control diet, significantly accelerated transplanted 2.51 cell tumor progression, as indicated by increased tumor mass in mice from DIO relative to control diet-fed animals. Genomic, biochemical, and immunological assessment of the tumor-enhancing effects of DIO in this model are ongoing. Conclusions: Herein, we report the development and characterization of a novel transplantable C3TAg mammary tumor model in C57BL/6J mice, highly responsive to energy balance. This work represents a promising new tool for preclinical studies of the interactions between diet, obesity, immunity and basal-like breast cancer. Funding: This work was supported by R35CA197627 to SDH. Citation Format: Meredith S. Carson, Elaine M. Glenny, Violet A. Kiesel, Ashlee Taylor, Daniel Roth, Jody Albright, Melissa VerHague, John E. French, Michael F. Coleman, Stephen D. Hursting. Characterization of a novel transplantable model of obesity-driven basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1336.

A novel cell line from human eccrine sweat gland duct cells for investigating sweating physiology.

Human eccrine sweat glands (eSG) represent vital components of the skin involved in regulating body temperature. Especially the eccrine duct, which opens directly into the skin surface and releases the aqueous sweat, constitutes the first contact point with topically applied substances. For scientific investigations and to understand the underlying sweating mechanism on a cellular level defined cellular material is beneficial. We, therefore, strived to generate a cell line derived from human eSG duct cells for identifying new mechanisms in sweating control, as such a standardized cell line is currently lacking. Isolated primary human eSG duct cells were transduced with simian virus 40large T antigen (SV40T) by lentiviral transduction. Successfully SV40T-transduced clones were selected by single-cell cloning with one clone, named 1D10, being particularly described in this work. In performed cellular investigations, SV40T-transduced duct-derived cells exhibited an extended lifespan with stable population doubling times suggesting its immortality. Besides, 1D10clonal cell culture demonstrated similarities with parental, primary duct cells regarding gene expression of selected sweat gland-related markers. When combined with primary coil cells in a hanging drop co-culture, those transduced duct-derived cells showed some duct cell-like features. Further, a certain degree of cellular communication and a specific reaction towards substance application was observed. Generated and herein described cell line derived from isolated human eSG duct cells is, based on the presented scientific findings, considered as immortal. Besides, this cell line shows some similarity with primary duct cells, although alterations from native glands were detected, among which is loss of expression of cystic fibrosis transmembrane conductance regulator. Provided some further investigations, presented SV40T-transduced duct-cell derived cell line seems a suited surrogate of primary eccrine duct cells.

Generation of reproductive transgenic pigs of a CRISPR-Cas9-based oncogene-inducible system by somatic cell nuclear transfer.

Alternative cancer models that are close to humans are required to create more valuable preclinical results during oncology studies. Here, a new onco-pig model via developing a CRISPR-Cas9-based Conditional Polycistronic gene expression Cassette (CRI-CPC) system to control the tumor inducing simian virus 40 large T antigen (SV40LT) and oncogenic HRASG12V . After conducting somatic cell nuclear transfer (SCNT), transgenic embryos were transplanted into surrogate mothers and five male piglets were born. Umbilical cord analysis confirmed that all piglets were transgenic. Two of them survived and they expressed a detectable green fluorescence. The test was made whether CRI-CPC models were naturally fertile and whether the CRI-CPC system was stably transferred to the offspring. By mating with a normal female pig, four offspring piglets were successfully produced. Among them, only three male piglets were transgenic. Finally, their applicability was tested as cancer models after transduction of Cas9 into fibroblasts from each CRI-CPC pig in vitro, resulting in cell acquisition of cancerous characteristics via the induction of oncogene expression. These results showed that our new CRISPR-Cas9-based onco-pig model was successfully developed.

Inhibition of T-antigen expression promoting glycogen synthase kinase 3 impairs merkel cell carcinoma cell growth.

Merkel cell carcinoma is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV). Since proliferation of MCPyV-positive MCC tumor cells strictly depends on expression of the virus-encoded T antigens (TA), these proteins theoretically represent ideal targets for different kinds of therapeutic approaches. Here we developed a cell-based assay to identify compounds which specifically inhibit growth of MCC cells by repressing TA expression. Applying this technique we screened a kinase inhibitor library and identified six compounds targeting glycogen synthase kinase 3 (GSK3) such as CHIR99021 as suppressors of TA transcription in MCC cells. Involvement of GSK3α and -β in the regulation of TA-expression was confirmed by combining GSK3A knockout with inducible GSK3B shRNA knockdown since double knockouts could not be generated. Finally, we demonstrate that CHIR99021 exhibits in vivo antitumor activity in an MCC xenograft mouse model suggesting GSK3 inhibitors as potential therapeutics for the treatment of MCC in the future.

A low-carbohydrate diet containing soy protein and fish oil reduces breast but not prostate cancer in C3(1)/Tag mice.

We recently showed that a low-carbohydrate (CHO) diet containing soy protein and fish oil dramatically reduces lung nodules in a mouse model of lung cancer when compared to a Western diet. To explore the universality of this finding, we herein compared this low-CHO diet to a Western diet on in preventing breast and prostate cancer using a mouse model that expresses the SV40 large T-antigen specifically in breast epithelia in females and prostate epithelia in males. We found that breast cancer was significantly reduced with this low-CHO diet and this correlated with a reduction in plasma levels of glucose, insulin, IL-6, TNFα and prostaglandin E2 (PGE2). This also corresponded with a reduction in the Ki67 proliferation index within breast tumors. On the other hand, this low-CHO diet did not reduce the incidence of prostate cancer in the male mice. Although it reduced both blood glucose and insulin to the same extent as in the female mice, there was no reduction in plasma IL-6, TNFα or PGE2 levels, or in the Ki67 proliferation index in prostate lesions. Based on immunohistochemistry studies with antibodies to 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), carnitine palmitoyltransferase Ia (CPT1a) and fatty acid synthase (FAS), it is likely that this difference in response of the two cancer types to this low-CHO diet reflects differences in the glucose dependence of breast and prostate cancer, with the former being highly dependent on glucose for energy and the latter being more dependent on fatty acids.

Generation of Mesenchymal Cell Lines Derived from Aged Donors.

Background: Mesenchymal stromal cells (MSCs) have the capacity for self-renewal and multi-differentiation, and for this reason they are considered a potential cellular source in regenerative medicine of cartilage and bone. However, research on this field is impaired by the predisposition of primary MSCs to senescence during culture expansion. Therefore, the aim of this study was to generate and characterize immortalized MSC (iMSC) lines from aged donors. Methods: Primary MSCs were immortalized by transduction of simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Proliferation, senescence, phenotype and multi-differentiation potential of the resulting iMSC lines were analyzed. Results: MSCs proliferate faster than primary MSCs, overcome senescence and are phenotypically similar to primary MSCs. Nevertheless, their multi-differentiation potential is unbalanced towards the osteogenic lineage. There are no clear differences between osteoarthritis (OA) and non-OA iMSCs in terms of proliferation, senescence, phenotype or differentiation potential. Conclusions: Primary MSCs obtained from elderly patients can be immortalized by transduction of SV40LT and hTERT. The high osteogenic potential of iMSCs converts them into an excellent cellular source to take part in in vitro models to study bone tissue engineering.

Stem cells immortalized by hTERT perform differently from those immortalized by SV40LT in proliferation, differentiation, and reconstruction of matrix microenvironment

Although matrix microenvironment has the potential to improve expanded stem cell proliferation and differentiation capacity, decellularized extracellular matrix (dECM) deposited by senescent cells does not contribute to the rejuvenation of adult stem cells, which has become a barrier to personalized stem cell therapy. Genetic modification is an effective strategy to protect cells from senescence but it carries the increased risk of malignant transformation and genetic instability. In this study, lentivirus carrying either human telomerase reverse transcriptase (hTERT) or simian virus 40 large T antigen (SV40LT) was used to transduce human infrapatellar fat pad-derived stem cells (IPFSCs). We found that virus transduction modified the proliferative, chondrogenic, and adipogenic abilities of IPFSCs. Interestingly, dECM deposited by immortalized cells significantly influenced replicative senescent IPFSCs in proliferation and differentiation preference, the effect of which is hinged on the approach of immortalization using either SV40LT or hTERT. Our findings indicate both dECM expansion and immortalization strategies can be used for replicative senescent adult stem cells’ proliferation and lineage-specific differentiation, which benefits future stem cell-based tissue regeneration. This approach may also work for adult stem cells with premature senescence in elderly/aged patients, which needs further investigation. Statement of significanceAdult stem cells are a promising solution for autologous cell-based therapy. Unfortunately, cell senescence due to donor age and/or ex vivo expansion prevents clinical application. Recent progress with decellularized extracellular matrix provides a potential for the rejuvenation of senescent stem cells by improving their proliferation and differentiation capacities. Given the fact that the young matrix can provide a healthy and energetic microenvironment, in this study, two approaches using lentivirus transduction of hTERT and SV40LT were compared. The goal was to immortalize donor cells for deposition of decellularized extracellular matrix. The matrix was demonstrated to contribute diverging effects on the chondrogenic and adipogenic differentiation of expanded stem cells and exhibited proliferation benefits as well. These findings provide an invaluable asset for stem cell-based tissue regeneration.

Insulin-like growth factor-1 induces IRE1-XBP1–dependent endoplasmic reticulum biogenesis in bovine mammary epithelial cells

Insulin-like growth factor-1 (IGF-1) plays a key role in proliferation and galactopoiesis in mammary epithelial cells (MEC), but its definitive functions on endoplasmic reticulum (ER) during protein synthesis remain unknown. The present study aimed to elucidate the effects of IGF-1 on ER biogenesis in MEC in vitro and examined the expression of ER biogenesis-associated genes in the mammary gland during early lactation. We treated mammary alveolar cells-large T antigen cells (immortalized bovine MEC line established via stable transfection with simian virus-40 large T-antigen) with IGF-1 and examined ER biogenesis using the fluorescence intensity of an ER tracker and quantitative real-time PCR. We found IGF-1 significantly increased ER tracker staining and upregulated mRNA levels of ER biogenesis-related genes, such as CHKA (choline kinase α), PCYT1A (choline-phosphate cytidylyltransferase A), and SURF4 (surfeit locus protein 4). We focused on unfolded protein response to explore molecular mechanisms by which IGF-1 induces ER biogenesis. We found IGF-1 significantly increased mRNA levels of the XBP1 splicing form (XBP1s). Based on western blot analysis, IGF-1 induced the expression of (inositol-requiring kinase 1 α) protein, upstream of XBP1s, and phosphorylated-IRE1α. The inhibition of IRE1 endoribonuclease activity with 4-methylumbelliferone 8-carbaldehyde (4μ8C) significantly suppressed the increase in ER tracker fluorescence and ER biogenesis-related gene expression induced by IGF-1. Also, IGF-1-induced XBP1s and ER biogenesis-associated gene expression was inhibited by rapamycin, an inhibitor of mTORC1 (mammalian target of rapamycin complex 1), indicating that IRE1-XBP1 activation by IGF-1 is mediated by mTORC1. Moreover, to clarify the expression of XBP1s and ER biogenesis-associated genes expression under normal physiological conditions, mammary gland tissue from biopsies of dairy cows during late gestation and lactation were analyzed. In vivo data highlighted the significant increases in the mRNA levels of XBP1s and ER biogenesis-related genes in mammary gland tissue immediately after calving through 6 wk of lactation. The mRNA levels of IGF1R (IGF-1 receptor) in mammary glands increased during 6 wk of lactation. Therefore, the present study indicated for the first time that IGF-1 induces ER biogenesis by activating the IRE1-XBP1 axis under the regulation of mTORC1 in bovine MEC line.

Abstract 2918: Characterization of a novel transplantable C3TAg breast cancer model in C57BL/6J mice

Abstract Introduction: In women in the United States, breast cancer (BC) is the second leading cause of cancer-related death. BC is a heterogeneous disease, characterized by several subtypes based on molecular markers including several hormone receptors. Transcriptomic analysis further stratifies the disease. The basal-like subtype, often triple-negative for hormone receptors, has the poorest prognosis partially due to the absence of targeted therapies. Obesity, a preventable cause of cancer, is an established risk factor for breast cancer, including basal-like breast cancers. Mouse models to study the interaction of BC and obesity, particularly the more aggressive basal-like BC, are crucial. The C3(1)/SV40 large T-antigen (TAg) transgenic mouse model is an established model of basal-like mammary cancer that recapitulates several key features of human breast cancer. However, this model was generated in FVB mice, which is not an ideal genetic background for studying obesity, immunology and other aspects of cancer research. The goal of this study was to develop and characterize a C3TAg transgenic mouse model on a C57BL/6 background. Therefore, the current study backcrossed C3TAg transgenic FVB mice with C57BL/6 mice, a model most commonly used to study obesity, for 10 generations to create a transplantable C3TAg model in C57BL/6 mice. Methods: Cell Line Generation: C3TAg transgenic FVB mice were backcrossed with C57BL/6 mice for 10 generations. Tumors from 6-month-old heterozygous female mammary tissue were harvested, dissociated, and subcloned by limited dilution. A total of 156 clonal lines were established from which a subset of 12 lines displaying differences in morphology, growth and expression of metabolic and EMT genes (by qPCR). In vivo: 96 female C57BL/6J mice were randomized into 12 groups (n=8/group) and orthotopically injected with three different cell densities (5*104, 1.5*105, 5*105 cells/mouse) from one of the four selected lines. Mice tumor growth was assessed using electronic calipers. In vitro: Proliferation and cellular respiration were assessed and molecular subtyping was conducted using Affymetrix microarray data. Results: All tested cell lines formed tumors in vivo, with macroscopic characteristics of basal-like tumors including central necrosis. In vitro growth and metabolism of tested lines showed modest dissimilarity; in contrast, tumor growth rates and metastatic outgrowth were starkly dissimilar among the tested lines. Transcriptomic data clustered these cell lines with human BC tumors. Conclusions: Here we report for the first time a transplantable C3TAg model in C57BL/6 mice with highly effective tumor engraftment and growth, and with variable phenotypes for tumor growth and metastasis. Thus, this model may serve as an attractive model for the study of the interactions of basal-like breast cancer and obesity. This work was supported by R35CA197627 to SH. Citation Format: Meredith S. Carson, Michael F. Colman, Daniel Roth, Jody Albright, Melissa VerHague, John E. French, Stephen D. Hursting. Characterization of a novel transplantable C3TAg breast cancer model in C57BL/6J mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2918.

RABL6A Promotes Pancreatic Neuroendocrine Tumor Angiogenesis and Progression In Vivo.

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet β cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.

Interactions of large T-Antigen (LT) protein of polyomaviruses with p53 unfold their cancerogenic potential

Polyomaviruses such as Simian Virus 40 (SV40) and John Cunningham Virus (JCV) have been extensively studied for their potential role in aiding oncogenic transformation. One of the mechanisms through which they do this is by inactivating p53, a known tumor suppressor, through one of their viral proteins, large T-antigen (LT). However, these two viruses represent only a fraction of existing polyomaviruses. Using Clustal Omega, we aligned the protein sequences of LT for 12 different polyomaviruses and found high similarity across polyomavirus LT. We then utilized Molecular Operating Environment (MOE) v2019.01 to compare the binding of SV40 LT to p53 and p53 to DNA to more precisely define the mechanism with which SV40 LT inactivates p53. By binding to p53 residues essential to DNA binding, SV40 LT prevents the proper interaction of p53 with DNA and consequently its fulfillment of transcription factor functions. To further explore the possibility for other polyomavirus LT to do the same, we either retrieved existing 3D structures from RCSB Protein Data Bank or generated 3D homology models of other polyomavirus LT and modeled their interactions with p53. These models interacted with p53 in a similar manner as SV40 LT and provide further evidence of the potential of other polyomavirus LT to inactivate p53. This work demonstrates the importance of investigating the oncogenic potential of polyomaviruses and elucidates future targets for cancer treatment. Communicated by Ramaswamy H. Sarma