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Zika virus (ZIKV) is transmitted mostly via mosquito bites and no vaccine is available, so it may reemerge. We and others previously demonstrated that neonatal infection of ZIKV results in heart failure and can be fatal. Animal models implicated ZIKV involvement in viral heart diseases. It is unknown whether and how ZIKV causes heart failure in adults. Herein, we studied the effects of ZIKV infection on the heart function of adult A129 mice. First, we found that ZIKV productively infects the rat-, mouse-, or human-originated heart cell lines and caused ubiquitination-mediated degradation of and distortive effects on connexin 43 (Cx43) protein that is important for communications between cardiomyocytes. Second, ZIKV infection caused 100% death of the A129 mice with decreasing body weight, worsening health score, shrugging fur, and paralysis. The viral replication was detected in multiple organs. In searching for the viral effects on heart of the A129 mice, we found that ZIKV infection resulted in the increase of cardiac muscle enzymes, implicating a viral acute myocardial injury. ZIKV-caused heart injury was also demonstrated by electrocardiogram (ECG) showing widened and fragmented QRS waves, prolonged PR interval, and slower heart rate. The intercalated disc (ICD) between two cardiomyocytes was destroyed, as shown by the electronic microscopy, and the Cx43 distribution in the ICDs was less organized in the ZIKV-infected mice compared to that in the phosphate-buffered saline (PBS)-treated mice. Consistently, ZIKV productively infected the heart of A129 mice and decreased Cx43 protein. Therefore, we demonstrated that ZIKV infection caused heart failure, which might lead to fatal sequelae in ZIKV-infected A129 mice.

As the global elderly population grows, it is socioeconomically and medically critical to provide diverse and effective means of mitigating the impact of aging on human health. Previous studies showed that the adeno-associated virus (AAV) vector induced overexpression of certain proteins, which can suppress or reverse the effects of aging in animal models. In our study, we sought to determine whether the high-capacity cytomegalovirus vector (CMV) can be an effective and safe gene delivery method for two such protective factors: telomerase reverse transcriptase (TERT) and follistatin (FST). We found that the mouse cytomegalovirus (MCMV) carrying exogenous TERT or FST (MCMV TERT or MCMV FST ) extended median lifespan by 41.4% and 32.5%, respectively. We report CMV being used successfully as both an intranasal and injectable gene therapy system to extend longevity. Specifically, this treatment significantly improved glucose tolerance, physical performance, as well as preventing body mass loss and alopecia. Further, telomere shortening associated with aging was ameliorated by TERT and mitochondrial structure deterioration was halted in both treatments. Intranasal and injectable preparations performed equally well in safely and efficiently delivering gene therapy to multiple organs, with long-lasting benefits and without carcinogenicity or unwanted side effects. Translating this research to humans could have significant benefits associated with quality of life and an increased health span.

Circular RNAs (circRNAs) are a newly recognized component of the transcriptome with critical roles in autoimmune diseases and viral pathogenesis. To address the importance of circRNA in RNA viral transcriptome, we systematically identified and characterized circRNAs encoded by the RNA genomes of betacoronaviruses using both bioinformatical and experimental approaches. We predicted 351, 224, and 2764 circRNAs derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus, respectively. We experimentally identified 75 potential SARS-CoV-2 circRNAs from RNA samples extracted from SARS-CoV-2-infected Vero E6 cells. A systematic comparison of viral and host circRNA features, including abundance, strand preference, length distribution, circular exon numbers, and breakpoint sequences, demonstrated that coronavirus-derived circRNAs had a spliceosome-independent origin. We further showed that back-splice junctions (BSJs) captured by inverse reverse-transcription polymerase chain reaction have different level of resistance to RNase R. Through northern blotting with a BSJ-spanning probe targeting N gene, we identified three RNase R-resistant bands that represent SARS-CoV-2 circRNAs that are detected cytoplasmic by single-molecule and amplified fluorescence in situ hybridization assays. Lastly, analyses of 169 sequenced BSJs showed that both back-splice and forward-splice junctions were flanked by homologous and reverse complementary sequences, including but not limited to the canonical transcriptional regulatory sequences. Our findings highlight circRNAs as an important component of the coronavirus transcriptome, offer important evaluation of bioinformatic tools in the analysis of circRNAs from an RNA genome, and shed light on the mechanism of discontinuous RNA synthesis.

As the global elderly population grows, it is socioeconomically and medically critical to have diverse and effective means of mitigating the impact of aging on human health. Previous studies showed that adenovirus-associated virus (AAV) vector induced overexpression of certain proteins can suppress or reverse the effects of aging in animal models. Here, we sought to determine whether the high-capacity cytomegalovirus vector can be an effective and safe gene delivery method for two such-protective factors: telomerase reverse transcriptase (TERT) and follistatin (FST). We found that the mouse cytomegalovirus (MCMV) carrying exogenous TERT or FST (MCMVTERT or MCMVFST) extended median lifespan by 41.4% and 32.5%, respectively. This is the first report of CMV being used successfully as both an intranasal and injectable gene therapy system to extend longevity. Treatment significantly improved glucose tolerance, physical performance, and prevented loss of body mass and alopecia. Telomere shortening seen with aging was ameliorated by TERT, and mitochondrial structure deterioration was halted in both treatments. Intranasal and injectable preparations performed equally well in safely and efficiently delivering gene therapy to multiple organs, with long-lasting benefits and without carcinogenicity or unwanted side effects. Translating this research to humans could have significant benefits associated with increased health span.

Coronavirus possesses the largest RNA genome among all the RNA viruses. Its genome encodes about 29 proteins. Most of the viral proteins are non-structural proteins (NSP) except envelop (E), membrane (M), nucleocapsid (N) and Spike (S) proteins that constitute the viral nucleocapsid, envelop and surface. We have recently cloned all the 29 SARS-CoV-2 genes into vectors for their expressions in mammalian cells except NSP11 that has only 14 amino acids (aa). We are able to express all the 28 cloned SARS-CoV-2 genes in human cells to characterize their subcellular distributions. The proteins of SARS-CoV-2 are mostly cytoplasmic but some are both cytoplasmic and nuclear. Those punctate staining proteins were further investigated by immunofluorescent assay (IFA) using specific antibodies or by co-transfection with an organelle marker-expressing plasmid. As a result, we found that NSP15, ORF6, M and ORF7a are related to Golgi apparatus, and that ORF7b, ORF8 and ORF10 colocalize with endoplasmic reticulum (ER). Interestingly, ORF3a distributes in cell membrane, early endosome, endosome, late endosome and lysosome, which suggests that ORF3a might help the infected virus to usurp endosome and lysosome for viral use. Furthermore, we revealed that NSP13 colocalized with SC35, a protein standing for splicing compartments in the nucleus. Our studies for the first time visualized the subcellular locations of SARS-CoV-2 proteins and might provide novel insights into the viral proteins’ biological functions.

Circular RNAs (circRNAs) encoded by DNA genomes have been identified across host and pathogen species as parts of the transcriptome. Accumulating evidences indicate that circRNAs play critical roles in autoimmune diseases and viral pathogenesis. Here we report that RNA viruses of the Betacoronavirus genus of Coronaviridae, SARS-CoV-2, SARS-CoV and MERS-CoV, encode a novel type of circRNAs. Through de novo circRNA analyses of publicly available coronavirus-infection related deep RNA-Sequencing data, we identified 351, 224 and 2,764 circRNAs derived from SARS-CoV-2, SARS-CoV and MERS-CoV, respectively, and characterized two major back-splice events shared by these viruses. Coronavirus-derived circRNAs are more abundant and longer compared to host genome-derived circRNAs. Using a systematic strategy to amplify and identify back-splice junction sequences, we experimentally identified over 100 viral circRNAs from SARS-CoV-2 infected Vero E6 cells. This collection of circRNAs provided the first line of evidence for the abundance and diversity of coronavirus-derived circRNAs and suggested possible mechanisms driving circRNA biogenesis from RNA genomes. Our findings highlight circRNAs as an important component of the coronavirus transcriptome.SummaryWe report for the first time that abundant and diverse circRNAs are generated by SARS-CoV-2, SARS-CoV and MERS-CoV and represent a novel type of circRNAs that differ from circRNAs encoded by DNA genomes.

The M112-113 gene is the first early gene of the murine cytomegalovirus (MCMV), and its expression is activated by the immediate-early 3 (IE3) protein during MCMV infection in permissive cells. At its 5&prime, terminus, a 10-bp motif, upstream of the TATA box of the M112-113 gene, was identified to bind to IE3, and it is necessary for IE3 to activate M112-113 gene expression (Perez KJ et al. 2013 JVI). At the 3&prime, terminus of the M112-113 gene, three poly(A) signals (PASs) are arranged closely, forming a PAS cluster. We asked whether it is necessary to have the PAS cluster for the M112-113 gene and wondered which PAS is required or important for M112-113 gene expression. In this study, we mutated one, two, or all three PASs in expressing plasmids. Then, we applied bacterial artificial chromosome (BAC) techniques to mutate PASs in viruses. Gene expression and viral replication were analyzed. We found that not all three PASs are needed for M112-113 gene expression. Moreover, we revealed that just one of the three poly(A)s is enough for MCMV replication. However, the deletion of all three PASs did not kill MCMV, although it significantly attenuated viral replication. Finally, an mRNA stability assay was performed and demonstrated that PASs are important to stabilize M112-113 mRNA. Therefore, we conclude that just one of the PASs of the M112-113 gene is sufficient and important for MCMV replication through the stabilization of M112-113 mRNA.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has been associated with neuropathology in fetuses and adults, imposing a serious health concern. Therefore, the development of a vaccine is a global health priority. Notably, neutralization tests have a significant value for vaccine development and virus diagnosis. The cytopathic effect (CPE)-based neutralization test (Nt-CPE) is a common neutralization method for ZIKV. However, this method has some drawbacks, such as being time-consuming and labor-intensive and having low-throughput, which precludes its application in the detection of large numbers of specimens. To improve this problem, we developed a neutralization test based on an enzyme-linked immunospot assay (Nt-ELISPOT) for ZIKV and performed the assay in a 96-well format. A monoclonal antibody (mAb), 11C11, with high affinity and reactivity to ZIKV was used to detect ZIKV-infected cells. To optimize this method, the infectious dose of ZIKV was set at a multiplicity of infection (MOI) of 0.0625, and a detection experiment was performed after incubating for 24 h. As a result, under these conditions, the Nt-ELISPOT had good consistency with the traditional Nt-CPE to measure neutralizing titers of sera and neutralizing antibodies. Additionally, three neutralizing antibodies against ZIKV were screened by this method. Overall, we successfully developed an efficient neutralization test for ZIKV that is high-throughput and rapid. This Nt-ELISPOT can potentially be applied to detecting neutralizing titers of large numbers of specimens in vaccine evaluation and neutralizing antibody screening for ZIKV.

Zika virus (ZIKV) encodes a precursor protein (also called polyprotein) of about 3424 amino acids that is processed by proteases to generate 10 mature proteins and a small peptide. In the present study, we characterized the chemical features, suborganelle distribution and potential function of each protein using Flag-tagged protein expression system. Western blot analysis revealed the molecular weight of the proteins and the polymerization of E, NS1, and NS3 proteins. In addition, we performed multi-labeled fluorescent immunocytochemistry and subcellular fractionation to determine the subcellular localization of these proteins in host cells. We found that 1) the capsid protein colocalizes with 3 different cellular organelles: nucleoli, Golgi apparatus, and lipid droplet; NS2b and NS4a are associated with the Golgi apparatus; 2) the capsid and NS1proteins distribute in both cytoplasm and nucleus, NS5 is a nuclear protein; 3) NS3 protein colocalizes with tubulin and affects Lamin A; 4) Envelope, PrM, and NS2a proteins co-localize with the endoplasmic reticulum; 5) NS1 is associated with autophagosomes and NS4b is related to early endosome; 6) NS5 forms punctate structures in the nucleus that associate with splicing compartments shown by SC35, leading to reduction of SC35 protein level and trafficking of SC35 from the nucleus to the cytoplasm. These data suggest that ZIKV generates 10 functional viral proteins that exhibit distinctive subcellular distribution in host cells.

The centrosome is a cytoplasmic nonenveloped organelle functioning as one of the microtubule-organizing centers and composing a centriole center surrounded by pericentriolar material (PCM) granules. PCM consists of many centrosomal proteins, including PCM1 and centrosomal protein 131 (CEP131), and helps maintain centrosome stability. Zika virus (ZIKV) is a flavivirus of the family Flaviviridae whose RNA and viral particles are replicated in the cytoplasm. However, how ZIKV interacts with host cell components during its productive infection stage is incompletely understood. Here, using several primate cell lines, we report that ZIKV infection disrupts and disperses the PCM granules. We demonstrate that PCM1- and CEP131-containing granules are dispersed in ZIKV-infected cells, whereas the centrioles remain intact. We found that ZIKV does not significantly alter cellular skeletal proteins, and, hence, these proteins may not be involved in the interaction between ZIKV and centrosomal proteins. Moreover, ZIKV infection decreased PCM1 and CEP131 protein, but not mRNA, levels. We further found that the protease inhibitor MG132 prevents the decrease in PCM1 and CEP131 levels and centriolar satellite dispersion. Therefore, we hypothesized that ZIKV infection induces proteasomal PCM1 and CEP131 degradation and thereby disrupts the PCM granules. Supporting this hypothesis, we show that ZIKV infection increases levels of mind bomb 1 (MIB1), previously demonstrated to be an E3 ubiquitin ligase for PCM1 and CEP131 and that ZIKV fails to degrade or disperse PCM in MIB1-ko cells. Our results imply that ZIKV infection activates MIB1-mediated ubiquitination that degrades PCM1 and CEP131, leading to PCM granule dispersion.

To countermeasure the host cellular intrinsic defense, cytomegalovirus (CMV) and herpes simplex viruses (HSV) have evolved the ability to disperse nuclear domain 10 (ND10, aka PML body). However, mechanisms underlying their action on ND10 differ. HSV infection produces ICP0, which degrades the ND10-forming protein PML. Human CMV (HCMV) infection expresses IE1 that deSUMOylates PML to result in dispersion of ND10. It has been demonstrated that HSV ICP0 degraded only the SUMOylated PML, so we hypothesized that HCMV IE1 can protect PML from degradation by ICP0. HCMV IE1-expressing cell lines (U-251 MG-IE1 and HELF-IE1) were used for infection of HSV-1 or transfection of ICP0-expressing plasmid. Multilabeling by immunocytochemistry assay and protein examination by Western blot assay were performed to determine the resultant fate of PML caused by ICP0 in the presence or absence of HCMV IE1. Here, we report that deSUMOylation of human PML (hPML) by HCMV IE1 was incomplete, as mono-SUMOylated PML remained in the IE1-expressing cells, which is consistent with the report by E. M. Schilling, M. Scherer, N. Reuter, J. Schweininger, et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). As expected, we found that IE1 protected PML from degradation by ICP0 or HSV-1 infection. An in vitro study found that IE1 with mutation of L174P failed to deSUMOylate PML and did not protect PML from degradation by ICP0; hence, we conclude that the deSUMOylation of PML is important for IE1 to protect PML from degradation by ICP0. In addition, we revealed that murine CMV failed to deSUMOylate and to protect the HSV-mediated degradation of hPML, and that HCMV failed to deSUMOylate and protect the HSV-mediated degradation of mouse PML. However, IE1-expressing cells did not enhance wild-type HSV-1 replication but significantly increased ICP0-defective HSV-1 replication at a low multiplicity of infection. Therefore, our results uncovered a host-virus functional interaction at the posttranslational level. IMPORTANCE Our finding that HCMV IE1 protected hPML from degradation by HSV ICP0 is important, because the PML body (aka ND10) is believed to be the first line of host intrinsic defense against herpesviral infection. How the infected viruses overcome the nuclear defensive structure (PML body) has not been fully understood. Herpesviral proteins, ICP0 of HSV and IE1 of CMV, have been identified to interact with PML. Here, we report that HCMV IE1 incompletely deSUMOylated PML, resulting in the mono-SUMOylated PML, which is consistent with the report of Schilling et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). The mono-SUMOylated PML was subjected to degradation by HSV ICP0. However, it was protected by IE1 from degradation by ICP0 or HSV-1 infection. In contrast, IE1 with L174P mutation lost the function of deSUMOylating PML and failed to protect the degradation of the mono-SUMOylated PML. Whether the mono-SUMOylated PML has any defensive function against viral infection will be further investigated.

The human cytomegalovirus (HCMV) major immediate early (MIE) gene is essential for viral replication. The most abundant products encoded by the MIE gene include IE1 and IE2. Genes of IE1 and IE2 share the MIE promoter (MIEP), the first 3 exons, and the first 2 introns. IE1 is expressed earlier than IE2 after CMV infection or MIE gene transfection. In this study, we identified 2 polypyrimidine (Py) tracts in intron 4 (between exons 4 and 5) that are responsible for transcriptional switching from IE1 to IE2. The first Py is important and the second one is essential for the splicing and expression of IE2. In searching for the mechanisms of MIE gene switching from IE1 to IE2, we found that the second Py was required for the IE2's fourth intron to bind to a splicing factor such as U2AF65, as determined by an RNA electrophoretic mobility shift assay and a chromatin immunoprecipitation (ChIP) assay, while the first Py enhanced the binding of U2AF65 with the intron. An HCMV BACmid with the second Py mutated failed to produce any virus, while the HCMV with the first Py mutated replicated with a defective phenotype. Furthermore, we designed a small RNA (scRNAPy) that is complementary to the intron RNA covering the two Pys. The scRNAPy interfered with the interaction of U2AF65 with the intron and repressed the IE2 expression. Therefore, our studies implied that IE2 gene splicing might be an anti-CMV target. IMPORTANCE CMV is a ubiquitous herpesvirus and a significant cause of disease and death in the immunocompromised and elderly. Insights into its gene regulation will provide clues in designing anti-CMV strategies. The MIE gene is one of the earliest genes of CMV and is essential for CMV replication. It is known that the MIE gene needs to be spliced to produce more than two proteins; however, how MIE gene splicing is regulated remains elusive. In the present studies, we identified two Pys in intron 4 and found that the first Py is important and the second is required for the splicing and expression of IE2. We further investigated the mechanisms of gene switching from IE1 to IE2 and found that the two Pys are responsible for U2AF65's binding with intron 4. Therefore, the Pys in intron 4 are the cis elements that determine the fate of IE2 splicing. Furthermore, we found that a small RNA that is complementary to intron 4 repressed IE2 expression. Hence, we provide the first piece of evidence for a unique mechanism of MIE gene regulation at the splicing level.

Varicella-zoster virus (VZV) is the causative agent of chickenpox and herpes zoster (shingles). After the primary infection, the virus remains latent in sensory ganglia and reactivates upon weakening of the cellular immune system due to various conditions, erupting from sensory neurons and infecting the corresponding skin tissue. The current varicella vaccine is highly attenuated in the skin and yet retains its neurovirulence and may reactivate and damage sensory neurons. The factors involved in neuronal invasion and establishment of latency are still elusive. Previously, we constructed a library of whole-gene deletion mutants carrying a bacterial artificial chromosome sequence and a luciferase marker in order to perform a comprehensive VZV genome functional analysis. Here, screening of dispensable gene deletion mutants in differentiated neuronal cells led to the identification of ORF7 as the first known, likely a main, VZV neurotropic factor. ORF7 is a virion component localized to the Golgi compartment in infected cells, whose deletion causes loss of polykaryon formation in epithelial cell culture. Interestingly, ORF7 deletion completely abolishes viral spread in human nervous tissue ex vivo and in an in vivo mouse model. This finding adds to our previous report that ORF7 is also a skin-tropic factor. The results of our investigation will not only lead to a better understanding of VZV neurotropism but could also contribute to the development of a neuroattenuated vaccine candidate against shingles or a vector for delivery of other antigens.

The significance of epigenetic changes in the etiology and pathogenesis of endometriosis is being progressively recognized. However, the function of lysine-histone epigenetic modifications and furthermore, their role in the pathology of this disease remains unclear. We lead a study aimed to identify specific epigenetic profiles and evaluate possible associations with gene expression to provide insights about the pathological processes occurring in endometriosis. Objective: Identify the methylation pattern of lysine-histones H4K20me3 and H3K79me3 and chromatin modulation by gene activation in endometrial tissues. Methods: We used endometrial tissues from 9 women, with and without endometriosis. Macro-dissection was achieved by following pathology protocols. Tissues were homogenized and disaggregated using a bullet blender. Total histone extraction was carried out using an Epiquick Kit (Epigentek) and then, western blots (WB) were performed. Both, the H4K20me3 and H3K79me3 were detected in all endometrial t...

Lung cancer is a leading cancer killer, lung adenocarcinoma (AC) being its most common type. Several studies suggest that the frequency or nature of genetic events driving AC differ among ethnic populations. These differences impact treatment decisions as targeted agents are under development for a number of genes that drive lung cancer. For example, mutations in the epidermal growth factor receptor (EGFR) represent a driving event in a substantial number of AC patients that can be targeted with tyrosine kinase inhibitors. With the funding of a U54 Partnership grant between the Ponce School of Medicine in Ponce, PR and the Moffitt Cancer Center in Tampa, Florida we are constructing a molecular database for Puerto Rican Lung Cancer patients. Sample collection includes acquisition of fresh frozen and paraffin-embedded tissues via the U54-funded Puerto Rico tissue bank. DNA analysis of these tissues includes ancestry markers and exome sequencing of genes commonly altered in AC, including KRAS, TP53, EGFR, STK11, CDKN1C and RB1. Initial results suggest that the frequency of KRAS mutations in this population are significantly lower than those seen in a white mainland cohort, whereas the rate of EGFR and STK11 mutations is elevated. Since both EFGR and STK11 are potentially targetable mutations these data suggest that Puerto Rican AC patients would greatly benefit from genetic screening. Our U54 project also probes the role of the retinoblastoma protein (Rb) in lung cancer. Rb was the first tumor suppressor to be discovered, and yet its potency as a tumor suppressor remains only partially explained. We are interested in characterizing how Rb loss contributes to the aggressiveness of small cell lung carcinomas (SCLC). SCLC are poorly differentiated tumors with early proclivity for metastasis. Prognosis is poor with only 10-20% of patients achieving long-term disease-free intervals. Still, SCLC aggressivity remains to be molecularly explained. Interestingly, SCLC show high rates of Rb inactivation, which led us to hypothesize that in SCLC, Rb loss during early tumorigenesis facilitates both over-proliferation and early metastasis. Supporting our hypothesis, we found that the Rb-deficient SCLC cell line H187 lacks expression of E-cadherin while overexpressing N-cadherin, relative to Rb-expressing non-small cell lung carcinoma cell lines H1666 and H1650. We confirmed the Rb-positive status of H1666 and H1650 and the Rb-negative status of H187 by immunoblot. These results suggest that Rb loss in lung cancer cell lines provokes a cadherin switch indicative of an epithelial-to-mesenchymal transition. Consistently, we found overexpression of the mesenchymal marker vimentin in H187 relative to H1666 and H1650. When compared to H1666 and H1650, H187 lacked expression of the adherens junction component α-catenin and of the cytoskeletal adapter Eplin. Furthermore, H187 cells show up-regulated levels of Pak1, a Rac1 and Cdc42 effector whose up-regulation is associated to adherens junction disruption. Tiam-1, a Rac1 and Cdc42 activator, is also up-regulated in H187 relative to H1666 and H1650. Together, our data in lung cell lines suggests that Rb loss results in adherens junction alterations possibly due to transcriptional alteration of cell adhesion genes, aberrant regulation of the Rho GTPases Rac1 and Cdc42, and disruption of the cortical cytoskeleton. Based on our data, we propose that Rb loss in SCLC, not only leads to an increased proliferative capacity during early tumorigenesis, but also exacerbates early tumor aggressiveness by perturbing cellular adhesion, which facilitates tumor cell detachment and metastasis. Our ultimate goal is to link the genetic alterations we found in Puerto Ricans to Rb function, cell adhesion, and tumor aggressivity. Citation Format: Pedro G. Santiago-Cardona, Brienne E. Engel, Jonathan González-Flores, Ruth Cruz-Cosme, Teresita Muñoz-Antonia, W. Douglas Cress. Molecular biology of lung cancer in Puerto Ricans. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C19. doi:10.1158/1538-7755.DISP13-C19

Osteosarcomas (OS) and small cell lung carcinomas (SCLC) are poorly differentiated, highly aggressive tumors, with an early proclivity to metastasize. Prognosis is poor for both, with only 10-20% of patients achieving long-term disease-free intervals. Still, OS and SCLC aggressivity remain to be molecularly explained. Interestingly, these cancer types show high rates of inactivation of the RB1 gene coding for the retinoblastoma protein (Rb), a known cell cycle repressor. This observation led us to hypothesize that in OS and SCLC, Rb loss during early tumorigenesis facilitates both increased proliferation and an early tendency for metastasis. Supporting our hypothesis, our previous studies in osteoblasts showed that Rb transcriptionally regulates a wide repertoire of cell adhesion genes, including those coding for adherens junction cadherins and integrins. We also found that adhrens junction assembly requires the Rb-dependent transcriptional repression of Pak1, a Rac1-binding kinase that destabilizes cell adhesion when up-regulated. Thus, the early proclivity for metastasis of OS and SCLC may be related to perturbations in cell adhesion due to Rb loss. Here we present new data using the SCLC line H187 and the non-small cell lung carcinoma (NSCLC) cell lines H1975 and H520. SCLC carcinomas have high rates (> 90%) of Rb loss while NSCLC are known to be Rb wild type. Therefore, comparing H1975 and H520 vs. H187 provided us with an Rb-proficient vs. Rb-deficient system similar to the one we previously used to study Rb-engendered effects in osteoblasts. Our immunoblots and qRT-PCR showed that the Rb-null H187 cells expressed dramatically reduced levels of E-cadherin mRNA and protein relative to H1975 and H520, confirming that Rb loss impairs cell adhesion due to adherens junction loss. Interestingly, N-cadherin is up-regulated in H187 cells, suggesting that Rb loss triggers a cadherin switch similar to the one associated with epithelial-to-mesenchymal transitions. Other cell adhesion-related genes we found down-regulated in Rb-null H187 cells relative to H1975 and H520, as determined by qRT-PCR, were α-catenin, β-catenin, OB-cadherin, and the cytoskeletal adapter proteins eplin, vinculin, formin-1, and α-actinin. Our immunoblots also showed decreased levels of merlin, an adherens junction-interacting protein, in H187 cells. We also studied the expression of Rac1-binding proteins, given their involvement in cell adhesion. Our immunoblots and qRT-PCR showed reduced levels of IQGAP1 in H187 cells relative to H1975 and H520 cells. Given that IQGAP1 is a GTPase activating protein (GAP) that represses Rac1 by promoting its GDP-bound inactive state, we postulate that Rb represses Rac1 function via IQGAP1. Beta-2-chimaerin, another Rac1 GAP was also down regulated at the protein level in H187 cells. Taken together, our data in lung cell lines recapitulate most of our findings in osteoblasts, both in regards to the effect of Rb on cell adhesion and to the need for the Rb-mediated repression of Rac1 for the establishment of cell adhesion. Based on our data, we propose that Rb loss in OS and SCLC, not only leads to an increased proliferative capacity during early tumorigenesis, but also exacerbates aggressiveness of tumors at early stages by perturbing cellular adhesion, which in turn facilitates tumor cell detachment and metastasis. Citation Format: Jonathan González-Flores, Ruth Cruz-Cosme, Yariana Rodríguez-Ortiz, Sarah Brady, Ricardo Fraticelli-Rosado, Brienne Engel, Douglas Cress, Pedro G. Santiago-Cardona. Molecular mechanisms of aggressiveness of Rb-deficient tumors. [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 C12.