Oncogenic Transformation Research Articles

Immunotherapy guided precision medicine in solid tumors.

Cancer is no longer recognized as a single disease but a collection of diseases each with its defining characteristics and behavior. Even within the same cancer type, there can be substantial heterogeneity at the molecular level. Cancer cells often accumulate various genetic mutations and epigenetic alterations over time, leading to a coexistence of distinct subpopulations of cells within the tumor. This tumor heterogeneity arises not only due to clonal outgrowth of cells with genetic mutations, but also due to interactions of tumor cells with the tumor microenvironment (TME). The latter is a dynamic ecosystem that includes cancer cells, immune cells, fibroblasts, endothelial cells, stromal cells, blood vessels, and extracellular matrix components, tumor-associated macrophages and secreted molecules. The complex interplay between tumor heterogeneity and the TME makes it difficult to develop one-size-fits-all treatments and is often the cause of therapeutic failure and resistance in solid cancers. Technological advances in the post-genomic era have given us cues regarding spatial and temporal tumor heterogeneity. Armed with this knowledge, oncologists are trying to target the unique genomic, epigenetic, and molecular landscape in the tumor cell that causes its oncogenic transformation in a particular patient. This has ushered in the era of personalized precision medicine (PPM). Immunotherapy, on the other hand, involves leveraging the body's immune system to recognize and attack cancer cells and spare healthy cells from the damage induced by radiation and chemotherapy. Combining PPM and immunotherapy represents a paradigm shift in cancer treatment and has emerged as a promising treatment modality for several solid cancers. In this chapter, we summarise major types of cancer immunotherapy and discuss how they are being used for precision medicine in different solid tumors.

Hypoxia-induced epigenetic regulation of breast cancer progression and the tumour microenvironment.

The events that control breast cancer progression and metastasis are complex and intertwined. Hypoxia plays a key role both in oncogenic transformation and in fueling the metastatic potential of breast cancer cells. Here we review the impact of hypoxia on epigenetic regulation of breast cancer, by interfering with multiple aspects of the tumour microenvironment. The co-dependent relationship between oxygen depletion and metabolic shift to aerobic glycolysis impacts on a range of enzymes and metabolites available in the cell, promoting posttranslational modifications of histones and chromatin, and changing the gene expression landscape to facilitate tumour development. Hormone signalling, particularly through ERα, is also tightly regulated by hypoxic exposure, with HIF-1α expression being a prognostic marker for therapeutic resistance in ER+ breast cancers. This highlights the strong need to understand the hypoxia-endocrine signalling axis and exploit it as a therapeutic target. Furthermore, hypoxia has been shown to enhance metastasis in TNBC cells, as well as promoting resistance to taxanes, radiotherapy and even immunotherapy through microRNA regulation and changes in histone packaging. Finally, several other mediators of the hypoxic response are discussed. We highlight a link between ionic dysregulation and hypoxia signalling, indicating a potential connection between HIF-1α and tumoural Na+ accumulation which would be worth further exploration; we present the role of Ca2+ in mediating hypoxic adaptation via chromatin remodelling, transcription factor recruitment and changes in signalling pathways; and we briefly summarise some of the findings regarding vesicle secretion and paracrine induced epigenetic reprogramming upon hypoxic exposure in breast cancer. By summarising these observations, this article highlights the heterogeneity of breast cancers, presenting a series of pathways with potential for therapeutic applications.

The oncogene cyclin D1 promotes bipolar spindle integrity under compressive force.

The mitotic spindle is the bipolar, microtubule-based structure that segregates chromosomes at each cell division. Aberrant spindles are frequently observed in cancer cells, but how oncogenic transformation affects spindle mechanics and function, particularly in the mechanical context of solid tumors, remains poorly understood. Here, we constitutively overexpress the oncogene cyclin D1 in human MCF10A cells to probe its effects on spindle architecture and response to compressive force. We find that cyclin D1 overexpression increases the incidence of spindles with extra poles, centrioles, and chromosomes. However, it also protects spindle poles from fracturing under compressive force, a deleterious outcome linked to multipolar cell divisions. Our findings suggest that cyclin D1 overexpression may adapt cells to increased compressive stress, possibly contributing to its prevalence in cancers such as breast cancer by allowing continued proliferation in mechanically challenging environments.

Choline kinase and miR-32-5p: A crucial interaction promoting apoptosis and delaying wound repair in cervical cancer cells

<p>Cervical cancer remains a significant global health challenge, especially in low- and middle-income countries. The elucidation of the molecular pathways underlying the carcinogenesis of cervical cancer is vital to develop targeted therapies. Our study investigates the regulatory effects of miR-32-5p on the choline kinase alpha (<italic>chka</italic>) gene in HeLa cells, a well-established cervical cancer cell line. Choline kinase alpha is recognized for its role in phosphatidylcholine biosynthesis, which is crucial for cell membrane formation, and is implicated in the oncogenic transformation of cells. Utilizing a combination of in silico prediction, luciferase assays, RT-qPCR, and Western blot analyses, we demonstrated that miR-32-5p directly targets the 3′ untranslated region (3′UTR) of <italic>chka</italic> mRNA, leading to a significant downregulation of <italic>chka</italic> expression. Our results demonstrate that miR-32-5p significantly downregulates <italic>chka</italic> at both the mRNA and protein levels, thus leading to decreased cellular proliferation and increased apoptosis. This was further confirmed by a cell cycle analysis, which showed a notable arrest in the G0/G1 phase. Additionally, scratch assays indicated a reduced migratory capacity in miR-32-5p-transfected cells, suggesting the potential anti-metastatic properties of miR-32-5p. These findings highlight the therapeutic potential of miR-32-5p as a biomarker and a target in cervical cancer treatment strategies. By modulating <italic>chka</italic> expression, miR-32-5p could serve as a novel approach to curb the progression and spread of cervical cancer, thus offering a promising avenue for future research and clinical applications. This study contributes to the growing understanding of miRNA-mediated gene regulation in cancer biology and underscores the importance of targeted genetic research in the development of cancer therapeutics.</p>

Exosomes Derived from Metastatic Colon Cancer Cells Induced Oncogenic Transformation and Migratory Potential of Immortalized Human Cells

Tiny vesicles, synthesized from cell membranes through endocytosis, form multivesicular bodies (MVBs). These MVBs can either undergo lysosomal degradation or be released to the extracellular environment. The vesicle that ranges in size from 30-300 nm that were positive for markers such as CD63 or CD9 are known as exosomes. Exosomes are shown to contain various biological macromolecules such as DNA, RNA, miRNA, proteins, and lipids. Releasing exosomes from cells to the extracellular environment is an important strategy to transfer information between the cells. Exosomes from primary colon cancer cells have been shown to induce the oncogenic transformation of normal cells. In this study, we have demonstrated that treating normal cells (HEK) with exosomes derived from the metastatic colon cancer cell line (SW620) increases cell proliferation, allowing the normal cells to grow anchored independently, suggesting the oncogenic transformation of normal cells. In addition, there was an increase in migration and invasion properties of HEK cells exposed to exosomes from SW620 cells. Our results suggest that following the treatment with metastatic colon cancer cell-derived exosomes, the HEK cells underwent an oncogenic transformation and Epithelial-Mesenchymal Transition (EMT) that will allow the cancerous cells to metastasize.

MET exon 14 skipping mutation drives cancer progression and recurrence via activation of SMAD2 signalling.

c-Met encoded by the proto-oncogene MET, also known as hepatocyte growth factor (HGF) receptor, plays a crucial role in cellular processes. MET exon 14 skipping alteration (METΔ14EX) is a newly discovered MET mutation. SMAD2 is an important downstream transcription factor in TGF-β pathway. Unfortunately, the mechanisms by which METΔ14EX leads to oncogenic transformation are scarcely understood. The relationship between METΔ14EX and SMAD2 has not been studied yet. We generate METΔ14EX models by CRISPR-Cas9. In vitro transwell, wound-healing, soft-agar assay, in vivo metastasis and subcutaneous recurrence assay were used to study the role of METΔ14EX in tumour progression. RNA-seq, Western blotting, co-immunoprecipitation (CO-IP) and immunofluorescent were performed to explore the interaction between c-Met and SMAD2. Our results demonstrated that METΔ14EX, independent of HGF, can prolong the constitutive activation of c-Met downstream signalling pathways by impeding c-Met degradation and facilitating tumour metastasis and recurrence. Meanwhile, METΔ14EX strengthens the interaction between c-Met and SMAD2, promoting SMAD2 phosphorylation. Therapeutically, MET inhibitor crizotinib impedes METΔ14EX-mediated tumour metastasis by decreasing SMAD2 phosphorylation. These data elucidated the previously unrecognised role of METΔ14EX in cancer progression via activation of SMAD2 independent of TGF-β, which helps to develop more effective therapies for such patients. METΔ14EX alteration significantly triggers tumour progression via activation of SMAD2 signalling that are involved in activating tumour invasion, metastasis and recurrence. On the left, in the MET wild-type (METWT), the juxtamembrane (JM) domain is involved in the regulation of tyrosine kinase activity, receptor degradation, and caspase cleavage. On the right, the METΔ14EX mutation leads to the loss of the juxtamembrane domain, resulting in an abnormal MET protein lacking a CBL-binding site. This causes the accumulation of truncated MET receptors followed by constitutive activation of the MET signalling pathway. Thus, the METΔ14EX-mutated protein has strong binding and phosphorylation to SMAD2, which results in the phosphorylation of a large number of SMAD2/3 proteins that combine with SMAD4 to form a complex in the nucleus, activating downstream signalling pathways, such as EMT and ECM remodelling, resulting in tumour progression and recurrence. TF transcription factor.

Transcriptomic analysis of neural stem and progenitor cells in comparison with glioblastoma stem cells

Introduction. There is currently no effective therapy for the treatment of glioblastoma. This is partly explained by the high degree of intra- and intertumor heterogeneity of GB, the source of which is believed to be glioblastoma stem cells (GSC). The question of the origin of GSC, which is important for improving clinical outcomes, still remains open. It is believed that GSCs can be formed as a result of oncogenic transformation of neural stem and progenitor cells (NSPcs), which have morphological and functional properties similar to them. Despite significant progress in elucidating the nature of GSCs, little is yet known about the specifically expressed genes and transcripts in these cells in comparison with NSPcs. In this regard, it becomes relevant to study the molecular mechanisms of gliomagenesis using model cell systems based on various clones of GSC.Aim. To conduct a comparative transcriptomic analysis of CD133+-NSPCs and CD133+-GSCs to study the molecular genetic differences between the phenotypes of these cells and identify potential targets for therapeutic effects on GSCs.Materials and methods. Used: highly sensitive transcriptomic analysis on high-density microarrays, cellular technologies, modern bioinformatics analysis.Results. Transcriptomic analysis of CD133+-GSCs and CD133+-NSPCs identified 1825 differentially expressed genes. The biological processes and signaling cascades activated in CD133+-GSCs have been established. It was shown that significant transcriptomic aberrations in CD133+-GSC compared to CD133+-NSPC are primarily due to a group of transcripts regulated by the Shh (Sonic hedgehog), mTOR (mammalian target of rapamycin), ALK (anaplastic lymphoma kinase) signaling cascades, transcription factors E2F1, PRC2, HOXA9, MYC, as well as oncogenes ERBB2 and KRAS. Six transcripts (AQP9, TOX15, HOXB2, STEAP3, TREM1, RFC2) highly expressed in CD133+-GSC and closely associated with the survival of patients with glioblastoma, which may be potential targets for therapeutic effects on CD133+-GSC associated with gliomagenesis, which may be potential targets for therapeutic effects on CD133+-GSC, have been identified and annotated.Conclusion. The data obtained indicate a number of significant molecular genetic differences between the two cell phenotypes, which can be used in the development of new therapeutic approaches for the treatment of glioblastoma.

The development of a hiPSC-based platform to identify tissue-dependencies of IDH1 R132H

The application of patient-derived (PD) in vitro tumor models represents the classical strategy for clinical translational oncology research. Using these cellular heterogeneous cultures for the isolation of cancer stem cells (CSCs), suggested to be the main driver for disease malignancy, relies on the use of surrogate biomarkers or is based on CSC-enriching culture conditions. However, the ability of those strategies to exclusively and efficiently enrich for CSC pool has been questioned. Here we present an alternative in vitro CSC model based on the oncogenic transformation of single clone-derived human induced pluripotent stem cells (hiPSC). Hotspot mutations in the DNA encoding for the R132 codon of the enzyme isocitrate dehydrogenase 1 (IDH1) and codon R175 of p53 are commonly occurring molecular features of different tumors and were selected for our transformation strategy. By choosing p53 mutant glial tumors as our model disease, we show that in vitro therapy discovery tests on IDH1-engineered synthetic CSCs (sCSCs) can identify kinases-targeting chemotherapeutics that preferentially target tumor cells expressing corresponding genetic alteration. In contrast, neural stem cells (NSCs) derived from the IDH1R132H overexpressing hiPSCs increase their resistance to the tested interventions indicating glial–to-neural tissue-dependent differences of IDH1R132H. Taken together, we provide proof for the potential of our sCSC technology as a potent addition to biomarker-driven drug development projects or studies on tumor therapy resistance. Moreover, follow-up projects such as comparing in vitro drug sensitivity profiles of hiPSC-derived tissue progenitors of different lineages, might help to understand a variety of tissue-related functions of IDH1 mutations.

Role of the Mitochondrial E3 Ubiquitin Ligases as Possible Therapeutic Targets in Cancer Therapy.

Ubiquitination is a post-translational modification that targets specific proteins on their lysine residues. Depending on the type of ubiquitination, this modification ultimately regulates the stability or degradation of the targeted proteins. Ubiquitination is mediated by three different classes of enzymes: the E1 ubiquitin-activating enzymes, the E2 ubiquitin-conjugating enzymes and, most importantly, the E3 ubiquitin ligases. E3 ligases are responsible for the final step of the ubiquitin cascade, interacting directly with the target proteins. E3 ligases can also be involved in DNA repair, cell cycle regulation and response to stress; alteration in their levels can be involved in oncogenic transformation and cancer progression. Of all the six hundred E3 ligases of the human genome, only three of them are specific to the mitochondrion: MARCH5, RNF185 and MUL1. Their alterations (that reflect on the alteration of the mitochondria functions) can be related to cancer progression, as underlined by the increasing research performed in recent years on these three mitochondrial enzymes. This review will focus on the function and mechanisms of the mitochondrial E3 ubiquitin ligases, as well as their important targets, in cancer development and progression, also highlighting their potential use for cancer therapy.

RNA-binding proteins regulating the CD44 alternative splicing.

Alternative splicing is often deregulated in cancer, and cancer-specific isoform switches are part of the oncogenic transformation of cells. Accumulating evidence indicates that isoforms of the multifunctional cell-surface glycoprotein CD44 play different roles in cancer cells as compared to normal cells. In particular, the shift of CD44 isoforms is required for epithelial to mesenchymal transition (EMT) and is crucial for the maintenance of pluripotency in normal human cells and the acquisition of cancer stem cells phenotype for malignant cells. The growing and seemingly promising use of splicing inhibitors for treating cancer and other pathologies gives hope for the prospect of using such an approach to regulate CD44 alternative splicing. This review integrates current knowledge about regulating CD44 alternative splicing by RNA-binding proteins.

Comprehensive Assessment of Albumin and Uric Acid Levels in Oral Submucous Fibrosis: A Comparative Case-Control Study Involving Different Risk Groups.

Background Oral submucous fibrosis (OSMF) is a premalignant disorder that impacts the oral cavity and pharynx. Major risk factors for OSMF are attributed to the consumption of betel nuts or tobacco. These substances harbor various carcinogens that trigger the production of free radicals and reactive oxygen species. Antioxidants are pivotal in preserving cellular integrity and impeding the oncogenic transformation of body cells. In this context, albumin and uric acid, being primary antioxidants present in body fluids, bestow a defensive effect against this condition. Thus, the present study is designed to elucidate the differential concentration of albumin and uric acid between patient cases and healthy cohorts. Methodology This case-control study was conducted to evaluate the albumin and uric acid levels in individuals diagnosed with OSMF (cases) and compare them with healthy controls for a period of six months. A cohort of 100 individuals was partitioned into four groups, with each group comprising 25 individuals: Group I was made up of healthy individuals; Group II consisted of individuals who chew tobacco and areca nuts but are not affected by OSMF; Group III included individuals who only use tobacco without areca nuts and are afflicted with OSMF; and Group IV comprised individuals who consume a combination of areca nuts and tobacco and are diagnosed with OSMF. Biochemical evaluation was carried out using the BS-380 chemistry autoanalyzer (Mindray, Shenzhen, China), and the quantification of serum albumin and uric acid was performed by the uricase-peroxidase (POD) method with dihydroxybenzene sulfonic acid (DHBS). Results The study cohort of 100 individuals was made up of 70 males and 30 females, with an average age of 42.51 (11.62) years. The comparison of the mean concentration of serum albumin across all groups revealed that healthy controls exhibited the highest serum albumin concentration of 4.284 (0.618), with a statistically significant p-value (0.001) across all groups. A comparison of the mean value of serum uric acid among all groups showed that healthy controls had the highest value of serum uric acid (5.26±1.161), with a considerable p-value (0.001) between all groups. Conclusion The present study concluded that serum biomarkers assessed were high in healthy individuals and consumption of areca nuts, tobacco, and their products was significantly associated with low levels of albumin and uric acid. Therefore, both albumin and uric acid can be used as important biomarkers for uncovering oral premalignant lesions and conditions ahead of time and can also be used in mass screening.

The Extracellular Matrix Dictates Regional Differences in Tumor Initiation.

Epidermal cells in different body regions are not equally sensitive to oncogenic transformation.

A reversible epigenetic memory of inflammatory injury controls lineage plasticity and tumor initiation in the mouse pancreas

Inflammation is essential to the disruption of tissue homeostasis and can destabilize the identity of lineage-committed epithelial cells. Here, we employ lineage-traced mouse models, single-cell transcriptomic and chromatin analyses, and CUT&TAG to identify an epigenetic memory of inflammatory injury in the pancreatic acinar cell compartment. Despite resolution of pancreatitis, our data show that acinar cells fail to return to their molecular baseline, with retention of elevated chromatin accessibility and H3K4me1 at metaplasia genes, such that memory represents an incomplete cell fate decision. Invivo, we find this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second insult but increased tumorigenesis with an oncogenic Kras mutation. The lowered threshold for oncogenic transformation, in turn, can be restored by blockade of MAPK signaling. Together, we define the chromatin dynamics, molecular encoding, and recall of a prolonged epigenetic memory of inflammatory injury that impacts future responses but remains reversible.

RAD51 Is Required for the Occupancy of Replication Licensing Factors on the Myeloma Genome

Genomic instability and extended replicative potential arise early during oncogenic transformation and contribute to the development and progression of cancer. Although chromosomal instability and replication are distinct pathways, they share certain proteins and activities. DNA replication starts by sequential loading of replication licensing factors at replication origins. We have observed that RAD51 - a keystone protein in stabilizing the genome through homologous recombination (HR) and a dependency in multiple myeloma (MM) - interacts with the MCM complex. In fact, RAD51 knockdown inhibits DNA replication in MM cells. Because RAD51 organizes the initial steps of homologous recombination, we investigated whether RAD51 plays a similar setup role in replication - namely, that RAD51 is required for the occupancy or proper assembly of replication licensing factors (ORC1-ORC6, CDC6, CDT1, MCM2-7) on the MM genome. To investigate this, we either inhibited (by knockdown or inhibitor) or overexpressed RAD51 in MM cells and monitored the occupancy of the replication complex on genomic DNA using ChIP with an antibody against MCM4. Subsequently, the proteins in the complex were released from DNA and identified by Western blotting. Although RAD51-KD (or inhibition) did not reduce the expression or stability of licensing factors, the occupancy of all these factors (MCM4, MCM2, MCM5, MCM6, MCM7, CDC6) on the MM genome was inhibited. Consistently, RAD51-overexpression led to an increase in the genomic occupancy of most of the licensing factors (ORC1, ORC2, MCM4, MCM2, MCM5, MCM6, MCM7, CDC6) tested. This was validated using antibodies against other members of the complex i.e., ORC1 and CDC6. Overall, we demonstrate that RAD51 in MM cells interacts with components of the replicative protein complexes (ORC1-ORC6, CDC6, CDT1, MCM2-7) that license and ensure DNA replication once per cell cycle. Our data also demonstrate that RAD51 is required for the occupancy of most of these factors on genomic DNA in MM cells, which may explain its ability to impact DNA replication in MM cells. We are currently trying to understand if RAD51 brings one or more of these factors to the origin of replication and if some of its activities (such as ATPase, DNA binding) are involved in loading these factors onto DNA. Based on its impact both on replication and HR, RAD51 is an attractive target in myeloma. Our inhibitor studies provide further rationale to study therapeutic strategy to target RAD51 in MM.

Overcoming Venetoclax Resistance in Leukemia with AI-Designed RNA Epigenetic Inhibitors

Background : Venetoclax resistance is a major obstacle to successful treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Its underlying mechanisms are multifaceted and have been linked to monocytic differentiation, reprograming mitochondrial metabolism, and increased expression of BRD4 and other BCL2-like proteins, etc. Our previous studies demonstrated that specific RNA cytosine methyltransferase, namely NSUN1 (aka NOL1, NPO2) and NSUN2, mediate the formation of drug-resistant active chromatin structures (ACS) at nascent RNAs to regulate drug resistance in leukemia cells (Cheng, J.X. et al. Nat Commun, 2018) ( Figure 1A). The Cancer Genome Atlas Program (TCGA) data demonstrated that elevated expression of NSUN1 and NSUN2 is correlated with significantly shorter patients' survival in almost all cancer subtypes. However, due to the lack of the crystal structures of NSUN1 and NSUN2 proteins, no NSUN1/2-targeting therapeutics have been developed yet. Methods: AI/supercomputing design of NSUN1/2-targeting drug libraries and analysis of structure activity relationship (SAR). Newly developed functional technologies, including NSUN1/2-targeting fluorescence releasing assay (FRA), 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). NSUN1/2-targeting functional drug screening (NSUN1/2-FDS) assay and drug growth inhibition (MTT) assay. H&E histology and immunohistochemistry (IHC). Enforced NSUN1/2 expression and knockout (KO) cell lines and syngeneic AML mouse models. Results: Identification of several ligan-binding modules/surfaces in NSUN1 and NSUN2 proteins and generation of small-molecule libraries to targe these ligand-binding surfaces/modules with AI/supercomputing structural prediction and ligand-receptor interaction simulation. Identification of several effective small-molecule inhibitors of NSUN1 and NSUN2 by functional screening of the AI-designed drug libraries in venetoclax-resistant human monocytic leukemia (SC), erythroid leukemia (K562) and lung cancer (H82) cells. Defining the binding modules of the identified NSUN1/2 inhibitors and selecting lead inhibitors through supercomputer/AI-directed SAR analysis. Elucidation of the action mechanisms of NSUN1/2 lead inhibitors thorough disruption of NSUN1/2-mediated ACS in the nucleus and translational complexes (TLC) in the ribosomes and mitochondria ( Figure 1A). In vivo validation of the efficacies of the identified lead inhibitors using a syngeneic AML mouse model. As shown in Figure 1B, NSUN1-17, one of the lead inhibitors, efficiently eliminated the AML cells/blasts from the blood vessels and parenchyma of the lungs of the syngeneic AML mice. In addition, our siRNA knock-down experiments demonstrated that downregulation of NSUN1/2 expression re-sensitizes venetoclax-resistant monocytic leukemia cells to a low dose of venetoclax. Our NIH/3T3 soft-agarose assay confirmed the oncogenic transformation capability of NSUN1 and NSUN2. We have also successfully established the homozygous ( Nsun2-/-) mouse model with markedly reduced body weight, but a normal lifespan, compared to the heterozygous mice ( Nsun2+/-). Conclusion: By leveraging the Argonne AI/supercomputer and our functional assays, we identified several small-molecule inhibitors that can disrupt the NSUN1/2-mediated ACS and TLC to overcome venetoclax resistance in AML cell lines and syngeneic mice. This study has not only illustrated the importance of NSUN1 and NSUN2 in maintaining oncogenic transcription and translation as well as venetoclax resistance in AML, but also paved the way to the development of novel RNA epigenetics-driven therapeutics for leukemia and other cancers.

Mitochondrial influences on smooth muscle phenotype.

Smooth muscle cells transition reversibly between contractile and noncontractile phenotypes in response to diverse influences, including many from mitochondria. Numerous molecules including myocardin, procontractile miRNAs, and the mitochondrial protein prohibitin-2 promote contractile differentiation; this is opposed by mitochondrial reactive oxygen species (mtROS), high lactate concentrations, and metabolic reprogramming induced by mitophagy and/or mitochondrial fission. A major pathway through which vascular pathologies such as oncogenic transformation, pulmonary hypertension, and atherosclerosis cause loss of vascular contractility is by enhancing mitophagy and mitochondrial fission with secondary effects on smooth muscle phenotype. Proproliferative miRNAs and the mitochondrial translocase TOMM40 also attenuate contractile differentiation. Hypoxia can initiate loss of contractility by enhancing mtROS and lactate production while simultaneously depressing mitochondrial respiration. Mitochondria can reduce cytosolic calcium by moving it across the inner mitochondrial membrane via the mitochondrial calcium uniporter, and then through mitochondria-associated membranes to and from calcium stores in the sarcoplasmic/endoplasmic reticulum. Through these effects on calcium, mitochondria can influence multiple calcium-sensitive nuclear transcription factors and genes, some of which govern smooth muscle phenotype, and possibly also the production of genomically encoded mitochondrial proteins and miRNAs (mitoMirs) that target the mitochondria. In turn, mitochondria also can influence nuclear transcription and mRNA processing through mitochondrial retrograde signaling, which is currently a topic of intensive investigation. Mitochondria also can signal to adjacent cells by contributing to the content of exosomes. Considering these and other mechanisms, it is becoming increasingly clear that mitochondria contribute significantly to the regulation of smooth muscle phenotype and differentiation.

VIPpred: a novel model for predicting variant impact on phosphorylation events driving carcinogenesis.

Disrupted protein phosphorylation due to genetic variation is a widespread phenomenon that triggers oncogenic transformation of healthy cells. However, few relevant phosphorylation disruption events have been verified due to limited biological experimental methods. Because of the lack of reliable benchmark datasets, current bioinformatics methods primarily use sequence-based traits to study variant impact on phosphorylation (VIP). Here, we increased the number of experimentally supported VIP events from less than 30 to 740 by manually curating and reanalyzing multi-omics data from 916 patients provided by the Clinical Proteomic Tumor Analysis Consortium. To predict VIP events in cancer cells, we developed VIPpred, a machine learning method characterized by multidimensional features that exhibits robust performance across different cancer types. Our method provided a pan-cancer landscape of VIP events, which are enriched in cancer-related pathways and cancer driver genes. We found that variant-induced increases in phosphorylation events tend to inhibit the protein degradation of oncogenes and promote tumor suppressor protein degradation. Our work provides new insights into phosphorylation-related cancer biology as well as novel avenues for precision therapy.

Alveolar type I cells can give rise to KRAS-induced lung adenocarcinoma

Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer and presents clinically with a high degree of biological heterogeneity and distinct clinical outcomes. The current paradigm of LUAD etiology posits alveolar epithelial type II (AT2) cells as the primary cell of origin, while the role of AT1 cells in LUAD oncogenesis remains unknown. Here, we examine oncogenic transformation in mouse Gram-domain containing 2 (Gramd2)+ AT1 cells via oncogenic KRASG12D. Activation of KRASG12D in AT1 cells induces multifocal LUAD, primarily of papillary histology. Furthermore, KRT8+ intermediate cell states were observed in both AT2- and AT1-derived LUAD, but SCGB3A2+, another intermediate cell marker, was primarily associated with AT1 cells, suggesting different mechanisms of tumor evolution. Collectively, our study reveals that Gramd2+ AT1 cells can serve as a cell of origin for LUAD and suggests that distinct subtypes of LUAD based on cell of origin be considered in the development of therapeutics.

CRISPR-Cas attack of HIV-1 proviral DNA can cause unintended deletion of surrounding cellular DNA.

Although HIV replication can be effectively inhibited by antiretroviral therapy, this does not result in a cure as the available drugs do not inactivate the integrated HIV-1 DNA in infected cells. Consequently, HIV-infected individuals need lifelong therapy to prevent viral rebound. Several preclinical studies indicate that CRISPR-Cas gene-editing systems can be used to achieve permanent inactivation of the viral DNA. It was previously shown that this inactivation was due to small inactivating mutations at the targeted sites in the HIV genome and to excision or inversion of the viral DNA fragment between two target sites. We, here, demonstrate that CRISPR-Cas treatment also causes large unintended deletions, which can include surrounding chromosomal sequences. As the loss of chromosomal sequences may cause oncogenic transformation of the cell, such unintended large deletions form a potential safety risk in clinical application of this antiviral application and possibly all CRISPR-Cas gene-editing approaches.

Single-cell profiling of MC1R-inhibited melanocytes.

The human red hair color (RHC) trait is caused by increased pheomelanin (red-yellow) and reduced eumelanin (black-brown) pigment in skin and hair due to diminished melanocortin 1 receptor (MC1R) function. In addition, individuals harboring the RHC trait are predisposed to melanoma development. While MC1R variants have been established as causative of RHC and are a well-defined risk factor for melanoma, it remains unclear mechanistically why decreased MC1R signaling alters pigmentation and increases melanoma susceptibility. Here, we use single-cell RNA sequencing (scRNA-seq) of melanocytes isolated from RHC mouse models to define a MC1R-inhibited Gene Signature (MiGS) comprising a large set of previously unidentified genes which may be implicated in melanogenesis and oncogenic transformation. We show that one of the candidate MiGS genes, TBX3, a well-known anti-senescence transcription factor implicated in melanoma progression, binds both E-box and T-box elements to regulate genes associated with melanogenesis and senescence bypass. Our results provide key insights into further mechanisms by which melanocytes with reduced MC1R signaling may regulate pigmentation and offer new candidates of study toward understanding how individuals with the RHC phenotype are predisposed to melanoma.