Oncogene induced senescence is a tumor suppressing defense mechanism, in which the cell cycle-dependent protein kinase (CDK) inhibitor p16INK4A (encoded by the CDKN2A gene) plays a key role. We previously reported that a transcriptional co-activator chromodomain helicase DNA binding protein 7 (CHD7) mediates oncogenic ras-induced senescence by inducing transcription of the p16INK4A gene. In the current study, we identified myeloid zinc finger 1 (MZF1) as the transcriptional factor that recruits CHD7 to the p16INK4A promoter, where it mediates oncogenic ras-induced p16INK4A transcription and senescence through CHD7, in primary human cells from multiple origins. Moreover, the expression of MZF1 is induced by oncogenic ras in senescent cells through the c-Jun and Ets1 transcriptional factors upon their activation by the Ras-Raf-1-MEK-ERK signaling pathway. In non-small cell lung cancer (NSCLC) and pancreatic adenocarcinoma (PAAD) where activating ras mutations occur frequently, reduced MZF1 expression is observed in tumors, as compared to corresponding normal tissues, and correlates with poor patient survival. Analysis of single cell RNA-sequencing data from PAAD patients revealed that among the tumor cells with normal RB expression levels, those with reduced levels of MZF1 are more likely to express lower p16INK4A levels. These findings have identified novel signaling components in the pathway that mediates induction of the p16INK4A tumor suppressor and the senescence response, and suggested that MZF1 is a potential tumor suppressor in at least some cancer types, the loss of which contributes to the inactivation of the p16INK4A/RB pathway and disruption of senescence in tumor cells with intact RB.

Nociceptive signals are usually transmitted to layer 4 neurons in somatosensory cortex via the spinothalamic-thalamocortical pathway. The layer 5 corticospinal neurons in sensorimotor cortex are reported to receive the output of neurons in superficial layers; and their descending axons innervate the spinal cord to regulate basic sensorimotor functions. Here, we show that a subset of layer 5 neurons receives spinal inputs through a direct spino-cortical circuit bypassing the thalamus, and thus define these neurons as spino-cortical recipient neurons (SCRNs). Morphological studies revealed that the branches from spinal ascending axons formed a kind of disciform structure with the descending axons from SCRNs in the basilar pontine nucleus (BPN). Electron microscopy and calcium imaging further confirmed that the axon terminals from spinal ascending neurons and SCRNs made functional synaptic contacts in the BPN, linking the ascending sensory pathway to the descending motor control pathway. Furthermore, behavioral tests indicated that the spino-cortical connection in the BPN was involved in nociceptive responses. In vivo calcium imaging showed that SCRNs responded to peripheral noxious stimuli faster than neighboring layer 4 cortical neurons in awake mice. Manipulating activities of SCRNs could modulate nociceptive behaviors. Therefore, this direct spino-cortical circuit represents a noncanonical pathway, allowing a fast sensory-motor transition of the brain in response to noxious stimuli.

DRG neurons are classified into distinct types to mediate the somatosensation with different modalities. Recently, transcriptional profilings of DRG neurons by single-cell RNA-sequencing have provided new insights into the neuron typing and functional properties. Zinc-finger CCHC domain-containing 12 (Zcchc12) was reported to be the representative marker for a subtype of galanin-positive (Gal+) DRG neurons. However, the characteristics and functions ofZcchc12+neurons are largely unknown. Here, we genetically labeledZcchc12+neurons inZcchc12-CreERT2::Ai9 mice, and verified thatZcchc12represented a new subpopulation of DRG neurons in both sexes.Zcchc12+neurons centrally innervated the superficial laminae in spinal dorsal horn, and peripherally terminated as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. In addition,Zcchc12+neurons also formed circumferential endings surrounding the hair follicles in hairy skin. Functionally,in vivocalcium imaging in DRGs revealed thatZcchc12+neurons were polymodal nociceptors and could be activated by mechanical and noxious thermal stimuli. Behavioral tests showed that selective ablation ofZcchc12+DRG neurons reduced the sensitivity to noxious heat in mice. Together, we identified a new subpopulation ofZcchc12+nociceptors essential for noxious heat sensation.SIGNIFICANCE STATEMENTZcchc12represents a new subpopulation of DRG neurons. The characteristics and functions ofZcchc12+neurons are largely unknown. Here we genetically labeledZcchc12+neurons, and showed that the fibers ofZcchc12+DRG neurons projected to superficial lamina at spinal dorsal horn, and innervated skin as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. Functionally,Zcchc12+DRG neurons responded to noxious mechanical and heat stimuli. Ablation ofZcchc12+DRG neurons impaired the sensation of noxious heat in mice. Therefore, we identify a new subpopulation of DRG neurons required for noxious heat sensation.

The AMP‐activated protein kinase (AMPK) is a molecular sensor to help maintain cellular energy homeostasis. AMPK is a heterotrimeric complex and its enzymatic catalytic subunit includes two isoforms: α1 and α2. Dysregulation of AMPK signaling is linked to neuronal diseases characterized with cognitive impairments. Emerging evidence also suggest isoform‐specific roles of AMPK in the brain. AMPK regulates protein synthesis, which is critical for memory formation and neuronal plasticity. However, the consequence of altering AMPK activity on the translation of specific proteins in the brain is unknown. Here, we use unbiased mass spectrometry‐based proteomics approach to analyze protein profile alterations in hippocampus and prefrontal cortex of transgenic mice in which the genes for the two AMPKα isoforms are conditionally deleted. The study revealed identities of proteins whose expression is sensitive to suppression of AMPKα1 and/or α2 isoform. These data may serve as a basis for future in‐depth study. Elucidation of the functional relevance of the alteration of specific proteins could provide insights into identification of novel therapeutic targets for neuronal disorders characterized with AMPK signaling dysregulation and impaired cellular energy metabolism.

MOTIVATION:The growth and survival of myeloma cells are greatly affected by their surrounding microenvironment. To understand the molecular mechanism and the impact of stiffness on the fate of myeloma-initiating cells (MICs), we develop a systems biological model to reveal the dynamic regulations by integrating reverse-phase protein array data and the stiffness-associated pathway. RESULTS:We not only develop a stiffness-associated signaling pathway to describe the dynamic regulations of the MICs, but also clearly identify three critical proteins governing the MIC proliferation and death, including FAK, mTORC1 and NFκB, which are validated to be related with multiple myeloma by our immunohistochemistry experiment, computation and manually reviewed evidences. Moreover, we demonstrate that the systematic model performs better than widely used parameter estimation algorithms for the complicated signaling pathway. AVAILABILITY AND IMPLEMENTATION:We can not only use the systems biological model to infer the stiffness-associated genetic signaling pathway and locate the critical proteins, but also investigate the important pathways, proteins or genes for other type of the cancer. Thus, it holds universal scientific significance. SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.

While mechanisms for metastasis were extensively studied in cancer cells from patients with detectable tumors, pathways underlying metastatic dissemination from early lesions before primary tumors appear are poorly understood. Her2 promotes breast cancer early dissemination by suppressing p38, but how Her2 downregulates p38 is unclear. Here, we demonstrate that in early lesion breast cancer models, Her2 inhibits p38 by inducing Skp2 through Akt-mediated phosphorylation, which promotes ubiquitination and proteasomal degradation of Tpl2, a p38 MAP3K. The early disseminating cells are Her2(+)Skp2(high)Tpl2(low)p-p38(low)E-cadherin(low)in the MMTV-Her2 breast cancer model. In human breast carcinoma, high Skp2 and low Tpl2 expression are associated with the Her2(+)status; Tpl2 expression positively correlates with that of activated p38; Skp2 expression negatively correlates with that of Tpl2 and activated p38. Moreover, the Her2-Akt-Skp2-Tpl2-p38 axis plays a key role in the disseminating phenotypes in early lesion breast cancer cells; inhibition of Tpl2 enhances early dissemination in vivo. These findings identify the Her2-Akt-Skp2-Tpl2-p38 cascade as a novel mechanism mediating breast cancer early dissemination and a potential target for novel therapies targeting early metastatic dissemination.

Peripheral nerve injury could lead to chronic neuropathic pain. Understanding transcriptional changes induced by nerve injury could provide fundamental insights into the complex pathogenesis of neuropathic pain. Gene expression profiles of dorsal root ganglia (DRG) in neuropathic pain condition have been studied. However, little is known about transcriptomic changes in individual DRG neurons after peripheral nerve injury. Here we performed single-cell RNA sequencing on dissociated mouse DRG cells after spared nerve injury (SNI). In addition to DRG neuron types that are found under physiological conditions, we identified three SNI-induced neuronal clusters (SNIICs) characterized by the expression of Atf3/Gfra3/Gal (SNIIC1), Atf3/Mrgprd (SNIIC2) and Atf3/S100b/Gal (SNIIC3). These SNIICs originated from Cldn9+/Gal+, Mrgprd+ and Trappc3l+ DRG neurons, respectively. Interestingly, SNIIC2 switched to SNIIC1 by increasing Gal and reducing Mrgprd expression 2 days after nerve injury. Inferring the gene regulatory networks after nerve injury, we revealed that activated transcription factors Atf3 and Egr1 in SNIICs could enhance Gal expression while activated Cpeb1 in SNIIC2 might suppress Mrgprd expression within 2 days after SNI. Furthermore, we mined the transcriptomic changes in the development of neuropathic pain to identify potential analgesic targets. We revealed that cardiotrophin-like cytokine factor 1, which activates astrocytes in the dorsal horn of spinal cord, was upregulated in SNIIC1 neurons and contributed to SNI-induced mechanical allodynia. Therefore, our results provide a new landscape to understand the dynamic course of neuron type changes and their underlying molecular mechanisms during the development of neuropathic pain.

AbstractWhen recruited to promoters, histone 3 lysine 4 (H3K4) methyltransferases KMT2 (KMT2A-D) activate transcription by opening chromatin through H3K4 methylation. Here, we report that KMT2 mutations occur frequently in non–small cell lung cancer (NSCLC) and are associated with high mutation loads and poor survival. KMT2C regulated DNA damage responses (DDR) through direct recruitment to DNA damage sites by Ago2 and small noncoding DNA damage response RNA, where it mediates H3K4 methylation, chromatin relaxation, secondary recruitment of DDR factors, and amplification of DDR signals along chromatin. Furthermore, by disrupting homologous recombination (HR)–mediated DNA repair, KMT2C/D mutations sensitized NSCLC to Poly(ADP-ribose) polymerase inhibitors (PARPi), whose efficacy is unclear in NSCLC due to low BRCA1/2 mutation rates. These results demonstrate a novel, transcription-independent role of KMT2C in DDR and identify high-frequency KMT2C/D mutations as much-needed biomarkers for PARPi therapies in NSCLC and other cancers with infrequent BRCA1/2 mutations.Significance:This study uncovers a critical role for KMT2C in DDR via direct recruitment to DNA damage sites, identifying high-frequency KMT2C/D mutations as biomarkers for response to PARP inhibition in cancer.

Cancer can metastasize from early lesions without detectable tumors. Despite extensive studies on metastasis in cancer cells from patients with detectable primary tumors, mechanisms for early metastatic dissemination are poorly understood. Her2 promotes breast cancer early dissemination by inhibiting p38, but the downstream pathway in this process was unknown. Using early lesion breast cancer models, we demonstrate that the effect of p38 suppression by Her2 on early dissemination is mediated by MK2 and heat shock protein 27 (Hsp27). The early disseminating cells in the MMTV-Her2 breast cancer model are Her2highp-p38lowp-MK2lowp-Hsp27low, which also exist in human breast carcinoma tissues. Suppression of p38 and MK2 by Her2 reduces MK2-mediated Hsp27 phosphorylation, and unphosphorylated Hsp27 binds to β-catenin and enhances its phosphorylation by Src, leading to β-catenin activation and disseminating phenotypes in early lesion breast cancer cells. Pharmacological inhibition of MK2 promotes, while inhibition of a p38 phosphatase Wip1 suppresses, early dissemination in vivo. These findings identify Her2-mediated suppression of the p38-MK2-Hsp27 pathway as a novel mechanism for cancer early dissemination, and provide a basis for new therapies targeting early metastatic dissemination in Her2+ breast cancer.

Many cancer risk loci act as expression quantitative trait loci (eQTLs) of transcripts including non-coding RNA. Long non-coding RNAs (lncRNAs) are implicated in various human cancers. However, the pathological and clinical impacts of the genetic determinants of lncRNAs in cancers remain largely unknown. In this study, we performed eQTL mapping of lncRNA expression (elncRNA) in 11 TCGA cancer types and characterized the biological processes of elncRNAs in the setting of genomic location, cancer treatment responses, and immune microenvironment. As a result, 10.86% of the cis-eQTLs and 1.67% of the trans-eQTLs of lncRNA were related to known genome-wide association studies (GWAS) cancer risk loci. The elncRNAs are significantly enriched for those which are previously annotated as predictive of drug sensitivities in cancer cell lines. We further revealed the downstream transcriptomic effectors of eQTL-elncRNA pairs. Our data specifically suggested that the genes affected by eQTL-elncRNA associations are enriched in the immune system processes and eQTL-elncRNA associations influence the constitution of tumor infiltrating lymphocytes. In ovarian cancer, the “rs34631313-AC092580.4” pair was associated with increased fraction of CD8+ T cells and M1 Macrophage; whereas in KIRC, the “rs9546285-LINC00426” pair was associated with increased fraction of CD8+ T cells and a decreased fraction of M2 macrophages. Our findings provide a systematic view of the transcriptomic impacts of the eQTL landscape of lncRNA in human cancers and suggest its strong potential relevance to cancer immunity and treatment.

Human embryogenesis is not well understood. Knowledge detailing human pre-gastrulation embryonic development including spatial self-organization and cell type ontogeny remains limited by available two-dimensional technological platforms. Here, we present a three-dimensional (3D) blastocyst-culture system, which enables human blastocyst development through primitive streak anlage (PSA). By the 3D-platform combined with immunofluorescence imaging and single-cell RNA-Seq, we reveal a developmental landscape of human pre-gastrulation embryos. Our protocol allows recording and analysis of embryo developmental landmarks and mechanisms from human blastocysts to pre-gastrulation stage (day 14 post- fertilization).

Multiple myeloma is a malignant still incurable plasma cell disorder. This is due to refractory disease relapse, immune impairment, and development of multi-drug resistance. The growth of malignant plasma cells is dependent on the bone marrow (BM) microenvironment and evasion of the host's anti-tumor immune response. Hence, we hypothesized that targeting tumor-stromal cell interaction and endogenous immune system in BM will potentially improve the response of multiple myeloma (MM). Therefore, we proposed a computational simulation of the myeloma development in the complicated microenvironment which includes immune cell components and bone marrow stromal cells and predicted the effects of combined treatment with multi-drugs on myeloma cell growth. We constructed a hybrid multi-scale agent-based model (HABM) that combines an ODE system and Agent-based model (ABM). The ODEs was used for modeling the dynamic changes of intracellular signal transductions and ABM for modeling the cell-cell interactions between stromal cells, tumor, and immune components in the BM. This model simulated myeloma growth in the bone marrow microenvironment and revealed the important role of immune system in this process. The predicted outcomes were consistent with the experimental observations from previous studies. Moreover, we applied this model to predict the treatment effects of three key therapeutic drugs used for MM, and found that the combination of these three drugs potentially suppress the growth of myeloma cells and reactivate the immune response. In summary, the proposed model may serve as a novel computational platform for simulating the formation of MM and evaluating the treatment response of MM to multiple drugs.

Hepatocellular carcinoma (HCC) is an aggressive tumor and the third leading cause of cancer-related death worldwide. Ovarian carcinoma immunoreactive antigen-like protein 2 (OCIAD2) has been found frequently methylated in various cancers, including HCC. The aim of the present study was to investigate the role of OCIAD2 in HCC progression. We analyzed liver hepatocellular carcinoma patients' data from the Cancer Genome Atlas (TCGA), including data extracted from 371 HCC tissues and 50 adjacent normal liver tissues. The RNA sequencing and DNA methylation data revealed that OCIAD2 were significantly hypermethylated and its expression level in the tumor tissues was much lower than that in the corresponding adjacent normal tissues. The methylation level in the promoter was negatively correlated with the expression level of OCAID2. Treatment of HCC cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycitydine (5-Aza) induced a significant increase in the OCIAD2 mRNA and protein. Knocking-down OCIAD2 led to an increased colony formation, migration and invasion dramatically, accompanying with an enhanced expression of MMP9 and activation of AKT and FAK. Inhibition of AKT signaling restored OCIAD2-mediated changes in HCC cell clonogenic growth, migration and invasion. Survival analysis of HCC patient's data indicated patients with a higher expression ratio of OCIAD2/MMP9 had a shorter overall survival than those with a lower expression ratio of OCIAD2/MMP9. Overall, our data indicate that reduced expression of OCIAD2 by DNA hypermethylation plays an important role in HCC tumor growth and invasion. Hypermethylation of OCIAD2 may contribute to HCC treatment development.

The current knowledge about heat nociception is mainly confined to the thermosensors, including the transient receptor potential cation channel V1 expressed in the nociceptive neurons of dorsal root ganglion (DRG). However, the loss of thermosensors only partially impairs heat nociception, suggesting the existence of undiscovered mechanisms. We found that the loss of an intracellular fibroblast growth factor (FGF), FGF13, in the mouse DRG neurons selectively abolished heat nociception. The noxious heat stimuli could not evoke the sustained action potential firing in FGF13-deficient DRG neurons. Furthermore, FGF13 interacted with the sodium channel Nav1.7 in a heat-facilitated manner. FGF13 increased Nav1.7 sodium currents and maintained the membrane localization of Nav1.7 during noxious heat stimulation, enabling the sustained firing of action potentials. Disrupting the FGF13/Nav1.7 interaction reduced the heat-evoked action potential firing and nociceptive behavior. Thus, beyond the thermosensors, the FGF13/Nav1.7 complex is essential for sustaining the transmission of noxious heat signals.

Sensory neurons are distinguished by distinct signaling networks and receptive characteristics. Thus, sensory neuron types can be defined by linking transcriptome-based neuron typing with the sensory phenotypes. Here we classify somatosensory neurons of the mouse dorsal root ganglion (DRG) by high-coverage single-cell RNA-sequencing (10 950 ± 1 218 genes per neuron) and neuron size-based hierarchical clustering. Moreover, single DRG neurons responding to cutaneous stimuli are recorded using an in vivo whole-cell patch clamp technique and classified by neuron-type genetic markers. Small diameter DRG neurons are classified into one type of low-threshold mechanoreceptor and five types of mechanoheat nociceptors (MHNs). Each of the MHN types is further categorized into two subtypes. Large DRG neurons are categorized into four types, including neurexophilin 1-expressing MHNs and mechanical nociceptors (MNs) expressing BAI1-associated protein 2-like 1 (Baiap2l1). Mechanoreceptors expressing trafficking protein particle complex 3-like and Baiap2l1-marked MNs are subdivided into two subtypes each. These results provide a new system for cataloging somatosensory neurons and their transcriptome databases.

Multiple myeloma (MM) is an incurable cancer characterized by clonal expansion of malignant plasma cells in the bone marrow and their egress into peripheral blood. The mechanisms of myeloma cells migration/invasion have remained unclear. Herein, we found SH3GL3 was highly expressed in the CD138-negative (CD138-) myeloma cells. The migration/invasion capability of CD138- cells was significantly higher than that in the CD138-positive (CD138+) cells. Silencing SH3GL3 using shRNA reduced myeloma cells migration/invasion. Conversely, overexpression of SH3GL3 increased myeloma cells migration/invasion. Moreover, SH3GL3 is also associated with the stemness and chemo-resistance of CD138- myeloma cells. Elevated expression of stem cell and multi-drug resistant markers were seen in the myeloma cells with overexpressed SH3GL3; while knocking-down SH3GL3 reduced the expression of these markers. A marked increase in p-PI3K and p-FAK was observed in the cells with overexpressed SH3GL3. To test if FAK/PI3K signaling pathway was involved in the SH3GL3-mediated myeloma cells migration, the cells transfected w/wo SH3GL3 cDNA were treated with FAK inhibitor 14 and PI3K inhibitor LY294002. Inhibition of FAK and PI3K attenuated SH3GL3-mediated migration /invasion. Our findings indicate that SH3GL3 plays an important role in myeloma cell migration/invasion, stemness and chemo-resistance. The SH3GL3-mediated myeloma cell migration/invasion is mediated by FAK/PI3K signaling pathway.

Research Background Currently, multiple myeloma is the second most common hematological malignancy in the U.S., constituting 1% of all cancers. With conventional treatment, the median survival time is typically 3–4 years, although it can be extended to 5–7 years or longer with advanced treatments. Recent research indicated that an increase in osteoclast (OC) activity is often associated withmultiple myeloma (MM) and that a decrease inosteoblast (OB) activity contributesto the osteolytic lesions in MM. Normally, the populations of OCs and OBs are inequilibrium, and an imbalance in this statecontributes to the development of lesions. Research procedures A multi-scale agent-based multiple myeloma model was developed to simulate the proliferation, migration and death of OBs and OCs. Subsequently, this model was employed to investigate the efficacy of thethree most commonly used drugs for MM treatment under the following two premises: the reduction in the progression of MM and the re-establishment of the equilibrium between OCs and OBs. Research purposes The simulated results not only demonstrated the capacity of the model to choose optimal combinations of the drugs but also showed that the optimal use of the three drugs can restore the balance between OCs and OBs as well as kill MMs. Furthermore, the drug synergism analysis function of the model revealed that restoring the balance between OBs and OCs can significantly increase the efficacy of drugs against tumor cells.

Abstract Objective. The aim of this study was determine whether intracavernosal injection (ICI) of insulin-like growth factor-1 (IGF-1) protein can improve corpus cavernosal smooth muscle relaxation in aging rats. Materials and methods. Ten young (4-month-old) and 30 old (24-month-old) Sprague–Dawley male rats were enrolled in the study. The old rats were divided into three groups: vehicle-only (n = 10), IGF-1 1 µg/kg (n = 10) and IGF-1 10 µg/kg treatment groups (n = 10). After 4 weeks of single IGF-1 injection treatment, strips of corporal tissue were precontracted with phenylephrine, and dose–response curves were generated to evaluate endothelial-dependent [acetylcholine (ACh)], endothelial-independent [sodium nitroprusside (SNP)] and electrical field stimulation (EFS) vasoreactivity. The changes in percentage of cavernosal smooth muscle and the concentration of nitric oxide (NO) in penile tissue were also evaluated. Results. After IGF-1 treatment, the vasoreactivity was significantly improved in both the 1 µg/kg and the 10 µg/kg treatment groups compared with the vehicle-only group at 4 weeks in response to ACh, SNP and EFS (all p < 0.05). The percentage of cavernosal smooth muscle was increased in the IGF-1 treatment groups. The NO concentrations were increased after IGF-1 treatment. Conclusions. These data demonstrate that ICI of IGF-1 can improve vasoreactivity via endothelium-dependent and endothelial-independent mechanisms in the corpus cavernosum of the aging rat.