Whole-Exome Sequencing Identifies a Novel Germline Variant in PTK7 Gene in Familial Colorectal Cancer

Colorectal cancer (CRC) is the third most frequently diagnosed malignancy worldwide. Only 5% of all CRC cases are due to germline mutations in known predisposition genes, and the remaining genetic burden still has to be discovered. In this study, we performed whole-exome sequencing on six members of a Polish family diagnosed with CRC and identified a novel germline variant in the protein tyrosine kinase 7 (inactive) gene (PTK7, ENST00000230419, V354M). Targeted screening of the variant in 1705 familial CRC cases and 1674 healthy elderly individuals identified the variant in an additional familial CRC case. Introduction of this variant in HT-29 cells resulted in increased cell proliferation, migration, and invasion; it also caused down-regulation of CREB, p21 and p53 mRNA and protein levels, and increased AKT phosphorylation. These changes indicated inhibition of apoptosis pathways and activation of AKT signaling. Our study confirmed the oncogenic function of PTK7 and supported its role in genetic predisposition of familial CRC.     

Identification of a Novel Mutation of β-Spectrin in Hereditary Spherocytosis Using Whole Exome Sequencing

Hereditary spherocytosis (HS), the most commonly inherited hemolytic anemia in northern Europeans, comprises a group of diseases whose heterogeneous genetic basis results in a variable clinical presentation. High-throughput genome sequencing methods have made a leading contribution to the recent progress in research on and diagnostics of inherited diseases and inspired us to apply whole exome sequencing (WES) to identify potential mutations in HS. The data presented here reveal a novel mutation probably responsible for HS in a single Polish family. Patients with clinical evidence of HS (clinical symptoms, hematological data, and EMA test) were enrolled in the study. The examination of the resulting WES data showed a number of polymorphisms in 71 genes associated with known erythrocyte pathologies (including membranopathies, enzymopathies, and hemoglobinopathies). Only a single SPTB gene variant indicated the possible molecular mechanism of the disease in the studied family. The new missense mutation p.C183Y was identified using WES in the SPTB gene, which is most likely the cause of clinical symptoms typical of hereditary spherocytosis (membranopathy) due to structural and functional impairments of human β-spectrin. This mutation allows for a better understanding of the molecular mechanism(s) of one of the membranopathies, hereditary spherocytosis.     

Read Count Patterns and Detection of Cancerous Copy Number Alterations in Plasma Cell-Free DNA Whole Exome Sequencing Data for Advanced Non-Small Cell Lung Cancer

Plasma cell-free DNA (cfDNA) sequencing data have been widely studied for early diagnosis and treatment response or recurrence monitoring of cancers because of the non-invasive benefits. In cancer studies, whole exome sequencing (WES) is mostly used for discovering single nucleotide variants (SNVs), but it also has the potential to detect copy number alterations (CNAs) that are mostly discovered by whole genome sequencing or microarray. In clinical settings where the quantity of the acquired blood from the patients is limited and where various sequencing experiments are not possible, providing various types of mutation information such as CNAs and SNVs using only WES will be helpful in the treatment decision. Here, we questioned whether the plasma cfDNA WES data for patients with advanced non-small cell lung cancer (NSCLC) could be exploited for CNA detection. When the read count (RC) signals of the WES data were investigated, a similar fluctuation pattern was observed among the signals of different samples, and it can be a major challenge hindering CNA detection. When these RC patterns among cfDNA were suppressed by the method we proposed, the cancerous CNAs were more distinguishable in some samples with higher cfDNA quantity. Although the potential to detect CNAs using the plasma cfDNA WES data for NSCLC patients was studied here, further studies with other cancer types, with more samples, and with more sophisticated techniques for bias correction are required to confirm our observation. In conclusion, the detection performance for cancerous CNAs can be improved by controlling RC bias, but it depends on the quantity of cfDNA in plasma.     

Molecular Mechanisms and Tumor Biological Aspects of 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells

5-Fluorouracil (5-FU) is a cornerstone drug used in the treatment of colorectal cancer (CRC). However, the development of resistance to 5-FU and its analogs remain an unsolved problem in CRC treatment. In this study, we investigated the molecular mechanisms and tumor biological aspects of 5-FU resistance in CRC HCT116 cells. We established an acquired 5-FU-resistant cell line, HCT116R^F10. HCT116R^F10 cells were cross-resistant to the 5-FU analog, fluorodeoxyuridine. In contrast, HCT116R^F10 cells were collaterally sensitive to SN-38 and CDDP compared with the parental HCT16 cells. Whole-exome sequencing revealed that a cluster of genes associated with the 5-FU metabolic pathway were not significantly mutated in HCT116 or HCT116R^F10 cells. Interestingly, HCT116R^F10 cells were regulated by the function of thymidylate synthase (TS), a 5-FU active metabolite 5-fluorodeoxyuridine monophosphate (FdUMP) inhibiting enzyme. Half of the TS was in an active form, whereas the other half was in an inactive form. This finding indicates that 5-FU-resistant cells exhibited increased TS expression, and the TS enzyme is used to trap FdUMP, resulting in resistance to 5-FU and its analogs.     

Cell Type-Specific Induction of Inflammation-Associated Genes in Crohn’s Disease and Colorectal Cancer

Based on the rapid increase in incidence of inflammatory bowel disease (IBD), the identification of susceptibility genes and cell populations contributing to this condition is essential. Previous studies suggested multiple genes associated with the susceptibility of IBD; however, due to the analysis of whole-tissue samples, the contribution of individual cell populations remains widely unresolved. Single-cell RNA sequencing (scRNA-seq) provides the opportunity to identify underlying cellular populations. We determined the enrichment of Crohn’s disease (CD)-induced genes in a publicly available Crohn’s disease scRNA-seq dataset and detected the strongest induction of these genes in innate lymphoid cells (ILC1), highly activated T cells and dendritic cells, pericytes and activated fibroblasts, as well as epithelial cells. Notably, these genes were highly enriched in IBD-associated neoplasia, as well as sporadic colorectal cancer (CRC). Indeed, the same six cell populations displayed an upregulation of CD-induced genes in a CRC scRNA-seq dataset. Finally, after integrating and harmonizing the CD and CRC scRNA-seq data, we demonstrated that these six cell types display a gradual increase in gene expression levels from a healthy state to an inflammatory and tumorous state. Together, we identified cell populations that specifically upregulate CD-induced genes in CD and CRC patients and could, therefore, contribute to inflammation-associated tumor development.     

Cellular Integrin α5β1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size−exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5β1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.     

Expression of Irisin/FNDC5 in Breast Cancer

Irisin is a myokine formed from fibronectin type III domain-containing protein 5 (FNDC5), which can be found in various cancer tissues. FNDC5 and irisin levels have been poorly studied in the tumor tissues of breast cancer (BC). The aim of this study was to determine the levels of irisin expression in BC tissues and compare them to clinicopathological factors and Ki-67 and PGC-1α expression levels. Tissue microarrays (TMAs) with 541 BC tissues and 61 samples of non-malignant breast disease (NMBD; control) were used to perform immunohistochemical reactions. FNDC5 gene expression was measured in 40 BC tissue samples, 40 samples from the cancer margin, and 16 NMBD samples. RT-PCR was performed for the detection of FNDC5 gene expression. Higher irisin expression was found in BC patients compared to normal breast tissue. FNDC5/irisin expression was higher in patients without lymph node metastases. Longer overall survival was observed in patients with higher irisin expression levels. FNDC5/irisin expression was increased in BC tissues and its high level was a good prognostic factor for survival in BC patients.     

Differentially Expressed Genes and Signaling Pathways Potentially Involved in Primary Resistance to Chemo-Immunotherapy in Advanced-Stage Gastric Cancer Patients

Recently, the combination of chemotherapy plus nivolumab (chemo-immunotherapy) has become the standard of care for advanced-stage gastric cancer (GC) patients. However, despite its efficacy, up to 40% of patients do not respond to these treatments. Our study sought to identify variations in gene expression associated with primary resistance to chemo-immunotherapy. Diagnostic endoscopic biopsies were retrospectively obtained from advanced GC patients previously categorized as responders (R) or non-responders (NR). Thirty-four tumor biopsies (R: n = 16, NR: n = 18) were analyzed by 3′ massive analysis of cDNA ends (3′MACE). We found >30 differentially expressed genes between R and NRs. Subsequent pathway enrichment analyses demonstrated that angiogenesis and the Wnt-β-catenin signaling pathway were enriched in NRs. Concomitantly, we performed next generation sequencing (NGS) analyses in a subset of four NR patients that confirmed alterations in genes that belonged to the Wnt/β-catenin and the phosphoinositide 3-kinase (PI3K) pathways. We speculate that angiogenesis, the Wnt, and the PI3K pathways might offer actionable targets. We also discuss therapeutic alternatives for chemo-immunotherapy-resistant advanced-stage GC patients.     

Association of Irisin/FNDC5 with ERRα and PGC-1α Expression in NSCLC

The rapid growth and division of cancer cells are associated with mitochondrial biogenesis or switching to glycolysis. ERRα, PGC-1α and irisin/FNDC5 are some of the proteins that can influence these processes. The aim of this study was to determine the correlation of these proteins in non-small cell lung cancer (NSCLC) and to investigate their association with clinicopathological parameters. Immunohistochemistry reactions were performed on tissue microarrays (860 NSCLC, 140 non-malignant lung tissue). The normal fibroblast cell line (IMR-90) and lung cancer cell lines (NCI-H1703 and NCI-H522) were used as co-cultures. The mRNA levels of FNDC5 and ESRRA (encoding ERRα) were assessed in IMR-90 cells after co-culture with lung cancer cells. We observed a decreased level of ERRα with an increase in tumor size (T), stages of the disease, and lymph node metastases (N). In the adenocarcinoma (AC) subtype, patients with a higher ERRα expression had significantly longer overall survival. A moderate positive correlation was observed between FNDC5 mRNA and ESRRA mRNA in NSCLCs. The expression of FNDC5 mRNA in IMR-90 cells increased after 24 h, and ESRRA gene expression increased after 48 h of co-culture. The ERRα receptor with PGC-1α participates in the control of FNDC5/irisin expression. Normal fibroblasts revealed an upregulation of the FNDC5 and ESRRA genes under the influence of lung cancer cells.     

Blocking Muscarinic Receptor 3 Attenuates Tumor Growth and Decreases Immunosuppressive and Cholinergic Markers in an Orthotopic Mouse Model of Colorectal Cancer

Tumor cells have evolved to express immunosuppressive molecules allowing their evasion from the host’s immune system. These molecules include programmed death ligands 1 and 2 (PD-L1 and PD-L2). Cancer cells can also produce acetylcholine (ACh), which plays a role in tumor development. Moreover, tumor innervation can stimulate vascularization leading to tumor growth and metastasis. The effects of atropine and muscarinic receptor 3 (M3R) blocker, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), on cancer growth and spread were evaluated in vitro using murine colon cancer cell line, CT-26, and in vivo in an orthotopic mouse model of colorectal cancer. In the in vitro model, atropine and 4-DAMP significantly inhibited CT-26 cell proliferation in a dose dependent manner and induced apoptosis. Atropine attenuated immunosuppressive markers and M3R via inhibition of EGFR/AKT/ERK signaling pathways. However, 4-DAMP showed no effect on the expression of PD-L1, PD-L2, and choline acetyltransferase (ChAT) on CT-26 cells but attenuated M3R by suppressing the phosphorylation of AKT and ERK. Blocking of M3R in vivo decreased tumor growth and expression of immunosuppressive, cholinergic, and angiogenic markers through inhibition of AKT and ERK, leading to an improved immune response against cancer. The expression of immunosuppressive and cholinergic markers may hold potential in determining prognosis and treatment regimens for colorectal cancer patients. This study’s results demonstrate that blocking M3R has pronounced antitumor effects via several mechanisms, including inhibition of immunosuppressive molecules, enhancement of antitumor immune response, and suppression of tumor angiogenesis via suppression of the AKT/ERK signaling pathway. These findings suggest a crosstalk between the cholinergic and immune systems during cancer development. In addition, the cholinergic system influences cancer evasion from the host’s immunity.     

Characteristics of TIMP1, CD63, and β1-Integrin and the Functional Impact of Their Interaction in Cancer

Tissue Inhibitor of Metalloproteases 1, also known as TIMP-1, is named for its well-established function of inhibiting the proteolytic activity of matrix metalloproteases. Given this function, many studies were carried out to verify if TIMP-1 was able to interrupt processes such as tumor cell invasion and metastasis. In contrast, many studies have shown that TIMP-1 expression is increased in several types of tumors, and this increase was correlated with a poor prognosis and lower survival in cancer patients. Later, it was shown that TIMP-1 is also able to modulate cell behavior through the induction of signaling pathways involved in cell growth, proliferation, and survival. The mechanisms involved in the regulation of the pleiotropic functions of TIMP-1 are still poorly understood. Thus, this review aimed to present literature data that show its ability to form a membrane complex with CD63 and β1-integrin, and point to N-glycosylation as a potential regulatory mechanism of the functions exerted by TIMP-1. This article reviewed the characteristics and functions performed individually by TIMP1, CD63, and β1-integrin, the roles of the TIMP-1/CD63/β1-integrin complex, both in a physiological context and in cancer, and the regulatory mechanisms involved in its assembly.     

PLA2G7/PAF-AH as Potential Negative Regulator of the Wnt Signaling Pathway Mediates Protective Effects in BRCA1 Mutant Breast Cancer

Past studies have confirmed that aberrant activation of the Wnt/β-catenin signaling is associated with tumorigenesis and metastasis in breast cancer, while the role of platelet-activating factor acetylhydrolase (PLA2G7/PAF-AH) in this signaling pathway remains unclear. In this study, we analyze the functional impact of PAF-AH on BRCA1 mutant breast cancer and explore its relationship to the Wnt signaling pathway. By performing immunohistochemistry, PAF-AH expression and β-catenin expression were examined in both BRCA1 WT and BRCA1 mutant breast cancer specimens. The BRCA1 mutant breast cancer cell line HCC1937 was used for in vitro experiments to assess the impact of PAF-AH on cellular functions. The intracellular distribution of β-catenin depending on PLA2G7/PAF-AH expression was investigated by immunocytochemistry. Significantly higher nuclear expression levels of PAF-AH were found in BRCA1 mutant tissue specimens than in BRCA1 WT samples. Cell viability, proliferation, and the motility rate of HCC1937 were significantly enhanced after PLA2G7 silencing, which indicated a protective role of PAF-AH in breast cancer. Nuclear PAF-AH expressed correlatedly with membranous β-catenin. PLA2G7 silencing provoked the β-catenin translocation from the membrane to the nucleus and activated Wnt signaling downstream genes. Our data showed a protective effect of high PAF-AH expression in BRCA1 mutant breast cancer. PAF-AH may achieve its protective effect by negatively regulating the Wnt pathway. In conclusion, our research sheds new light on the regulatory pathways in BRCA1 mutant breast cancer.     

Concomitant Antihyperalgesic and Antitumor Effects of Gabapentin in a Murine Cancer Pain Model

Cancer pain may be the consequence of physical nerve compression by a growing tumor. We employed a murine model to study whether gabapentin was able to regulate tumor growth, in addition to controlling hyperalgesic symptoms. A fluorescent melanoma cell line (B16–BL6/Zs green) was inoculated into the proximity of the sciatic nerve in male C57BL/6 mice. The tumor gradually compressed the nerve, causing hypersensitivity. Tumor growth was characterized via in vivo imaging techniques. Every other day, gabapentin (100 mg/Kg) or saline was IP administered to each animal. In the therapeutic protocol, gabapentin was administered once the tumor had induced increased nociception. In the preventive protocol, gabapentin was administered before the appearance of the positive signs. Additionally, in vitro experiments were performed to determine gabapentin’s effects on cell-line proliferation, the secretion of the chemokine CCL2, and calcium influx. In the therapeutically treated animals, baseline responses to noxious stimuli were recovered, and tumors were significantly reduced. Similarly, gabapentin reduced tumor growth during the preventive treatment, but a relapse was noticed when the administration stopped. Gabapentin also inhibited cell proliferation, the secretion of CCL2, and calcium influx. These results suggest that gabapentin might represent a multivalent strategy to control cancer-associated events in painful tumors.     

Extracellular Hsp90α Promotes Tumor Lymphangiogenesis and Lymph Node Metastasis in Breast Cancer

Early detection and discovery of new therapeutic targets are urgently needed to improve the breast cancer treatment outcome. Here we conducted an official clinical trial with cross-validation to corroborate human plasma Hsp90α as a novel breast cancer biomarker. Importantly, similar results were noticed in detecting early-stage breast cancer patients. Additionally, levels of plasma Hsp90α in breast cancer patients were gradually elevated as their clinical stages of regional lymph nodes advanced. In orthotopic breast cancer mouse models, administrating with recombinant Hsp90α protein increased both the primary tumor lymphatic vessel density and sentinel lymph node metastasis by 2 and 10 times, respectively. What is more, Hsp90α neutralizing antibody treatment approximately reduced 70% of lymphatic vessel density and 90% of sentinel lymph node metastasis. In the in vitro study, we demonstrated the role of extracellular Hsp90α (eHsp90α) as a pro-lymphangiogenic factor, which significantly enhanced migration and tube formation abilities of lymphatic endothelial cells (LECs). Mechanistically, eHsp90α signaled to the AKT pathway through low-density lipoprotein receptor-related protein 1 (LRP1) to upregulate the expression and secretion of CXCL8 in the lymphangiogenic process. Collectively, this study proves that plasma Hsp90α serves as an auxiliary diagnosis biomarker and eHsp90α as a molecular mediator promoting lymphangiogenesis in breast cancer.     

Role of Glycated High Mobility Group Box-1 in Gastric Cancer

Advanced glycation end products (AGEs) are produced in response to a high-glucose environment and oxidative stress and exacerbate various diseases. N^ε-(Carboxymethyl)lysine (CML) is an AGE that is produced by the glycation of lysine residues of proteins. There are a few reports on alterations in protein function due to CML modification; however, its association with cancer is not clear. We investigated the significance of CML modification in high mobility group box protein-1 (HMGB1), a cytokine that is significantly associated with cancer progression. Treatment of the gastric cancer cell lines TMK1 and MKN74 with glyoxal or glucose resulted in increased CML modification compared to untreated cells. CML-HMGB1 was modified via oxidation and more pronouncedly activated the receptor for AGE and downstream AKT and NF-κB compared to naïve HMGB1 and oxidized HMGB1. CML-HMGB1 bound with reduced affinity to DNA and histone H3, resulting in enhanced extranuclear translocation and extracellular secretion. Treatment of gastric cancer cells with CML-HMGB1 enhanced cell proliferation and invasion, sphere formation, and protection from thapsigargin-induced apoptosis, and decreased 5-FU sensitivity in comparison to HMGB1. Further, CML-HMGB1 was detected at various levels in all the 10 gastric cancer tumor specimens. HMGB1 levels correlated with primary tumor progression and distant metastasis, whereas CML-HMGB1 levels were associated with primary tumor progression, lymph node metastasis, distant metastasis, and stage. In addition, CML-HMGB1 levels correlated with oxidative stress in cancer tissues and resistance to neoadjuvant therapy. Therefore, CML modification of HMGB1 enhanced the cancer-promoting effect of HMGB1. In this study, CML-HMGB1 has been highlighted as a new therapeutic target, and analysis of the molecular structure of CML-HMGB1 is desired in the future.     

Secreted HSP90α-LRP1 Signaling Promotes Tumor Metastasis and Chemoresistance in Pancreatic Cancer

The extracellular heat shock protein 90α (eHSP90α) has been reported to promote cancer cell motility. However, whether pancreatic cancer (PC) cells expressed membrane-bound or secreted HSP90α, as well as its underlying mechanism for PC progression, were still unclear. Our study demonstrated that the amounts of secreted HSP90α proteins were discrepant in multiple PC cells. In addition, highly invasive Capan-2 cells have a higher level of secreted HSP90α compared with those of less invasive PL45 cells. The conditioned medium of Capan-2 cells or recombinant HSP90α treatment stimulated the migration and invasion of PC cells, which could be prevented with a neutralizing anti-HSP90α antibody. Furthermore, secreted HSP90α promoted elements of epithelial–mesenchymal transition in PL45 cells, including increases in vimentin and Snail expressions, decreases in E-cadherin expression, and changes in cell shape towards a mesenchymal phenotype, but these phenomena were reversed by the anti-HSP90α antibody in Capan-2 cells. In addition, high levels of low-density lipoprotein receptor-related protein 1 (LRP1) were associated with worsened patient survival in pancreatic adenocarcinoma. We demonstrated LRP1 as a receptor of eHSP90α for its stimulatory role in metastasis, by activating the AKT pathway. In addition, silencing LRP1 enhanced the chemosensitivity to gemcitabine and doxorubicin in Capan-2 cells. Therefore, our study indicated that blocking secreted HSP90α underlies an aspect of metastasis and chemoresistance in PC.     

Propolin G-Suppressed Epithelial-to-Mesenchymal Transition in Triple-Negative Breast Cancer Cells via Glycogen Synthase Kinase 3β-Mediated Snail and HDAC6-Regulated Vimentin Degradation

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer with a poor prognosis. The incidence and mortality rate of TNBC are frequently found in younger women. Due to the absence of a good therapeutic strategy, effective remedies for inhibiting TNBC have been developed for improving the cure rate. Epithelial-to-mesenchymal transition (EMT) is a critical mechanism to regulate cancer cell motility and invasion. Furthermore, ectopic expression of EMT molecules correlates with the metastasis and poor prognosis of TNBC. Targeting EMT might be a strategy for the therapy and prevention of TNBC. Propolin G, an active c-prenylflavanone in Taiwanese propolis, has been shown to possess anti-cancer activity in many cancers. However, the anti-metastasis activity of propolin G on TNBC is still unclear. The present study showed that the migration and invasion activities of TNBC cells was suppressed by propolin G. Down-regulated expression of Snail and vimentin and up-regulated expression of E-cadherin were dose- and time-dependently observed in propolin G-treated MDA-MB-231 cells. Propolin G inhibited Snail and vimentin expressions via the signaling pathways associated with post-translational modification. The activation of glycogen synthase kinase 3β (GSK-3β) by propolin G resulted in increasing GSK-3β interaction with Snail. Consequently, the nuclear localization and stability of Snail was disrupted resulting in promoting the degradation. Propolin G-inhibited Snail expression and the activities of migration and invasion were reversed by GSK-3β inhibitor pretreatment. Meanwhile, the outcomes also revealed that histone deacetylase 6 (HDAC6) activity was dose-dependently suppressed by propolin G. Correspondently, the amounts of acetyl-α-tubulin, a down-stream substrate of HDAC6, were increased. Dissociation of HDAC6/Hsp90 with vimentin leading to increased vimentin acetylation and degradation was perceived in the cells with the addition of propolin G. Moreover, up-regulated expression of acetyl-α-tubulin by propolin G was attenuated by HDAC6 overexpression. On the contrary, down-regulated expression of vimentin, cell migration and invasion by propolin G were overturned by HDAC6 overexpression. Conclusively, restraint cell migration and invasion of TNBC by propolin G were activated by the expression of GSK-3β-suppressed Snail and the interruption of HDAC6-mediated vimentin protein stability. Aiming at EMT, propolin G might be a potential candidate for TNBC therapy.     

PKR-Mediated Phosphorylation of eIF2a and CHK1 Is Associated with Doxorubicin-Mediated Apoptosis in HCC1143 Triple-Negative Breast Cancer Cells

Triple-negative breast cancer is more aggressive than other types of breast cancer. Protein kinase R (PKR), which is activated by dsRNA, is known to play a role in doxorubicin-mediated apoptosis; however, its role in DNA damage-mediated apoptosis is not well understood. In this study, we investigated the roles of PKR and its downstream players in doxorubicin-treated HCC1143 triple-negative breast cancer cells. Doxorubicin treatment induces DNA damage and apoptosis. Interestingly, doxorubicin treatment induced the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) via PKR, whereas the inhibition of PKR with inhibitor C16 reduced eIF2α phosphorylation. Under these conditions, doxorubicin-mediated DNA fragmentation, cell death, and poly(ADP ribose) polymerase and caspase 7 levels were recovered. In addition, phosphorylation of checkpoint kinase 1 (CHK1), which is known to be involved in doxorubicin-mediated DNA damage, was increased by doxorubicin treatment, but blocked by PKR inhibition. Protein translation was downregulated by doxorubicin treatment and upregulated by blocking PKR phosphorylation. These results suggest that PKR activation induces apoptosis by increasing the phosphorylation of eIF2α and CHK1 and decreasing the global protein translation in doxorubicin-treated HCC1143 triple-negative breast cancer cells.