Genomic Heterogeneity and Clonal Evolution in Gastroesophageal Junction Cancer Revealed by Single Cell DNA Sequencing.

Gastroesophageal junction (GEJ) cancer is a tumor that occurs at the junction of stomach and esophagus anatomically. GEJ cancer frequently metastasizes to lymph nodes, however the heterogeneity and clonal evolution process are unclear. This study is the first of this kind to use single cell DNA sequencing to determine genomic variations and clonal evolution related to lymph node metastasis. Multiple Annealing and Looping Based Amplification Cycles (MALBAC) and bulk exome sequencing were performed to detect single cell copy number variations (CNVs) and single nucleotide variations (SNVs) respectively. Four GEJ cancer patients were enrolled with two (Pt.3, Pt.4) having metastatic lymph nodes. The most common mutation we found happened in the TTN gene, which was reported to be related with the tumor mutation burden in cancers. Significant intra-patient heterogeneity in SNVs and CNVs were found. We identified the SNV subclonal architecture in each tumor. To study the heterogeneity of CNVs, the single cells were sequenced. The number of subclones in the primary tumor was larger than that in lymph nodes, indicating the heterogeneity of primary site was higher. We observed two patterns of multi-station lymph node metastasis: one was skip metastasis and the other was to follow the lymphatic drainage. Taken together, our single cell genomic analysis has revealed the heterogeneity and clonal evolution in GEJ cancer.

A heterogeneous cellular response to ionizing radiation revealed by single cell transcriptome sequencing.

Our understanding on transcriptional regulation of tumour cells responding to ionizing radiation (IR) has mostly come from bulk sequencing. However, due to the heterogeneity of tumour, how each individual cell responds to IR differently is unclear. We report here a heterogeneous cellular response to IR by single cell transcriptome sequencing. We utilized the barcoded Smart-seq2 single cell transcriptome sequencing technology in breast cancer cell line MDA-MB-231 both without and with IR treatment. To further understand how ATM, a major hub protein required for an optimal DNA damage response, affected the heterogeneous IR response, we also knocked down ATM gene for single cell transcriptome sequencing. Single cell t-SNE analysis showed four clusters of cells responding to IR in distinctive ways: Cluster 1 changed the least; Cluster 2 responded to IR by upregulating ribosome associated genes, while Cluster 4 upregulated both ribosome and G1/S phase associated genes; Cluster 3 was a new cluster, which appeared only in irradiated cells. In the absence of ATM kinase, cells displayed much less transcriptional changes after IR. And Cluster 4 in wild-type cells, which had the greatest change in response to IR, was not present in the ATM knock-down cells. We also selected three IR-induced genes for functional validation in both MDA-MB-231 and an additional breast cancer cell line to demonstrate their importance in radiation sensitivity. Taken together, our single cell transcriptome analysis has revealed a heterogeneous cellular response to DNA damage induced by IR and identified potential biomarkers of radiation sensitivity.

ROS/KRAS/AMPK Signaling Contributes to Gemcitabine-Induced Stem-like Cell Properties in Pancreatic Cancer.

Poor prognosis in pancreatic cancer (PanCa) is partially due to chemoresistance to gemcitabine (GEM). Glucose metabolism has been revealed to contribute to the therapeutic resistance and pluripotent state of PanCa cells. However, few studies have focused on the effects of GEM on cancer cell metabolism, stemness of tumor cells, and molecular mechanisms that critically influence PanCa treatment. We demonstrate that GEM treatment induces metabolic reprogramming, reducing mitochondrial oxidation and upregulating aerobic glycolysis, and promotes stem-like behaviors in cancer cells. Inhibiting aerobic glycolysis suppresses cancer cell stemness and strengthens GEM's cytotoxicity. GEM-induced metabolic reprogramming is KRAS dependent, as knockdown of KRAS reverses the metabolic shift. GEM-induced metabolic reprogramming also activates AMP-activated protein kinase (AMPK), which promotes glycolytic flux and cancer stemness. In addition, GEM-induced reactive oxygen species (ROS) activate the KRAS/AMPK pathway. This effect was validated by introducing exogenous hydrogen peroxide (H2O2). Taken together, these findings reveal a counterproductive GEM effect during PanCa treatment. Regulating cellular redox, targeting KRAS/AMPK signaling, or reversing metabolic reprogramming might be effective approaches to eliminate cancer stem cells (CSCs) and enhance chemosensitivity to GEM to improve the prognosis of PanCa patients.

Combination chemotherapy of valproic acid (VPA) and gemcitabine regulates STAT3/Bmi1 pathway to differentially potentiate the motility of pancreatic cancer cells.

BACKGROUND: Gemcitabine is the standard first-line chemotherapy regimen for pancreatic cancer. However, its therapeutic value is substantially limited in pancreatic cancer patients due to occurrence of resistance towards gemcitabine. A strategy of combined chemo-regimens is widely employed in clinical settings in attempt to reduce the chance of developing therapeutic resistance. Valproic acid (VPA) has been reported as a promising anticancer drug in various clinical trials and studies. However, the clinical value and potential dose-effect of VPA in combination with gemcitabine for pancreatic cancer treatment are under investigated. RESULTS: In this study, we determined the synergistic effect of VPA and gemcitabine and found that high-dose VPA significantly and dose-dependently enhanced the sensitivity of pancreatic cancer cells to gemcitabine. Intriguingly, low-dose VPA potentiated the migration and invasion of pancreatic cancer cells that already showed gemcitabine-induced motility. Moreover, low-dose VPA increased the reactive oxygen species (ROS) production, which activated AKT to further stimulate the activation of STAT3, Bmi1 expression and eventually promoted the migration and invasion of pancreatic cancer cells induced by gemcitabine. Whereas high-dose VPA stimulated excessive ROS accumulation that promoted p38 activation, which suppressed the activation of STAT3 and Bmi1. CONCLUSION: Pancreatic cancer cells respond differentially towards low- or high-dose of VPA in combination with gemcitabine, and a low VPA further potentiate pancreatic cancer cell to migrate and invade. Our results suggest that STAT3/Bmi1 signaling cascade, which is regulated by ROS-dependent, AKT- or p38-modulated pathways, primarily mediated the sensitivity and motility of pancreatic cancer cells towards combined gemcitabine and VPA regimen. These findings suggest a highly clinically relevant new mechanism of developing resistance against combined chemo-regimens, warranting further mechanistic and translational exploration for VPA in combination with gemcitabine and other chemotherapies.

High glucose promotes pancreatic cancer cells to escape from immune surveillance via AMPK-Bmi1-GATA2-MICA/B pathway.

BACKGROUND: Modulation of cell surface expression of MHC class I chain-related protein A/B (MICA/B) has been proven to be one of the mechanisms by which tumor cells escape from NK cell-mediated killing. Abnormal metabolic condition, such as high glucose, may create a cellular stress milieu to induce immune dysfunction. Hyperglycemia is frequently presented in the majority of pancreatic cancer patients and is associated with poor prognosis. In this study, we aimed to detect the effects of high glucose on NK cell-mediated killing on pancreatic cancer cells through reduction of MICA/B expression. METHODS: The lysis of NK cells on pancreatic cancer cells were compared at different glucose concentrations through lactate dehydrogenase release assay. Then, qPCR, Western Blot, Flow cytometry and Immunofluorescence were used to identify the effect of high glucose on expression of MICA/B, Bmi1, GATA2, phosphorylated AMPK to explore the underlying mechanisms in the process. Moreover, an animal model with diabetes mellitus was established to explore the role of high glucose on NK cell-mediated cytotoxicity on pancreatic cancer in vivo. RESULTS: In our study, high glucose protects pancreatic cancer from NK cell-mediated killing through suppressing MICA/B expression. Bmi1, a polycomb group (PcG) protein, was found to be up-regulated by high glucose, and mediated the inhibition of MICA/B expression through promoting GATA2 in pancreatic cancer. Moreover, high glucose inhibited AMP-activated protein kinase signaling, leading to high expression of Bmi1. CONCLUSION: Our findings identify that high glucose may promote the immune escape of pancreatic cancer cells under hyperglycemic tumor microenvironment. In this process, constitutive activation of AMPK-Bmi1-GATA2 axis could mediate MICA/B inhibition, which may serve as a therapeutic target for further intervention of pancreatic cancer immune evasion.

Mechanistic Evaluation and Translational Signature of Gemcitabine-induced Chemoresistance by Quantitative Phosphoproteomics Analysis with iTRAQ Labeling Mass Spectrometry.

One of the main causations of the poor prognosis of pancreatic cancer is the lack of effective chemotherapies. Gemcitabine is a widely used chemotherapeutic drug, but limited therapeutic efficacy is achieved due to chemoresistance. Recent studies demonstrated that the presence of cancer stem cells may lead to the failure of chemotherapy. Moreover, gemcitabine can promote the stemness of pancreatic cancer cells. We detected the alterations in protein phosphorylation and signaling pathways in pancreatic cancer cells after gemcitabine treatment using iTRAQ labeling LC-MS/MS, because it was featured with the advantages of strong separation ability and analysis range. A total of 232 differentially expressed phosphorylated proteins were identified in this study. Gene Ontology analysis revealed that nuclear lumen, nuclear part and organelle lumen were enriched for cell components and protein binding, poly (A) RNA binding and RNA binding were enriched for molecular function. A variety of signaling pathways were enriched based on KEGG analysis. AMPK, mTOR and PI3K/Akt pathways were verified after gemcitabine exposure. Moreover, we found there were complex interactions of phosphorylated proteins in modulating cancer stemness induced by gemcitabine exposure based on PPIs map. Our experiments may identify potential targets and strategies for sensitizing pancreatic cancer cells to gemcitabine.

Up-regulation of glycolysis promotes the stemness and EMT phenotypes in gemcitabine-resistant pancreatic cancer cells.

Cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT)-type cells are considered as underlying causes of chemoresistance, tumour recurrence and metastasis in pancreatic cancer. We aimed to describe the mechanisms - particularly glycolysis - involved in the regulation of the CSC and EMT phenotypes. We used a gemcitabine-resistant (GR) Patu8988 cell line, which exhibited clear CSC and EMT phenotypes and showed reliance on glycolysis. Inhibition of glycolysis using 2-deoxy-D-glucose (2-DG) significantly enhanced the cytotoxicity of gemcitabine and inhibited the CSC and EMT phenotypes in GR cells both in vitro and in vivo. Intriguingly, the use of the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) restored the CSC and EMT phenotypes. H2 O2 produced changes similar to those of 2-DG, indicating that ROS were involved in the acquired cancer stemness and EMT phenotypes of GR cells. Moreover, doublecortin-like kinase 1 (DCLK1), a pancreatic CSC marker, was highly expressed and regulated the stemness and EMT phenotypes in GR cell. Both 2-DG and H2 O2 treatment suppressed DCLK1 expression, which was also rescued by NAC. Together, these findings revealed that glycolysis promotes the expression of DCLK1 and maintains the CSC and EMT phenotypes via maintenance of low ROS levels in chemoresistant GR cells. The glycolysis-ROS-DCLK1 pathway may be potential targets for reversing the malignant behaviour of pancreatic cancer.

Gemcitabine treatment promotes pancreatic cancer stemness through the Nox/ROS/NF-κB/STAT3 signaling cascade.

Gemcitabine, the standard chemotherapy drug for advanced pancreatic cancer, has shown limited benefits because of profound chemoresistance. However, the mechanism involved remains unclear. Cancer stem cells exhibit great tumorigenicity and are closely correlated with drug resistance and tumor relapse. In this study, we demonstrated that certain doses of gemcitabine increased the ratios of CD24+ and CD133+ cells and the expression of stemness-associated genes such as Bmi1, Nanog, and Sox2. The enhancement of stemness after gemcitabine treatment was accompanied by increased cell migration, chemoresistance, and tumorigenesis. Moreover, we found that gemcitabine promoted the binding of phosphorylated STAT3 to the promoter of Bmi1, Nanog, and Sox2 genes. Furthermore, inhibition of STAT3 partially reversed gemcitabine-induced sphere formation, migration, chemoresistance, and tumor relapse. We also demonstrated that the activation of STAT3 and gemcitabine-enhanced stemness was NADPH oxidase (Nox)-generated, ROS-dependent, and NF-κB partially mediated the process. Together, our results suggest a pivotal role of pancreatic cancer stem cells in developing chemoresistance toward gemcitabine treatment through the Nox/ROS/NF-κB/STAT3 signaling pathway. These findings will provide new insight for identifying potential targets that can be used to sensitize pancreatic cancer cells to chemotherapy.

Bmi1 inhibition enhances the sensitivity of pancreatic cancer cells to gemcitabine.

As the standard therapy for pancreatic cancer, gemcitabine shows limited efficacy in pancreatic cancer patients because of chemoresistance. Aberrant expression of Bmi1 has been reported to activate multiple growth-regulatory pathways and confer anti-apoptotic abilities to many cancer cells. However, the role of Bmi1 in response of pancreatic cancer cells towards gemcitabine resistance remains elusive. In this study, we found that certain dose of gemcitabine treatment induced Bmi1 expression in pancreatic cancer cells. Knockdown of Bmi1 enhanced ROS production and promoted the cytotoxic effect of gemcitabine. The increased oxidative stress upon gemcitabine treatment could disrupt mitochondrial membrane and decrease mitochondrial membrane potential, eventually leading to apoptosis. Bmi1 inhibition also suppressed the activation of NF-κB signaling and the expressions of downstream molecules in pancreatic cancer cells treated with gemcitabine. Moreover, we observed Bmi1 inhibition sensitized the pancreatic xenograft tumors to gemcitabine in vivo. Taken together, our study demonstrated that Bmi1 could decrease the sensitivity of pancreatic cancer cells to gemcitabine through increasing oxidative stress and inhibiting NF-κB signaling, thus Bmi1 may serve as a promising target for sensitizing pancreatic cancer cells to chemotherapy.