Polyamine Anabolism Promotes Chemotherapy-Induced Breast Cancer Stem Cell Enrichment.

Breast cancer patients may initially benefit from cytotoxic chemotherapy but experience treatment resistance and relapse. Chemoresistant breast cancer stem cells (BCSCs) play a pivotal role in cancer recurrence and metastasis, however, identification and eradication of BCSC population in patients are challenging. Here, an mRNA-based BCSC signature is developed using machine learning strategy to evaluate cancer stemness in primary breast cancer patient samples. Using the BCSC signature, a critical role of polyamine anabolism in the regulation of chemotherapy-induced BCSC enrichment, is elucidated. Mechanistically, two key polyamine anabolic enzymes, ODC1 and SRM, are directly activated by transcription factor HIF-1 in response to chemotherapy. Genetic inhibition of HIF-1-controlled polyamine anabolism blocks chemotherapy-induced BCSC enrichment in vitro and in xenograft mice. A novel specific HIF-1 inhibitor britannin is identified through a natural compound library screening, and demonstrate that coadministration of britannin efficiently inhibits chemotherapy-induced HIF-1 transcriptional activity, ODC1 and SRM expression, polyamine levels, and BCSC enrichment in vitro and in xenograft and autochthonous mouse models. The findings demonstrate the key role of polyamine anabolism in BCSC regulation and provide a new strategy for breast cancer treatment.

Unveiling a novel entry gate: Insect foregut as an alternative infection route for fungal entomopathogens.

Insects and their natural microbial pathogens are intertwined in constant arms races, with pathogens continually seeking entry into susceptible hosts through distinct routes. Entomopathogenic fungi are primarily believed to infect host insects through external cuticle penetration. Here, we report a new variety, Beauveria bassiana var. majus (Bbm), that can infect insects through the previously unrecognized foregut. Dual routes of infection significantly accelerate insect mortality. The pH-responsive transcription factor PacC in Bbm exhibits rapid upregulation and efficient proteolytic processing via PalC for alkaline adaptation in the foregut. Expression of PalC is regulated by the adjacent downstream gene Aia. Compared to non-enteropathogenic strains such as ARSEF252, Aia in Bbm lacks a 249-bp fragment, resulting in its enhanced alkaline-induced expression. This induction promotes PalC upregulation and facilitates PacC activation. Expressing the active form of BbmPacC in ARSEF252 enables intestinal infection. This study uncovers the pH-responsive Aia-PalC-PacC cascade enhancing fungal alkaline tolerance for intestinal infection, laying the foundation for developing a new generation of fungal insecticides to control destructive insect pests.

Hypoxia-inducible factor 1 recruits FACT and RNF20/40 to mediate histone ubiquitination and transcriptional activation of target genes.

Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying enzymes leading to changes in histone acetylation, citrullination, and methylation at target genes. Here, we demonstrate that hypoxia-inducible gene expression in estrogen receptor (ER)-positive MCF7 and ER-negative SUM159 human breast cancer cells requires the histone H2A/H2B chaperone facilitates chromatin transcription (FACT) and the H2B ubiquitin ligase RING finger protein 20/40 (RNF20/40). Knockdown of FACT or RNF20/40 expression leads to decreased transcription initiation and elongation at HIF-1 target genes. Mechanistically, FACT and RNF20/40 are recruited to hypoxia response elements (HREs) by HIF-1 and stabilize binding of HIF-1 (and each other) at HREs. Hypoxia induces the monoubiquitination of histone H2B at lysine 120 at HIF-1 target genes in an HIF-1-dependent manner. Together, these findings delineate a cooperative molecular mechanism by which FACT and RNF20/40 stabilize multiprotein complex formation at HREs and mediate histone ubiquitination to facilitate HIF-1 transcriptional activity.

The advances of E2A-PBX1 fusion in B-cell acute lymphoblastic Leukaemia.

The E2A-PBX1 gene fusion is a common translocation in B-cell acute lymphoblastic leukaemia. Patients harbouring the E2A-PBX1 fusion gene typically exhibit an intermediate prognosis. Furthermore, minimal residual disease has unsatisfactory prognostic value in E2A-PBX1 B-cell acute lymphoblastic leukaemia. However, the mechanism of E2A-PBX1 in the occurrence and progression of B-cell acute lymphoblastic leukaemia is not well understood. Here, we mainly review the roles of E2A and PBX1 in the differentiation and development of B lymphocytes, the mechanism of E2A-PBX1 gene fusion in B-cell acute lymphoblastic leukaemia, and the potential therapeutic approaches.

NARF is a hypoxia-induced coactivator for OCT4-mediated breast cancer stem cell specification.

Hypoxia is a key characteristic of the breast cancer microenvironment that promotes expression of the transcriptional activator hypoxia-inducible factor 1 (HIF-1) and is associated with poor patient outcome. HIF-1 increases the expression or activity of stem cell pluripotency factors, which control breast cancer stem cell (BCSC) specification and are required for cancer metastasis. Here, we identify nuclear prelamin A recognition factor (NARF) as a hypoxia-inducible, HIF-1 target gene in human breast cancer cells. NARF functions as an essential coactivator by recruiting the histone demethylase KDM6A to OCT4 bound to genes encoding the pluripotency factors NANOG, KLF4, and SOX2, leading to demethylation of histone H3 trimethylated at lysine-27 (H3K27me3), thereby increasing the expression of NANOG, KLF4, and SOX2, which, together with OCT4, mediate BCSC specification. Knockdown of NARF significantly decreased the BCSC population in vitro and markedly impaired tumor initiation capacity and lung metastasis in orthotopic mouse models.

Establishment and validation of a carbohydrate metabolism-related gene signature for prognostic model and immune response in acute myeloid leukemia.

Introduction: The heterogeneity of treatment response in acute myeloid leukemia (AML) patients poses great challenges for risk scoring and treatment stratification. Carbohydrate metabolism plays a crucial role in response to therapy in AML. In this multicohort study, we investigated whether carbohydrate metabolism related genes (CRGs) could improve prognostic classification and predict response of immunity and treatment in AML patients. Methods: Using univariate regression and LASSO-Cox stepwise regression analysis, we developed a CRG prognostic signature that consists of 10 genes. Stratified by the median risk score, patients were divided into high-risk group and low-risk group. Using TCGA and GEO public data cohorts and our cohort (1031 non-M3 patients in total), we demonstrated the consistency and accuracy of the CRG score on the predictive performance of AML survival. Results: The overall survival (OS) was significantly shorter in high-risk group. Differentially expressed genes (DEGs) were identified in the high-risk group compared to the low-risk group. GO and GSEA analysis showed that the DEGs were mainly involved in immune response signaling pathways. Analysis of tumor-infiltrating immune cells confirmed that the immune microenvironment was strongly suppressed in high-risk group. The results of potential drugs for risk groups showed that inhibitors of carbohydrate metabolism were effective. Discussion: The CRG signature was involved in immune response in AML. A novel risk model based on CRGs proposed in our study is promising prognostic classifications in AML, which may provide novel insights for developing accurate targeted cancer therapies.

Comprehensive analysis of FRAS1/FREM family as potential biomarkers and therapeutic targets in renal clear cell carcinoma.

Background: FRAS1 (Fraser syndrome protein 1), together with FREM1 (the Fras1-related extracellular matrix proteins 1) and FREM2, belonging to the FRAS1/FREM extracellular matrix protein family, are considered to play essential roles in renal organogenesis and cancer progression. However, their roles in kidney renal clear cell carcinoma (KIRC) remain to be elucidated. Methods: FRAS1/FREM RNA expression analysis was performed using TCGA/GTEx databases, and valided using GEO databases and real-time PCR. Protein expression was peformed using CPTAC databases. Herein, we employed an array of bioinformatics methods and online databases to explore the potential oncogenic roles of FRAS1/FREM in KIRC. Results: We found that FRAS1, FREM1 and FREM2 genes and proteins expression levels were significantly decreased in KIRC tissues than in normal tissues. Decreased FRAS1/FREM expression levels were significantly associated with advanced clinicopathological parameters (pathological stage, grade and tumor metastasis status). Notably, the patients with decreased FRAS1/FREM2 expression showed a high propensity for metastasis and poor prognosis. FRAS1/FREM were correlated with various immune infiltrating cells, especially CD4+ T cells and its corresponding subsets (Th1, Th2, Tfh and Tregs). FRAS1 and FREM2 had association with DNA methylation and their single CpG methylation levels were associated with prognosis. Moreover, FRAS1/FREM might exert antitumor effects by functioning in key oncogenic signalling pathways and metabolic pathways. Drug sensitivity analysis indicated that high FRAS1 and FREM2 expression can be a reliable predictor of targeted therapeutic drug response, highlighting the potential as anticancer drug targets. Conclusion: Together, our results indicated that FRAS1/FREM family members could be potential therapeutic targets and valuable prognostic biomarkers of KIRC.

Chemotherapy-induced adenosine A2B receptor expression mediates epigenetic regulation of pluripotency factors and promotes breast cancer stemness.

Rationale: Triple-negative breast cancer (TNBC) is characterized by its unique molecular profile, aggressive nature and lack of targeted therapy. Chemotherapy induces expression of pluripotency factors and mediates an active induction of breast cancer stem cells (BCSCs) in TNBC, which potentiates the risk of tumor recurrence and metastasis and increases patient mortality. Adenosine receptor 2B (A2BR) expression and activation of its downstream signaling pathway has been implied to promote breast cancer metastasis. This study is to investigate the role of A2BR in the regulation of chemotherapy-induced BCSC enrichment. Methods: We generated shRNA-mediated A2BR knockdown subclones in TNBC cell lines and evaluated the effect on the BCSC phenotype by Aldefluor and mammosphere assays in vitro. We performed chromatin immunoprecipitation (ChIP) assay to investigate recruitment of transcription factor FOXO3 and histone modification enzymes KDM6A and p300 to the regulatory regions of pluripotency factors, as well as levels of histone modification marks H3K27ac and H3K27me3 on these regions. We employed both xenograft model and genetically engineered, autochthonous breast cancer model to evaluate the effect of A2BR on chemotherapy-induced BCSC enrichment in vivo. Results: We demonstrated that chemotherapy increased protein level of A2BR, which contributed to chemotherapy-induced pluripotency factor expression and BCSC enrichment in TNBC. A2BR mediated activation of p38 MAPK and nuclear translocation of chromatin remodeling factor SMARCD3, which interacted and recruited histone demethylase KDM6A and histone acetyltransferase p300 specifically to the pluripotency factor genes NANOG, SOX2 and KLF4. Recruitment of KDM6A and p300 decreased histone H3K27me3 and increases H3K27ac marks, and increased transcription factor FOXO3 binding to NANOG, SOX2 and KLF4 genes, leading to transcriptional activation of these genes and BCSC specification. Genetic or pharmacological inhibition of A2BR blocked chemotherapy-mediated epigenetic activation of pluripotency factor genes and BCSC enrichment in vitro and in vivo, and delayed tumor recurrence after chemotherapy was discontinued. Conclusion: Chemotherapy-induced A2BR expression mediates epigenetic activation of pluripotency factors and promotes breast cancer stemness. Targeting A2BR in combination with chemotherapy may block BCSC enrichment and improve outcome in TNBC.

HIF-1 Interacts with TRIM28 and DNA-PK to release paused RNA polymerase II and activate target gene transcription in response to hypoxia.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a regulator of oxygen (O2) homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions but pauses after approximately 30-60 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1α and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain as well as the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies reveal a molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.

HIF-1 recruits NANOG as a coactivator for TERT gene transcription in hypoxic breast cancer stem cells.

Breast cancer stem cells (BCSCs) play essential roles in tumor formation, drug resistance, relapse, and metastasis. NANOG is a protein required for stem cell self-renewal, but the mechanisms by which it performs this function are poorly understood. Here, we show that hypoxia-inducible factor 1α (HIF-1α) is required for NANOG-mediated BCSC enrichment. Mechanistically, NANOG is recruited by HIF-1 to cooperatively activate transcription of the TERT gene encoding the telomerase reverse transcriptase that maintains telomere length, which is required for stem cell self-renewal. NANOG stimulates HIF-1 transcriptional activity by recruitment of the deubiquitinase USP9X, which inhibits HIF-1α protein degradation, and by stabilizing HIF-1α interaction with the coactivator p300, which mediates histone acetylation. Our results delineate a cooperative transcriptional mechanism by which HIF-1 and NANOG mediate BCSC self-renewal.

Histone citrullination by PADI4 is required for HIF-dependent transcriptional responses to hypoxia and tumor vascularization.

Hypoxia-inducible factors (HIFs) activate transcription of target genes by recruiting coactivators and chromatin-modifying enzymes. Peptidylarginine deiminase 4 (PADI4) catalyzes the deimination of histone arginine residues to citrulline. Here, we demonstrate that PADI4 expression is induced by hypoxia in a HIF-dependent manner in breast cancer and hepatocellular carcinoma cells. PADI4, in turn, is recruited by HIFs to hypoxia response elements (HREs) and is required for HIF target gene transcription. Hypoxia induces histone citrullination at HREs that is PADI4 and HIF dependent. RNA sequencing revealed that almost all HIF target genes in breast cancer cells are PADI4 dependent. PADI4 is required for breast and liver tumor growth and angiogenesis in mice. PADI4 expression is correlated with HIF-1α expression and vascularization in human breast cancer biopsies. Thus, HIF-dependent recruitment of PADI4 to target genes and local histone citrullination are required for transcriptional responses to hypoxia.

Hypoxia-inducible factor-dependent ADAM12 expression mediates breast cancer invasion and metastasis.

Breast cancer patients with increased expression of hypoxia-inducible factors (HIFs) in primary tumor biopsies are at increased risk of metastasis, which is the major cause of breast cancer-related mortality. The mechanisms by which intratumoral hypoxia and HIFs regulate metastasis are not fully elucidated. In this paper, we report that exposure of human breast cancer cells to hypoxia activates epidermal growth factor receptor (EGFR) signaling that is mediated by the HIF-dependent expression of a disintegrin and metalloprotease 12 (ADAM12), which mediates increased ectodomain shedding of heparin-binding EGF-like growth factor, an EGFR ligand, leading to EGFR-dependent phosphorylation of focal adhesion kinase. Inhibition of ADAM12 expression or activity decreased hypoxia-induced breast cancer cell migration and invasion in vitro, and dramatically impaired lung metastasis after orthotopic implantation of MDA-MB-231 human breast cancer cells into the mammary fat pad of immunodeficient mice.

Chemotherapy-induced S100A10 recruits KDM6A to facilitate OCT4-mediated breast cancer stemness.

Breast cancer stem cells (BCSCs) play a critical role in cancer recurrence and metastasis. Chemotherapy induces BCSC specification through increased expression of pluripotency factors, but how their expression is regulated is not fully understood. Here, we delineate a pathway controlled by hypoxia-inducible factor 1 (HIF-1) that epigenetically activates pluripotency factor gene transcription in response to chemotherapy. Paclitaxel induces HIF-1-dependent expression of S100A10, which forms a complex with ANXA2 that interacts with histone chaperone SPT6 and histone demethylase KDM6A. S100A10, ANXA2, SPT6, and KDM6A are recruited to OCT4 binding sites and KDM6A erases H3K27me3 chromatin marks, facilitating transcription of genes encoding the pluripotency factors NANOG, SOX2, and KLF4, which along with OCT4 are responsible for BCSC specification. Silencing of S100A10, ANXA2, SPT6, or KDM6A expression blocks chemotherapy-induced enrichment of BCSCs, impairs tumor initiation, and increases time to tumor recurrence after chemotherapy is discontinued. Pharmacological inhibition of KDM6A also impairs chemotherapy-induced BCSC enrichment. These results suggest that targeting HIF-1/S100A10-dependent and KDM6A-mediated epigenetic activation of pluripotency factor gene expression in combination with chemotherapy may block BCSC enrichment and improve clinical outcome.

Rational combination with PDK1 inhibition overcomes cetuximab resistance in head and neck squamous cell carcinoma.

Cetuximab, an EGFR-blocking antibody, is currently approved for treatment of metastatic head and neck squamous cell carcinoma (HNSCC), but its response rate is limited. In addition to blocking EGFR-stimulated cell signaling, cetuximab can induce endocytosis of ASCT2, a glutamine transporter associated with EGFR in a complex, leading to glutathione biosynthesis inhibition and cellular sensitization to ROS. Pyruvate dehydrogenase kinase-1 (PDK1), a key mitochondrial enzyme overexpressed in cancer cells, redirects glucose metabolism from oxidative phosphorylation toward aerobic glycolysis. In this study, we tested the hypothesis that targeting PDK1 is a rational approach to synergize with cetuximab through ROS overproduction. We found that combination of PDK1 knockdown or inhibition by dichloroacetic acid (DCA) with ASCT2 knockdown or with cetuximab treatment induced ROS overproduction and apoptosis in HNSCC cells, and this effect was independent of effective inhibition of EGFR downstream pathways but could be lessened by N-acetyl cysteine, an anti-oxidative agent. In several cetuximab-resistant HNSCC xenograft models, DCA plus cetuximab induced marked tumor regression, whereas either agent alone failed to induce tumor regression. Our findings call for potentially novel clinical trials of combining cetuximab and DCA in patients with cetuximab-sensitive EGFR-overexpressing tumors and patients with cetuximab-resistant EGFR-overexpressing tumors.

Hypoxia-inducible factor 1-dependent expression of adenosine receptor 2B promotes breast cancer stem cell enrichment.

Breast cancer stem cells (BCSCs), which are characterized by a capacity for unlimited self-renewal and for generation of the bulk cancer cell population, play a critical role in cancer relapse and metastasis. Hypoxia is a common feature of the cancer microenvironment that stimulates the specification and maintenance of BCSCs. In this study, we found that hypoxia increased expression of adenosine receptor 2B (A2BR) in human breast cancer cells through the transcriptional activity of hypoxia-inducible factor 1. The binding of adenosine to A2BR promoted BCSC enrichment by activating protein kinase C-δ, which phosphorylated and activated the transcription factor STAT3, leading to increased expression of interleukin 6 and NANOG, two key mediators of the BCSC phenotype. Genetic or pharmacological inhibition of A2BR expression or activity decreased hypoxia- or adenosine-induced BCSC enrichment in vitro, and dramatically impaired tumor initiation and lung metastasis after implantation of MDA-MB-231 human breast cancer cells into the mammary fat pad of immunodeficient mice. These data provide evidence that targeting A2BR might be an effective strategy to eradicate BCSCs.

Reciprocal Regulation of DUSP9 and DUSP16 Expression by HIF1 Controls ERK and p38 MAP Kinase Activity and Mediates Chemotherapy-Induced Breast Cancer Stem Cell Enrichment.

Triple-negative breast cancer (TNBC) has a poor prognosis due to its aggressive characteristics and lack of targeted therapies. Cytotoxic chemotherapy may reduce tumor bulk, but leaves residual disease due to the persistence of chemotherapy-resistant breast cancer stem cells (BCSC), which are critical for tumor recurrence and metastasis. Here, we demonstrate that hypoxia-inducible factor (HIF)-1-dependent regulation of mitogen-activated protein kinase (MAPK) signaling pathways contributes to chemotherapy-induced BCSC enrichment. Chemotherapy increased DUSP9 expression and decreased DUSP16 expression in a HIF1-dependent manner, leading to inhibition of ERK and activation of p38 signaling pathways, respectively. Inhibition of ERK caused transcriptional induction of the pluripotency factor Nanog through decreased inactivating phosphorylation of FoxO3, while activation of p38 stabilized Nanog and Klf4 mRNA through increased inactivating phosphorylation of RNA-binding protein ZFP36L1, both of which promoted specification of the BCSC phenotype. Inhibition of HIF1 or p38 signaling blocked chemotherapy-induced pluripotency factor expression and BCSC enrichment. These surprising results delineate a mechanism by which a transcription factor switches cells from ERK to p38 signaling in response to chemotherapy and suggest that therapeutic targeting of HIF1 or the p38 pathway in combination with chemotherapy will block BCSC enrichment and improve outcome in TNBC.Significance: These findings provide a molecular mechanism that may account for the increased relapse rate of women with TNBC who are treated with cytotoxic chemotherapy and suggest that combining chemotherapy with an inhibitor of HIF1 or p38 activity may increase patient survival. Cancer Res; 78(15); 4191-202. ©2018 AACR.

Chemotherapy-Induced Ca2+ Release Stimulates Breast Cancer Stem Cell Enrichment.

Breast cancer stem cells (BCSCs) play a critical role in tumor recurrence and metastasis. Exposure of breast cancer cells to chemotherapy leads to an enrichment of BCSCs. Here, we find that chemotherapy induces the expression of glutathione S-transferase omega 1 (GSTO1), which is dependent on hypoxia-inducible factor 1 (HIF-1) and HIF-2. Knockdown of GSTO1 expression abrogates carboplatin-induced BCSC enrichment, decreases tumor initiation and metastatic capacity, and delays tumor recurrence after chemotherapy. GSTO1 interacts with the ryanodine receptor RYR1 and promotes calcium release from the endoplasmic reticulum. Increased cytosolic calcium levels activate PYK2 → SRC → STAT3 signaling, leading to increased expression of pluripotency factors and BCSC enrichment. HIF inhibition blocks chemotherapy-induced GSTO1 expression and BCSC enrichment. Combining HIF inhibitors with chemotherapy may improve clinical outcome in breast cancer.

Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells.

Exposure of breast cancer cells to hypoxia increases the percentage of breast cancer stem cells (BCSCs), which are required for tumor initiation and metastasis, and this response is dependent on the activity of hypoxia-inducible factors (HIFs). We previously reported that exposure of breast cancer cells to hypoxia induces the ALKBH5-mediated demethylation of N6-methyladenosine (m6A) in NANOG mRNA leading to increased expression of NANOG, which is a pluripotency factor that promotes BCSC specification. Here we report that exposure of breast cancer cells to hypoxia also induces ZNF217-dependent inhibition of m6A methylation of mRNAs encoding NANOG and KLF4, which is another pluripotency factor that mediates BCSC specification. Although hypoxia induced the BCSC phenotype in all breast-cancer cell lines analyzed, it did so through variable induction of pluripotency factors and ALKBH5 or ZNF217. However, in every breast cancer line, the hypoxic induction of pluripotency factor and ALKBH5 or ZNF217 expression was HIF-dependent. Immunohistochemistry revealed that expression of HIF-1α and ALKBH5 was concordant in all human breast cancer biopsies analyzed. ALKBH5 knockdown in MDA-MB-231 breast cancer cells significantly decreased metastasis from breast to lungs in immunodeficient mice. Thus, HIFs stimulate pluripotency factor expression and BCSC specification by negative regulation of RNA methylation.