Stem-like Characteristics Research Articles

Characterization of gastric cancer stem-like molecular features, immune and pharmacogenomic landscapes.

Cancer stem cells (CSCs) actively reprogram their tumor microenvironment (TME) to sustain a supportive niche, which may have a dramatic impact on prognosis and immunotherapy. However, our knowledge of the landscape of the gastric cancer stem-like cell (GCSC) microenvironment needs to be further improved. A multi-step process of machine learning approaches was performed to develop and validate the prognostic and predictive potential of the GCSC-related score (GCScore). The high GCScore subgroup was not only associated with stem cell characteristics, but also with a potential immune escape mechanism. Furthermore, we experimentally demonstrated the upregulated infiltration of CD206+ tumor-associated macrophages (TAMs) in the invasive margin region, which in turn maintained the stem cell properties of tumor cells. Finally, we proposed that the GCScore showed a robust capacity for prediction for immunotherapy, and investigated potential therapeutic targets and compounds for patients with a high GCScore. The results indicate that the proposed GCScore can be a promising predictor of prognosis and responses to immunotherapy, which provides new strategies for the precision treatment of GCSCs.

Gastric Organoids: Progress and Remaining Challenges.

The stomach is a complex and physiologically necessary organ, yet large differences in physiology between mouse and human stomachs have impeded translation of physiological discoveries and drug screens performed using murine gastric tissues. Gastric cancer (GC) is a global health threat, with a high mortality rate and limited treatment options. The heterogeneous nature of GC makes it poorly suited for current “one size fits all” standard treatments. In this review, we discuss the rapidly evolving field of gastric organoids, with a focus on studies expanding cultures from primary human tissues and describing the benefits of mouse organoid models. We introduce the differing methods for culturing healthy gastric tissue from adult tissues or pluripotent stem cells, discuss the promise these systems have for preclinical drug screens, and highlight applications of organoids for precision medicine. Finally, we discuss the limitations of these models and look to the future to present potential ways gastric organoids will advance treatment options for patients with GC.

P04.03 Different role of integrin a6 in glioblastoma proneural and mesenchymal stem-like cells

Abstract BACKGROUND The plasticity of tumoral cells and the presence of cells displaying stem-like features are two interrelated traits of Glioblastoma (GBM) lesions and both concur in defining its heterogeneity. Particularly, GBM stem-like cells (GSC) can be classified according to trascriptional data in different subgroups, being the Proneural (PN-GSC) and the Mesenchymal (MES-GSC) the most consolidated clusters. GSC are responsible of most of the malignant characteristics of GBM, including therapeutic resistance and tumor recurrence. Therefore, a better understanding of the mechanisms regulating GSC responsiveness to therapy taking into account GSC molecular heterogeneity may help to improve patient’s outcome. Integrin a6 is a commonly used marker for GSC capable to enrich for GSC population and sustain stemness. We investigate the role of integrin a6 in both PN and MES GSC on stemness and radioresistance. MATERIAL AND METHODS The expression of integrin a6 was analyzed in GSC cultures obtained from post-surgical specimens either displaying PN or MES trascriptional traits. Using cell sorting to enrich for integrin a6 expression (integrin a6-high and a6-low) and gene silencing with lentiviral-based shRNA, integrin a6 impact on both GSC cultures was tested. Also, silenced MES-GSC were analysed by means of RNA-seq. The major pathways found altered by integrin a6 silencing were validated at functional level using gliomasphere-based clonogenic assay, extreme limiting dilution assay and gamma-H2AX to monitor DNA damage repair kinetics. RESULTS After sorting GSC cultures by integrin a6 expression, PN-GSC a6-low showed a significant reduction in clonogenic capability and gliomasphere size when compared to a6-high (p<0.0001). On the contrary, sorted MES-GSC did not display any differences. Similar results were obtained following integrin a6 lentiviral silencing. However, RNAseq on silenced MES-GSC revealed a significant impact on cell cycle regulation and DNA damage repair pathways. Indeed, Integrin a6 trascriptional inhibition in MES-GSCs impaired the capacity to clear gamma-H2AX foci after ionizing radiation (p<0.001) and significantly alters MES-GSC capacity to recover from radiation treatment in gliomasphere formation assay (p<0.01). According to the interpretation of the curves with the linear quadratic model, integrin a6 silenced cells displayed higher alpha- and beta- values and lower alpha/beta ratio. The obtained values demonstrate increased radiosensitivity and impaired capacity to repair sublethal DNA damage, in addition to an enhanced sensitivity to fractionated doses. CONCLUSION The data obtained showed that Integrin a6 regulates proliferation and stemness-related features in PN-GSC while supports radioresistance of MES-GSCs. Altogether, we reveal that integrin a6 controls different stem-associated features in GSCs depending on the molecular subtype.

Apatinib triggers autophagic and apoptotic cell death via VEGFR2/STAT3/PD-L1 and ROS/Nrf2/p62 signaling in lung cancer

BackgroundRecently, a variety of clinical trials have shown that apatinib, a small-molecule anti-angiogenic drug, exerts promising inhibitory effects on multiple solid tumors, including non-small cell lung cancer (NSCLC). However, the underlying molecular mechanism of apatinib on NSCLC remains unclear.MethodsMTT, EdU, AO/EB staining, TUNEL staining, flow cytometry, colony formation assays were performed to investigate the effects of apatinib on cell proliferation, cell cycle distribution, apoptosis and cancer stem like properties. Wound healing and transwell assays were conducted to explore the role of apatinib on migration and invasion. The regulation of apatinib on VEGFR2/STAT3/PD-L1 and ROS/Nrf2/p62 signaling were detected. Furthermore, we collected conditioned medium (CM) from A549 and H1299 cells to stimulate phorbol myristate acetate (PMA)-activated THP-1 cells, and examined the effect of apatinib on PD-L1 expression in macrophages. The Jurkat T cells and NSCLC cells co-culture model was used to assess the effect of apatinib on T cells activation. Subcutaneous tumor formation models were established to evaluate the effects of apatinib in vivo. Histochemical, immunohistochemical staining and ELISA assay were used to examine the levels of signaling molecules in tumors.ResultsWe showed that apatinib inhibited cell proliferation and promoted apoptosis in NSCLC cells in vitro. Apatinib induced cell cycle arrest at G1 phase and suppressed the expression of Cyclin D1 and CDK4. Moreover, apatinib upregulated Cleaved Caspase 3, Cleaved Caspase 9 and Bax, and downregulated Bcl-2 in NSCLC cells. The colony formation ability and the number of CD133 positive cells were significantly decreased by apatinib, suggesting that apatinib inhibited the malignant and stem-like features of NSCLC cells. Mechanistically, apatinib inhibited PD-L1 and c-Myc expression by targeting VEGFR2/STAT3 signaling. Apatinib also inhibited PD-L1 expression in THP-1 derived macrophages stimulated by CM from NSCLC cells. Furthermore, apatinib pretreatment increased CD69 expression and IFN-γ secretion in stimulated Jurkat T cells co-cultured with NSCLC cells. Apatinib also promoted ROS production and inhibited Nrf2 and p62 expression, leading to the autophagic and apoptotic cell death in NSCLC. Moreover, apatinib significantly inhibited tumor growth in vivo.ConclusionOur data indicated that apatinib induced autophagy and apoptosis in NSCLC via regulating VEGFR2/STAT3/PD-L1 and ROS/Nrf2/p62 signaling.

Green Synthesized Honokiol Transfersomes Relieve the Immunosuppressive and Stem-Like Cell Characteristics of the Aggressive B16F10 Melanoma.

BackgroundHonokiol (HK) is a natural bioactive compound with proven antineoplastic properties against melanoma. However, it shows very low bioavailability when administered orally. Alternatively, topical administration may offer a promising route. The objective of the current study was to fabricate HK transfersomes (HKTs) for topical treatment of melanoma. As an ultradeformable carrier system, transfersomes can overcome the physiological barriers to topical treatment of melanoma: the stratum corneum and the anomalous tumor microenvironment. Moreover, the immunomodulatory and stemness-regulation roles of HKTs were the main interest of this study.MethodsTFs were prepared using the modified scalable heating method. A three-factor, three-level Box–Behnken design was utilized for the optimization of the process and formulation variables. Intracellular uptake and cytotoxicity of HKTs were evaluated in nonactivated and stromal cell–activated B16F10 melanoma cells to investigate the influence of the complex tumor microenvironment on the efficacy of HK. Finally, ELISA and Western blot were performed to evaluate the expression levels of TGF-β and clusters of differentiation (CD47 and CD133, respectively).ResultsThe optimized formula exhibited a mean size of 190 nm, highly negative surface charge, high entrapment efficiency, and sustained release profile. HKTs showed potential to alleviate the immunosuppressive characteristics of B16F10 melanoma in vitro via downregulation of TGF-β signaling. In addition, HKTs reduced expression of the “do not eat me” signal — CD47. Moreover, HKTs possessed additional interesting potential to reduce the expression of the stem-like cell marker CD133. These outcomes were boosted upon combination with metformin, an antihyperglycemic drug recently reported to possess different functions in cancer, while combination with collagenase, an extracellular matrix–depleting enzyme, produced detrimental effects.ConclusionHKTs represent a promising scalable formulation for treatment of the aggressive B16F10 melanoma, which is jam-packed with immunosuppressive and stem-like cell markers.

ETV7 regulates breast cancer stem-like cell features by repressing IFN-response genes

Cancer stem cells (CSCs) represent a population of cells within the tumor able to drive tumorigenesis and known to be highly resistant to conventional chemotherapy and radiotherapy. In this work, we show a new role for ETV7, a transcriptional repressor member of the ETS family, in promoting breast cancer stem-like cells plasticity and resistance to chemo- and radiotherapy in breast cancer (BC) cells. We observed that MCF7 and T47D BC-derived cells stably over-expressing ETV7 showed reduced sensitivity to the chemotherapeutic drug 5-fluorouracil and to radiotherapy, accompanied by an adaptive proliferative behavior observed in different culture conditions. We further noticed that alteration of ETV7 expression could significantly affect the population of breast CSCs, measured by CD44+/CD24low cell population and mammosphere formation efficiency. By transcriptome profiling, we identified a signature of Interferon-responsive genes significantly repressed in cells over-expressing ETV7, which could be responsible for the increase in the breast CSCs population, as this could be partially reverted by the treatment with IFN-β. Lastly, we show that the expression of the IFN-responsive genes repressed by ETV7 could have prognostic value in breast cancer, as low expression of these genes was associated with a worse prognosis. Therefore, we propose a novel role for ETV7 in breast cancer stem cells’ plasticity and associated resistance to conventional chemotherapy and radiotherapy, which involves the repression of a group of IFN-responsive genes, potentially reversible upon IFN-β treatment. We, therefore, suggest that an in-depth investigation of this mechanism could lead to novel breast CSCs targeted therapies and to the improvement of combinatorial regimens, possibly involving the therapeutic use of IFN-β, with the aim of avoiding resistance development and relapse in breast cancer.

Phytomedicines Targeting Cancer Stem Cells: Therapeutic Opportunities and Prospects for Pharmaceutical Development.

The presence of small subpopulations of cells within tumor cells are known as cancer stem cells (CSCs). These cells have been the reason for metastasis, resistance with chemotherapy or radiotherapy, and tumor relapse in several types of cancers. CSCs underwent to epithelial–mesenchymal transition (EMT) and resulted in the development of aggressive tumors. CSCs have potential to modulate numerous signaling pathways including Wnt, Hh, and Notch, therefore increasing the stem-like characteristics of cancer cells. The raised expression of drug efflux pump and suppression of apoptosis has shown increased resistance with anti-cancer drugs. Among many agents which were shown to modulate these, the plant-derived bioactive agents appear to modulate these key regulators and were shown to remove CSCs. This review aims to comprehensively scrutinize the preclinical and clinical studies demonstrating the effects of phytocompounds on CSCs isolated from various tumors. Based on the available convincing literature from preclinical studies, with some clinical data, it is apparent that selective targeting of CSCs with plants, plant preparations, and plant-derived bioactive compounds, termed phytochemicals, may be a promising strategy for the treatment of relapsed cancers.

MOMC-1. Employing the Zika Virus to kill paediatric nervous system tumour cells

Malignant paediatric nervous system tumours, such as Medulloblastoma, Neuroblastoma and ATRT commonly harbour tumour cells with stem-like features which are highly tumorigenic and resistant to conventional cancer therapies. These tumours can exhibit high lethality and may result in severe sequelae, including cognitive and motor deficits that significantly affect patients’ quality of life. Oncolytic virotherapy is a novel therapy class that exploits viruses that preferentially infect and destroy tumour cells. These viruses present a unique advantage in targeting highly heterogeneous cancers, such as nervous system tumours, as they possess a secondary mechanism of action through which they induce a tumour-specific immune response. Clinical studies employing oncolytic virotherapy have in general reported low toxicity and minimal adverse effects, deeming oncolytic virotherapy as a potentially attractive and safer intervention against paediatric tumours.The Zika virus (ZIKV) is capable of infecting and destroying neural stem-like cancer cells from human embryonal Central Nervous System (CNS) tumours in vitro and in vivo. Infection of CNS tumour cells with ZIKV effectively inhibits tumour metastasis in mice and, in some cases, induces complete tumour remission. Neuroblastoma arises from immature nerve cells and multiple Neuroblastoma cell lines are susceptible to ZIKV infection and oncolysis. These initial findings have demonstrated the potential for a ZIKV-based virotherapy against paediatric nervous system tumours and warrants examination into the molecular mechanisms through which ZIKV executes its oncolytic ability. My research goal is to elucidate the mechanisms which are of paramount importance for ZIKV-induced oncolysis of brain tumour and Neuroblastoma cells. Utilising global expression omics profiling of ZIKV infection and mapping of viral protein-host protein interactions will identify these mechanisms both at the cellular pathway and molecular levels. These collectively will inform our understanding of how we can employ a future ZIKV-based virotherapy against paediatric nervous system tumours.

Abstract 2155: BRCAness in HGSC cancer initiation

Abstract Introduction: The luteal phase of BRCA mutation carrier tissues has distinct profiles of differentially expressed genes compared to wild-type fimbria. High Grade Serous Ovarian Carcinoma (HGSOC) is one of the most lethal gynecological diseases and presents at late tumor stage. Studies focusing on the early events of the disease have implicated the fimbriated end of the fallopian tube as the high-risk zone for cancer development. In this study, we aim to identify BRCA transcriptomic signatures in the fimbria of patients with a BRCA mutation and compare the luteal and follicular phases to capture the effect of the extrinsic milieu in response to stressors. Experimental Procedures: Laser capture microdissection (LCM) was performed on 68 formalin fixed paraffin embedded (FFPE) fallopian tube samples based on BRCA mutation status and ovulatory cycle: Normal-Follicular, Normal-Luteal, BRCA-Follicular, BRCA-Luteal. Nine HGSC samples were derived from patients with a mutation in BRCA1. Six-Twelve, 10um FFPE sections were cut and stained with hematoxylin prior to LCM. RNA was extracted using the Roche High Pure FFPE Micro Kit and samples and were processed using Illumina Tru-Seq Stranded Total RNA Kit with RiboGold ready and sequenced on the Illumina Hi-seq 2000 V3. Bioinformatic analysis performed on multiple comparisons highlighted differentially expressed genes. Gene Set Enrichment Analysis (GSEA) performed on subgroups highlighted pathways of interest for each comparison. Genes highly expressed or strongly suppressed were shortlisted for further analysis. Western blot analysis performed on wildtype patients (WT), BRCA1 mutation and BRCA2 mutation showed protein expression of selected genes. Results: The fimbriae compared to ampullae have more stem-like features, have up-regulated antioxidant/xenobiotic genes, and have more inflammatory cells intercalated within the epithelia. The fimbria of FTE-BRCA have distinctly up-regulated genes involved in DDR (DNA damage response) which are exacerbated in the luteal phase of the ovulatory cycle. BRCA1/2 luteal versus follicular phases are involved in endoplasmic reticulum stress, migration, MAPKK. Whereas decreased differentially expressed genes are involved with G2/M checkpoint, DNA replication and ER-associated degradation pathway. Post-ovulatory phase of the ovarian cycle drives inflammation, differentiation and DNA damage-repair in FTE cells. Furthermore, GSEA analysis of the luteal and follicular phases showed positive enrichment of the inflammatory response pathway and negative enrichment of oxidative phosphorylation in the luteal phase. Conclusions: Our results highlight distinct pathways related to repair and inflammation for the BRCA1 mutation carriers and luteal phase of the fimbria. These results are consistent with previous examination of the fallopian tube and provide insight into targetable genes associated with the development of HGSC. Citation Format: Ramlogan Sowamber, Iru Paudel, Alex Sanchez, Alexandra Diaz, Patricia Shaw, Matthew Schlumbrecht, Sophia George. BRCAness in HGSC cancer initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2155.

Pan-cancer network disorders revealed by overall and local signaling entropy.

Tumor development is a process involving loss of the differentiation phenotype and acquisition of stem-like characteristics, which is driven by intracellular rewiring of signaling network. The measurement of network reprogramming and disorder would be challenging due to the complexity and heterogeneity of tumors. Here, we proposed signaling entropy (SR) to assess the degree of tumor network disorder. We calculated SR for 33 tumor types in The Cancer Genome Atlas database based on transcriptomic and proteomic data. The SR of tumors was significantly higher than that of normal samples and was highly correlated with cell stemness, cancer type, tumor grade, and metastasis. We further demonstrated the sensitivity and accuracy of using local SR in prognosis prediction and drug response evaluation. Overall, SR could reveal cancer network disorders related to tumor malignant potency, clinical prognosis, and drug response.

3D Multicellular Stem-Like Human Breast Tumor Spheroids Enhance Tumorigenicity of Orthotopic Xenografts in Athymic Nude Rat Model.

Simple SummaryBreast cancer presents a unique clinical problem because of the variety of cellular subtypes present, including cancer stem cells (CSCs). Breast CSCs can induce the formation of new blood vessels at the site of tumor growth and a develop metastatic phenotype by enhancing a stromal cell response, similar to that of the primary breast cancer. The aim of this study was to investigate breast cancer cells cultured in stromal stem cell factor-supplemented media to generate 3D spheroids that exhibit increased stem-like properties. These 3D stem-like spheroids reproducibly and efficiently established orthotopic breast cancer xenografts in the athymic nude rat. This approach enables a means to develop orthotopic tumors with a stem-like phenotype in a larger athymic rat rodent model of human breast cancer.Therapeutic targeting of stem cells needs to be strategically developed to control tumor growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes present, including cancer stem cells (CSCs). The development of 3D stem-like properties of human breast tumor spheroids in stem cell factor conditioned media was investigated in orthotopic xenografts for enhanced tumorgenicity in the athymic nude rat model. MCF-7, ZR-75-1, and MDA-MB-231 breast cancer cell lines were cultured in serum-free, stem cell factor-supplemented medium under non-adherent conditions and passaged to generate 3rd generation spheroids. The spheroids were co-cultured with fetal lung fibroblast (FLF) cells before orthotopic heterotransplantation into the mammary fat pads of athymic nude rats. Excised xenografts were assessed histologically by H&E staining and immunohistochemistry for breast cancer marker (ERB1), proliferation marker (Ki67), mitotic marker (pHH3), hypoxia marker (HIF-2α), CSC markers (CD47, CD44, CD24, and CD133), and vascularization markers (CD31, CD34). Breast cancer cells cultured in stem cell factor supplemented medium generated 3D spheroids exhibited increased stem-like characteristics. The 3D stem-like spheroids co-cultured with FLF as supporting stroma reproducibly and efficiently established orthotopic breast cancer xenografts in the athymic nude rat.

Jorunnamycin A Suppresses Stem-Like Phenotypes and Sensitizes Cisplatin-Induced Apoptosis in Cancer Stem-Like Cell-Enriched Spheroids of Human Lung Cancer Cells

It has been recognized that cancer stem-like cells (CSCs) in tumor tissue crucially contribute to therapeutic failure, resulting in a high mortality rate in lung cancer patients. Due to their stem-like features of self-renewal and tumor formation, CSCs can lead to drug resistance and tumor recurrence. Herein, the suppressive effect of jorunnamycin A, a bistetrahydroisoquinolinequinone isolated from Thai blue sponge Xestospongia sp., on cancer spheroid initiation and self-renewal in the CSCs of human lung cancer cells is revealed. The depletion of stemness transcription factors, including Nanog, Oct-4, and Sox2 in the lung CSC-enriched population treated with jorunnamycin A (0.5 μM), resulted from the activation of GSK-3β and the consequent downregulation of β-catenin. Interestingly, pretreatment with jorunnamycin A at 0.5 μM for 24 h considerably sensitized lung CSCs to cisplatin-induced apoptosis, as evidenced by upregulated p53 and decreased Bcl-2 in jorunnamycin A-pretreated CSC-enriched spheroids. Moreover, the combination treatment of jorunnamycin A (0.5 μM) and cisplatin (25 μM) also diminished CD133-overexpresssing cells presented in CSC-enriched spheroids. Thus, evidence on the regulatory functions of jorunnamycin A may facilitate the development of this marine-derived compound as a novel chemotherapy agent that targets CSCs in lung cancer treatment.

Expanded ATLAS™-identified neoantigen-specific peripheral T cells (NPTs) demonstrate considerable neoantigen breadth, polyfunctionality, and effector function.

Abstract Adoptive T cell therapy using ex vivo expanded autologous tumor-infiltrating lymphocytes (TIL) has resulted in notable tumor regression across multiple solid tumor indications. Despite their success, TIL harvest requires invasive surgery and sterile isolation. In addition, extracted cells from an immunosuppressive tumor microenvironment (TME) often have impaired functions, low neoantigen-specificity and require high dose cytokine for expansion. All possible reasons for non-responders or post-treatment relapse. Generating T cell therapies specifically expanded against neoantigen targets and derived from peripheral blood lymphocytes may overcome these limitations. The ATLAS bioassay identifies patient-specific neoantigen targets of T cells, and Inhibigens™, tumor antigens that are detrimental to protective immune responses. Using PLANET™, a robust and scalable closed manufacturing process, T cells specific for up to 30 ATLAS-identified neoantigens (excluding Inhibigens) are expanded, providing considerable breadth of tumor recognition and the potential to limit tumor escape. Use of peripheral T cells leverages robust cells not suppressed by the TME and eliminates the need for invasive tumor resection. Our data show that NPTs are specific for up to 89% of targeted neoantigens, consist of non-exhausted effector and central memory cells, and express proliferative and tissue homing markers. NPTs are highly polyfunctional, secreting multiple combinations of IFNγ, Granzyme B, TNFα, and MIP1α in response to specific neoantigens. Evaluation of markers for memory-progenitor stem-like features in NPTs are being explored and will be discussed. The TITAN™ clinical trial evaluating GEN-011 NPTs is ongoing (NCT04596033).

TET2 controls differentiation of terminally exhausted CD8 T cells

Abstract CD8 T cells are critical for clearing infections and immune surveillance of cancers. However, in the setting of chronic viral infections and tumor growth, CD8 T cells become exhausted. T cell exhaustion (TEX) is characterized by a progressive increase of inhibitory receptor expression and concomitant diminishment of effector function. This diminished function renders the host unable to clear the virus or cancer. The TEX population is heterogenous and can be divided broadly into TEX progenitor and TEX terminal populations. TEX progenitor are self-renewing, responsive to PD-1 blockade and give rise to the later TEX subsets, including short-lived TEX terminal cells. Control of DNA demethylation is required in multiple settings for maintenance or loss of stem-like characteristics. We report that TET2, a methylcytosine dioxygenase that mediates active DNA demethylation, is required for TEX progenitors to differentiate fully into TEX terminal cells. TET2-deficiency during persistent viral infection resulted in a numerical loss of TEX terminal cells, while maintaining the TEX progenitor population. The TEX terminal cells that escaped this differentiation block retained some features of TEX progenitors, including decreased expression of several inhibitory receptors. TET2-deficient TEX progenitors responded to PD-1 blockade but were unable to establish expansion of TEX cells thought to be required for viral control. These data highlight the role of DNA methylation and TET2 in establishing TEX diversity.

Transcriptional repressor BACH2 is a key regulator of stem-like CD8 T cells during chronic viral infection

Abstract During chronic viral infection and cancer, a CD8 T cell subset with stem-like characteristics maintains long-term T cell immunity and mediates the response induced by immune checkpoint blockade. The transcriptional and epigenetic programs of stem-like CD8 T cells diverged from those of other CD8 subsets early in the immune response. Using mice with chronic lymphocytic choriomeningitis virus (LCMV) infection, we found that the gene encoding transcriptional repressor BACH2 was highly expressed in stem-like CD8 T cells but was epigenetically silenced in terminally exhausted CD8 T cells early after infection. Ectopic expression of BACH2 enforced the transcriptional and epigenetic programs of stem-like CD8 T cells and restrained antiviral CD8 T cells in the lymphoid tissues. BACH2 deficiency impaired the differentiation of stem-like CD8 T cells and dysregulated their molecular program. We further demonstrated that BACH2 promoted stem-like CD8 T cell differentiation by suppressing BLIMP1 and RUNX3 pathways. In summary, our results showed that transcriptional repressor BACH2 drove stem-like CD8 T cell differentiation by guarding them against alternative cell fate.

Tyrosine Kinase c-MET as Therapeutic Target for Radiosensitization of Head and Neck Squamous Cell Carcinomas.

Simple SummaryThe overall five-year survival rate of patients with loco-regional advanced head and neck squamous cell carcinomas (HNSCC) is only around 40%. The curability of HNSCC with radiochemotherapy was previously found to be associated with clinical and biological parameters including tumor volume, hypoxia, epidermal growth factor receptor expression, and human papillomavirus infection status. Different signaling pathways, e.g., constitutively activated receptor tyrosine kinase signaling, increased DNA damage repair and intracellular defense against reactive oxygen species were identified as factors driving HNSCC progression and its resistance to therapy. c-MET was found to be hyperactivated in HNSCC and has been reported to drive tumor progression, therapy resistance, and metastatic spread. Here, we investigated the therapeutic potential of c-MET targeting strategies for HNSCC radiosensitization and discovered putative resistance mechanisms impeeding success of therapeutic intervention. This study highlights the importance of detailed knowledge about biological mechanisms and regulatory networks for future patient stratification and individualized treatment concepts.The receptor tyrosine kinase c-MET activates intracellular signaling and induces cell proliferation, epithelial-to-mesenchymal-transition and migration. Within the present study, we validated the prognostic value of c-MET in patients with head and neck squamous cell carcinoma (HNSCC) treated with radio(chemo)therapy using the Cancer Genome Atlas database and found an association of increased MET gene expression and protein phosphorylation with reduced disease-specific and progression-free survival. To investigate the role of c-MET-dependent radioresistance, c-MET-positive cells were purified from established HNSCC cell lines and a reduced radiosensitivity and enhanced sphere-forming potential, compared to the c-MET-depleted cell population, was found in two out of four analyzed cell lines pointing to regulatory heterogeneity. We showed that c-MET is dynamically regulated after irradiation in vitro and in vivo. Interestingly, no direct impact of c-MET on DNA damage repair was found. The therapeutic potential of eight c-MET targeting agents in combination with irradiation demonstrated variable response rates in six HNSCC cell lines. Amongst them, crizotinib, foretinib, and Pha665752 exhibited the strongest radiosensitizing effect. Kinase activity profiling showed an association of crizotinib resistance with compensatory PI3K/AKT and MAP kinase signaling. Overall, our results indicate that c-MET is conferring radioresistance in HNSCC through modulation of intracellular kinase signaling and stem-like features.

Roles of microRNAs in Regulating Cancer Stemness in Head and Neck Cancers.

Simple SummaryHead and neck squamous cell carcinomas (HNSCCs) are highly heterogeneous human malignancies associated with genetic and environmental factors. In HNSCCs, cancer stem cells (CSCs) provide the plasticity for cancer cell progression, metastasis, therapeutic resistance, and recurrence. During carcinogenesis, microRNAs (miRNAs) play important roles in regulating the maintenance and acquisition of cancer stem cell features. Therefore, in this review, we summarize the roles of miRNAs in regulating the cancer stemness of HNSCCs to provide potential therapeutic applications.Head and neck squamous cell carcinomas (HNSCCs) are epithelial malignancies with 5-year overall survival rates of approximately 40–50%. Emerging evidence indicates that a small population of cells in HNSCC patients, named cancer stem cells (CSCs), play vital roles in the processes of tumor initiation, progression, metastasis, immune evasion, chemo-/radioresistance, and recurrence. The acquisition of stem-like properties of cancer cells further provides cellular plasticity for stress adaptation and contributes to therapeutic resistance, resulting in a worse clinical outcome. Thus, targeting cancer stemness is fundamental for cancer treatment. MicroRNAs (miRNAs) are known to regulate stem cell features in the development and tissue regeneration through a miRNA–target interactive network. In HNSCCs, miRNAs act as tumor suppressors and/or oncogenes to modulate cancer stemness and therapeutic efficacy by regulating the CSC-specific tumor microenvironment (TME) and signaling pathways, such as epithelial-to-mesenchymal transition (EMT), Wnt/β-catenin signaling, and epidermal growth factor receptor (EGFR) or insulin-like growth factor 1 receptor (IGF1R) signaling pathways. Owing to a deeper understanding of disease-relevant miRNAs and advances in in vivo delivery systems, the administration of miRNA-based therapeutics is feasible and safe in humans, with encouraging efficacy results in early-phase clinical trials. In this review, we summarize the present findings to better understand the mechanical actions of miRNAs in maintaining CSCs and acquiring the stem-like features of cancer cells during HNSCC pathogenesis.

Cancer Cell Intrinsic and Immunologic Phenotypes Determine Clinical Outcomes in Basal-like Breast Cancer.

Basal-like breast cancer (BLBC) is a particularly aggressive intrinsic molecular subtype of breast cancer that lacks targeted therapies. There is also no clinically useful test to risk stratify patients with BLBC. We hypothesized that a transcriptome-based phenotypic characterization of BLBC tumors and their microenvironments may overcome these challenges. We conducted a retrospective correlative genomic sequencing study using a matched pairs design with validation in five independent cohorts. The study was conducted on a large population-based prospective cohort of the major molecular subtypes of breast cancer conducted in the greater Seattle-Puget Sound metropolitan area. Cases consisted of women 20-69 years of age first diagnosed with invasive breast cancer identified through the population-based Surveillance Epidemiology and End Results program. Patients for this analysis (n = 949) were identified from the 1,408 patients with stage I-III triple-negative breast cancer [estrogen receptor-negative (ER-), progesterone receptor-negative (PR-), HER2-]. Of the 949 women, 248 developed a recurrence after their initial diagnosis. A matched set of 67 recurrent and nonrecurrent BLBC tumors was subjected to transcriptome sequencing. Through RNA sequencing of the matched sets of recurrent and nonrecurrent BLBC tumors, we aimed to identify prognostic phenotypes.To identify nonredundant and uncorrelated prognostic genes, we used an ensemble of variable selection algorithms, which resulted in a ranking of genes on the basis of their expected utility in classification. Using leave-one-out cross-validation, we trained a random forest classifier on the basis of the top 21 genes (BRAVO-DX). Validations were performed in five independent triple-negative or BLBC cohorts, and biomarker robustness and transferability were demonstrated by employing real-time PCR. We found that cancer cell intrinsic and immunologic phenotypes are independent predictors of recurrence. By simultaneously interrogating the tumor and its microenvironment, we developed a compound risk model that stratified patients into low-, medium-, and high-risk groups, with a 14%/56%/74% chance of recurrence, respectively. Biologically, the primary tumors of patients who developed a recurrence had increased growth factor signaling and stem-like features, while nonrecurrent tumors showed high lymphocyte infiltration with clonal expansion of T and B cells, as well as antitumor polarization of macrophages. We validated our model in five independent cohorts, including three large cohorts, where BRAVO-DX was highly informative in identifying patients with disease recurrence [HR, 6.79 (95% confidence interval (CI), 1.89-24.37); HR, 3.45 (95% CI, 2.41-4.93); and HR, 1.69 (95% CI, 1.17-2.46)]. A smaller gene set focused on the tumor immunophenotype, BRAVO-IMMUNE, was highly prognostic in all five cohorts. Together, these results indicate that phenotypic characteristics of BLBCs and their microenvironment are associated with recurrence-free survival and demonstrate the utility of intrinsic and extrinsic phenotypes as independent prognostic biomarkers in BLBC. Pending further evaluation and validation, our prognostic model has the potential to inform clinical decision-making for patients with BLBC as it identifies those at high risk of rapidly progressing on standard chemotherapy, as well as those who may benefit from alternative first-line therapies.

Integrated Metabolomics and Transcriptomics Analysis of Monolayer and Neurospheres from Established Glioblastoma Cell Lines.

Simple SummaryGlioblastomas are very aggressive tumours without efficient treatment, where cancer stem-like cells are thought to be responsible for relapse. This pilot study investigated the metabolic discrepancies between monolayer and neurosphere cultures of two glioblastoma cell lines using transcriptomics and metabolomics. We show that the two culture systems display substantial differences regarding their metabolome and transcriptome. Specifically, we found that metabolic reactions connected to arginine biosynthesis are crucial to support the different metabolic needs of neurospheres from the two cell lines. By identifying metabolic vulnerabilities in different glioblastoma subpopulations, new therapeutic strategies may be emerging that can be explored to treat this disease. Moreover, this data set may be of great value as a resource for the scientific community.Altered metabolic processes contribute to carcinogenesis by modulating proliferation, survival and differentiation. Tumours are composed of different cell populations, with cancer stem-like cells being one of the most prominent examples. This specific pool of cells is thought to be responsible for cancer growth and recurrence and plays a particularly relevant role in glioblastoma (GBM), the most lethal form of primary brain tumours. Here, we have analysed the transcriptome and metabolome of an established GBM cell line (U87) and a patient-derived GBM stem-like cell line (NCH644) exposed to neurosphere or monolayer culture conditions. By integrating transcriptome and metabolome data, we identified key metabolic pathways and gene signatures that are associated with stem-like and differentiated states in GBM cells, and demonstrated that neurospheres and monolayer cells differ substantially in their metabolism and gene regulation. Furthermore, arginine biosynthesis was identified as the most significantly regulated pathway in neurospheres, although individual nodes of this pathway were distinctly regulated in the two cellular systems. Neurosphere conditions, as opposed to monolayer conditions, cause a transcriptomic and metabolic rewiring that may be crucial for the regulation of stem-like features, where arginine biosynthesis may be a key metabolic pathway. Additionally, TCGA data from GBM patients showed significant regulation of specific components of the arginine biosynthesis pathway, providing further evidence for the importance of this metabolic pathway in GBM.

Aurora-A kinase oncogenic signaling mediates TGF-\u03b2-induced triple-negative breast cancer plasticity and chemoresistance

Triple-negative breast cancer (TNBCs) account for 15–20% of all breast cancers and represent the most aggressive subtype of this malignancy. Early tumor relapse and progression are linked to the enrichment of a sub-fraction of cancer cells, termed breast tumor-initiating cells (BTICs), that undergo epithelial to mesenchymal transition (EMT) and typically exhibit a basal-like CD44high/CD24low and/or ALDH1high phenotype with critical cancer stem-like features such as high self-renewal capacity and intrinsic (de novo) resistance to standard of care chemotherapy. One of the major mechanisms responsible for the intrinsic drug resistance of BTICs is their high ALDH1 activity leading to inhibition of chemotherapy-induced apoptosis. In this study, we demonstrated that aurora-A kinase (AURKA) is required to mediate TGF-β-induced expression of the SNAI1 gene, enrichment of ALDH1high BTICs, self-renewal capacity, and chemoresistance in TNBC experimental models. Significantly, the combination of docetaxel (DTX) with dual TGF-β and AURKA pharmacologic targeting impaired tumor relapse and the emergence of distant metastasis. We also showed in unique chemoresistant TNBC cells isolated from patient-derived TNBC brain metastasis that dual TGF-β and AURKA pharmacologic targeting reversed cancer plasticity and enhanced the sensitivity of TNBC cells to DTX-based-chemotherapy. Taken together, these findings reveal for the first time the critical role of AURKA oncogenic signaling in mediating TGF-β-induced TNBC plasticity, chemoresistance, and tumor progression.