Cancer Stem Cell Quiescence Research Articles

Identification of SETD4 as an Onco-Immunological Biomarker Encompassing the Tumor Microenvironment, Prognoses, and Therapeutic Responses in Various Human Cancers.

SET domain-containing protein 4 (SETD4) is a histone methyltransferase that has been shown to modulate cell proliferation, differentiation, and inflammatory responses by regulating histone H4 trimethylation (H4K20me3). Previous reports have demonstrated its function in the quiescence of cancer stem cells as well as drug resistance in several cancers. A limited number of systematic studies have examined SETD4's role in the tumor microenvironment, pathogenesis, prognosis, and therapeutic response. Utilizing The Cancer Genome Atlas database, and other publicly accessible platforms, we comprehensively analyzed SETD4 gene expression, methylation patterns, and prognostic significance. Furthermore, we investigated its association with cancer-related pathways, the immune microenvironment, immunotherapy markers, and drug resistance signatures of chemotherapy. Additionally, qRT-PCR was performed to validate SETD4 expression in clinical specimens. The expression of SETD4 was abnormal across a variety of cancer types and the expression of SETD4 in colorectal cancer tissues was verified in clinical specimens. The upregulation of SETD4 may be a prognostic risk factor predicting poor overall survival and progression-free survival. The analysis revealed that the mRNA level of SETD4 was modulated by promoter methylation, and patients with lower methylation levels showed shorter survival times. Pathway analysis showed that SETD4 influenced several key cell cycle pathways, including the G2M checkpoint, and mitotic spindle pathways. In addition, SETD4 negatively affects immune cell infiltration in most cancers, including B cells, CD8 T cells, and macrophages. The correlation between SETD4 and cancer stemness as well as homologous recombination deficiency varied across tumor types, suggesting that SETD4 may play a multifaceted role in tumor resistance. Notably, we identified several potential agents targeting SETD4. This study demonstrates that SETD4 is an immune-oncogenic molecule in multiple cancers, with the potential to be a diagnosis, prognosis, and targeted therapy marker.

Emerging Role of Autophagy in Governing Cellular Dormancy, Metabolic Functions, and Therapeutic Responses of Cancer Stem Cells.

Tumors are composed of heterogeneous populations of dysregulated cells that grow in specialized niches that support their growth and maintain their properties. Tumor heterogeneity and metastasis are among the major hindrances that exist while treating cancer patients, leading to poor clinical outcomes. Although the factors that determine tumor complexity remain largely unknown, several genotypic and phenotypic changes, including DNA mutations and metabolic reprograming provide cancer cells with a survival advantage over host cells and resistance to therapeutics. Furthermore, the presence of a specific population of cells within the tumor mass, commonly known as cancer stem cells (CSCs), is thought to initiate tumor formation, maintenance, resistance, and recurrence. Therefore, these CSCs have been investigated in detail recently as potential targets to treat cancer and prevent recurrence. Understanding the molecular mechanisms involved in CSC proliferation, self-renewal, and dormancy may provide important clues for developing effective therapeutic strategies. Autophagy, a catabolic process, has long been recognized to regulate various physiological and pathological processes. In addition to regulating cancer cells, recent studies have identified a critical role for autophagy in regulating CSC functions. Autophagy is activated under various adverse conditions and promotes cellular maintenance, survival, and even cell death. Thus, it is intriguing to address whether autophagy promotes or inhibits CSC functions and whether autophagy modulation can be used to regulate CSC functions, either alone or in combination. This review describes the roles of autophagy in the regulation of metabolic functions, proliferation and quiescence of CSCs, and its role during therapeutic stress. The review further highlights the autophagy-associated pathways that could be used to regulate CSCs. Overall, the present review will help to rationalize various translational approaches that involve autophagy-mediated modulation of CSCs in controlling cancer progression, metastasis, and recurrence.

Opportunities in Cancer Therapies: Deciphering the Role of Cancer Stem Cells in Tumour Repopulation.

Tumour repopulation during treatment is a well acknowledged yet still challenging aspect of cancer management. The latest research results show clear evidence towards the existence of cancer stem cells (CSCs) that are responsible for tumour repopulation, dissemination, and distant metastases in most solid cancers. Cancer stem cell quiescence and the loss of asymmetrical division are two powerful mechanisms behind repopulation. Another important aspect in the context of cancer stem cells is cell plasticity, which was shown to be triggered during fractionated radiotherapy, leading to cell dedifferentiation and thus reactivation of stem-like properties. Repopulation during treatment is not limited to radiotherapy, as there is clinical proof for repopulation mechanisms to be activated through other conventional treatment techniques, such as chemotherapy. The dynamic nature of stem-like cancer cells often elicits resistance to treatment by escaping drug-induced cell death. The aims of this scoping review are (1) to describe the main mechanisms used by cancer stem cells to initiate tumour repopulation during therapy; (2) to present clinical evidence for tumour repopulation during radio- and chemotherapy; (3) to illustrate current trends in the identification of CSCs using specific imaging techniques; and (4) to highlight novel technologies that show potential in the eradication of CSCs.

The Interaction between DNMT1 and High-Mannose CD133 Maintains the Slow-Cycling State and Tumorigenic Potential of Glioma Stem Cell.

The quiescent/slow‐cycling state preserves the self‐renewal capacity of cancer stem cells (CSCs) and leads to the therapy resistance of CSCs. The mechanisms maintaining CSCs quiescence remain largely unknown. Here, it is demonstrated that lower expression of MAN1A1 in glioma stem cell (GSC) resulted in the formation of high‐mannose type N‐glycan on CD133. Furthermore, the high‐mannose type N‐glycan of CD133 is necessary for its interaction with DNMT1. Activation of p21 and p27 by the CD133–DNMT1 interaction maintains the slow‐cycling state of GSC, and promotes chemotherapy resistance and tumorigenesis of GSCs. Elimination of the CD133–DNMT1 interaction by a cell‐penetrating peptide or MAN1A1 overexpression inhibits the tumorigenesis of GSCs and increases the sensitivity of GSCs to temozolomide. Analysis of glioma samples reveals that the levels of high‐mannose type N‐glycan are correlated with glioma recurrence. Collectively, the high mannose CD133–DNMT1 interaction maintains the slow‐cycling state and tumorigenic potential of GSC, providing a potential strategy to eliminate quiescent GSCs.

Autophagy and senescence: Insights from normal and cancer stem cells.

Autophagy is a fundamental cellular process, which allows cells to adapt to metabolic stress through the degradation and recycling of intracellular components to generate macromolecular precursors and produce energy. Autophagy is also critical in maintaining cellular/tissue homeostasis, as well preserving immunity and preventing human disease. Deregulation of autophagic processes is associated with cancer, neurodegeneration, muscle and heart disease, infectious diseases and aging. Research on a variety of stem cell types establish that autophagy plays critical roles in normal and cancer stem cell quiescence, activation, differentiation, and self-renewal. Considering its critical function in regulating the metabolic state of stem cells, autophagy plays a dual role in the regulation of normal and cancer stem cell senescence, and cellular responses to various therapeutic strategies. The relationships between autophagy, senescence, dormancy and apoptosis frequently focus on responses to various forms of stress. These are interrelated processes that profoundly affect normal and abnormal human physiology that require further elucidation in cancer stem cells. This review provides a current perspective on autophagy and senescence in both normal and cancer stem cells.

Stem cell quiescence and its clinical relevance.

Quiescent state has been observed in stem cells (SCs), including in adult SCs and in cancer SCs (CSCs). Quiescent status of SCs contributes to SC self-renewal and conduces to averting SC death from harsh external stimuli. In this review, we provide an overview of intrinsic mechanisms and extrinsic factors that regulate adult SC quiescence. The intrinsic mechanisms discussed here include the cell cycle, mitogenic signaling, Notch signaling, epigenetic modification, and metabolism and transcriptional regulation, while the extrinsic factors summarized here include microenvironment cells, extracellular factors, and immune response and inflammation in microenvironment. Quiescent state of CSCs has been known to contribute immensely to therapeutic resistance in multiple cancers. The characteristics and the regulation mechanisms of quiescent CSCs are discussed in detail. Importantly, we also outline the recent advances and controversies in therapeutic strategies targeting CSC quiescence.

Tuning Cancer Fate: Tumor Microenvironment's Role in Cancer Stem Cell Quiescence and Reawakening.

Cancer cell dormancy is a common feature of human tumors and represents a major clinical barrier to the long-term efficacy of anticancer therapies. Dormant cancer cells, either in primary tumors or disseminated in secondary organs, may reawaken and relapse into a more aggressive disease. The mechanisms underpinning dormancy entry and exit strongly resemble those governing cancer cell stemness and include intrinsic and contextual cues. Cellular and molecular components of the tumor microenvironment persistently interact with cancer cells. This dialog is highly dynamic, as it evolves over time and space, strongly cooperates with intrinsic cell nets, and governs cancer cell features (like quiescence and stemness) and fate (survival and outgrowth). Therefore, there is a need for deeper insight into the biology of dormant cancer (stem) cells and the mechanisms regulating the equilibrium quiescence-versus-proliferation are vital in our pursuit of new therapeutic opportunities to prevent cancer from recurring. Here, we review and discuss microenvironmental regulations of cancer dormancy and its parallels with cancer stemness, and offer insights into the therapeutic strategies adopted to prevent a lethal recurrence, by either eradicating resident dormant cancer (stem) cells or maintaining them in a dormant state.

SET Domain-Containing Protein 4 Epigenetically Controls Breast Cancer Stem Cell Quiescence.

Quiescent cancer stem cells (CSC) play important roles in tumorigenesis, relapse, and resistance to chemoradiotherapy. However, the determinants of CSC quiescence and how they sustain themselves to generate tumors and relapse beyond resistance to chemoradiotherapy remains unclear. Here, we found that SET domain-containing protein 4 (SETD4) epigenetically controls breast CSC (BCSC) quiescence by facilitating heterochromatin formation via H4K20me3 catalysis. H4K20me3 localized to the promoter regions and regulated the expression of a set of genes in quiescent BCSCs (qBCSC). SETD4-defined qBCSCs were resistant to chemoradiotherapy and promoted tumor relapse in a mouse model. Upon activation, a SETD4-defined qBCSC sustained itself in a quiescent state by asymmetric division and concurrently produced an active daughter cell that proliferated to produce a cancer cell population. Single-cell sequence analysis indicated that SETD4+ qBCSCs clustered together as a distinct cell type within the heterogeneous BCSC population. SETD4-defined quiescent CSCs were present in multiple cancer types including gastric, cervical, ovarian, liver, and lung cancers and were resistant to chemotherapy. SETD4-defined qBCSCs had a high tumorigenesis potential and correlated with malignancy and chemotherapy resistance in clinical breast cancer patients. Taken together, the results from our previous study and current study on six cancer types reveal an evolutionarily conserved mechanism of cellular quiescence epigenetically controlled by SETD4. Our findings provide insights into the mechanism of tumorigenesis and relapse promoted by SETD4-defined quiescent CSCs and have broad implications for clinical therapies. SIGNIFICANCE: These findings advance our knowledge on the epigenetic determinants of quiescence in cancer stem cell populations and pave the way for future pharmacologic developments aimed at targeting drug-resistant quiescent stem cells.

Involvement of Polo-like kinase 1 (Plk1) in quiescence regulation of cancer stem-like cells of the gastric cancer cell lines.

Cancer stem cells (CSCs) have been hypothesized to initiate tumor growth and be resistant to chemoradiotherapy, and these processes appear to be closely related to CSC quiescence. Here, a CSC-like cell population with a high level of CD44 expression was obtained from the human gastric cancer cell lines MKN45 and MKN74. Using a PKH26-labeling retention assay, quiescent CSC-like cells with low levels of Ki67 and PCNA expression were found in spheres formed in serum-free medium, and exhibited resistance to drug and radiation treatments. Polo-like kinase 1 (Plk1) and ribosomal S6 kinase 1 (RSK1) were silenced in the quiescent CSC-like cells. The Plk1-specific inhibitors inhibited the activation of RSK1 and induced quiescence in the CSC-like cells, but increased RSK1 activity and resulted in apoptosis in non-CSCs. Furthermore, RSK1 silencing by inhibitors activated Plk1 and had no effect on the growth of spheres in the CSC-like cells, but did not affect phosphorylation of Plk1 and led to decreased proliferation in non-CSCs. Our results showed that Plk1 and RSK1 play important roles in the conversion of CSCs between active and quiescent states.

The quintessential quiescence of cancer stem cells: a struggle towards better treatment

The quintessential quiescence of cancer stem cells: a struggle towards better treatment

Cancer Stem Cell Quiescence and Plasticity as Major Challenges in Cancer Therapy.

Cells with stem-like properties, tumorigenic potential, and treatment-resistant phenotypes have been identified in many human malignancies. Based on the properties they share with nonneoplastic stem cells or their ability to initiate and propagate tumors in vivo, such cells were designated as cancer stem (stem-like) or tumor initiating/propagating cells. Owing to their implication in treatment resistance, cancer stem cells (CSCs) have been the subject of intense investigation in past years. Comprehension of CSCs' intrinsic properties and mechanisms they develop to survive and even enhance their aggressive phenotype within the hostile conditions of the tumor microenvironment has reoriented therapeutic strategies to fight cancer. This report provides selected examples of malignancies in which the presence of CSCs has been evidenced and briefly discusses methods to identify, isolate, and functionally characterize the CSC subpopulation of cancer cells. Relevant biological targets in CSCs, their link to treatment resistance, proposed targeting strategies, and limitations of these approaches are presented. Two major aspects of CSC physiopathology, namely, relative in vivo quiescence and plasticity in response to microenvironmental cues or treatment, are highlighted. Implications of these findings in the context of the development of new therapies are discussed.

Abstract B35: BMP regulation of cancer stem cell quiescence is responsible for chemotherapeutic resistance in glioblastoma

Abstract Introduction: Cancer stem cells (CSCs) represent a distinct cellular population that has been implicated in tumor recurrence following chemotherapy. Accumulating evidence indicates that BMP signaling plays an important role in the regulation of CSCs, and may be involved in the resistance of CSCs to cytotoxic drugs. The role of BMP pathway regulation of the CSC phenotype responsible for treatment resistance in glioblastoma (GBM) has not been explored. Methods: Paraffin-embedded (FFPE) samples from GBM patients were used to evaluate the expression of markers for BMP signaling (pSMAD1/5/8), proliferation (PCNA), and stemness (SOX2) by immunostaining. We tested the self-renewal capacity of primary patient-derived glioma stem cell (GSC) lines using a sphere formation assay. Expression of stemness markers SOX2 and Bmi-1 in BMP4 treated and untreated GSCs, was analyzed by Western blotting. We also performed xenograft transplantation, whereby GSCs were injected intracranially into immunedeficient mice. When tumor formation was confirmed post-injection, mice were administrated EdU and sacrificed at successive time points thereafter. The frequency of all EdU positive cells as well as ID-1positive/ EdU positive cells was quantified from the primary tumor sections throughout the labeling and chase. To study chemoresistance in glioblastoma, GSC lines were treated with graded concentrations of temozolomide (TMZ). Results: We found that pSMAD1/5/8 positive cells in primary patient tumors were largely PCNA-negative. BMP4 treatment inhibited GSC proliferation and self-renewal (sphere-formation), but did not abolish expression of markers of stemness, such as Sox2 and Bmi-1, or tumorigenicity. In our in vivo model, we found ID-1 positive cells retain EdU following a long-term chase. Taken together, these results demonstrate that BMP4-enriched cells possess the characteristics of a quiescent stem cell population. BMP4 treatment protects GSCs against TMZ-induced growth inhibition. Furthermore, pSMAD1/5/8-expressing cells do not possess the DNA damage marker gamma-H2AX following TMZ treatment, suggesting that activation of the BMP pathway protects GSCs against TMZ-induced DNA damage. Conclusion: This study establishes that the BMP signaling pathway is involved in the maintenance of GSC quiescence and may play a role in glioblastoma chemoresistance. Citation Format: Megan YiJun Wu, Angela Celebre, Jeffrey Chan, Jennifer Guan, Karan Dhand, James Loukides, Jason Karamchandani, Sunit Das. BMP regulation of cancer stem cell quiescence is responsible for chemotherapeutic resistance in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B35.

ΔNp63α induces quiescence and downregulates the BRCA1 pathway in estrogen receptor-positive luminal breast cancer cell line MCF7 but not in other breast cancer cell lines

Despite apparent resection of tumors, breast cancer patients often suffer relapse due to remnant dormant tumor cells. Although quiescence of cancer stem cells is thought as one of the mechanisms regulating dormancy, the mechanism underlying quiescence is unclear. Since ΔNp63α, an isoform of p51/p63, is crucial in the maintenance of stem cells within mammary epithelium, we investigated its roles in the regulation of dormancy in normal and malignant breast cells. Inducible expression of ΔNp63α in MCF7 estrogen receptor positive (ER+) luminal breast cancer cells led to quiescence and acquisition of progenitor-like properties. Judging from mRNA-microRNA microarray analysis, activation of bone morphogenetic protein (BMP) signaling and inhibition of Wnt signaling emerged as prominent mechanisms underlying ΔNp63α-dependent induction of quiescence and acquisition of stemness in MCF7. More interestingly, through Ingenuity Pathway analysis, we found for the first time that BRCA1 pathway was the most significantly downregulated pathway by ΔNp63α expression in quiescent MCF7 cells, where miR-205 was a downstream mediator. Furthermore, ΔNp63α-expressing MCF7 cells exhibited resistance to paclitaxel and doxorubicin. Expression of ΔNp63α in normal MCF10A basal cells increased proliferation and stemness, but did not affect more aggressive luminal (T47D) and basal (MDA-MB-231) cells with p53 mutation. Gene expression datasets analyses suggested that ΔNp63 expression is associated with relapse-free survival of luminal A/B-type patients, but not of the other subtypes. Our results established a cell type-specific function of ΔNp63α in induction of quiescence and downregulation of the BRCA1 pathway which suggested a role of ΔNp63α in the dormancy of luminal breast cancers.

OC-008 A 3D primary tumour drug screen identifies hedgehog signalling as a key regulator of intestinal cancer stem cell quiescence and tumorigenesis

<h3>Introduction</h3> Tumour dormancy and cellular quiescence have recently been strongly implicated in disease recurrence after adjuvant therapy in colorectal cancer (CRC). CRC is increasingly recognised as a heterogeneous disease with significant implications for medical and surgical management. Little is known about the control of cellular quiescence and the molecular features of dormant cells in CRC. Here we present the molecular and functional features of dormant intestinal tumour cells and describe the paracrine signalling pathways that govern their behaviour. <h3>Method</h3> Transgenic Apc^1322t/H2BYFP mice were generated to allow the characterisation, both functionally and molecularly of dormant tumour cells. A drug screen was performed on <i><i>ex vivo</i></i>3D matrigel-cultured tumour spheroids. Proliferation kinetics, growth characteristics and molecular alterations were correlated with known signalling pathways implicated in intestinal tumorigenesis. Selected compounds were then advanced to validation using six human colorectal cancer cell lines, that represent the three main subtypes of CRC, in 2D and 3D culture experiments. <h3>Results</h3> Dormant tumour cells represent a small population of tumour cells that are highly clonogenic, chemoresistant and share many features similar to reserve intestinal stem cells. The Hedgehog and BMP pathways play a dominant role in controlling the growth of tumour spheroids in culture. Antagonism of the Hedgehog pathway causes spheroid growth collapse, the activation of dormant tumour cells and yet no cytotoxicity. The effects of Hedgehog antagonism appears to be CRC sub-type specific; with the most marked effects in the commonest CCSN1 (CIN) subtype of CRC. <h3>Conclusion</h3> Here we provide the first molecular and functional characterisation of dormant tumour cells, showing them to represent the hypothesised quiescent cancer stem cell. Interestingly, our data confirms that of others, in demonstrating Wnt inactivation fails to effect the growth of Apc-mutant tumour cells. However we show that the Hedgehog pathway appears to control not only spheroid growth at a global level but also and uniquely the activation of dormant tumour cells. Manipulation of the Hedgehog pathway in combination with standard-of-care chemotherapy may provide significant benefits for patients with classical Apc-mutated and Wnt driven CRC. <h3>Disclosure of interest</h3> None Declared. <h3>References</h3> Buczacki SJ, Zecchini HI, Nicholson AM, <i>et al</i>. Intestinal label-retaining cells are secretory precursors expressing Lgr5. Nature 2013;495(7439):65–9 Buczacki SJA, Davies RJ. The confounding effects of tumour heterogeneity and cellular plasticity on personalized surgical management of colorectal cancer. Colorectal Dis. 2014;16(5):329–31

HIF1α Regulates mTOR Signaling and Viability of Prostate Cancer Stem Cells.

Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSC), were recently identified and characterized in prostate cancer. A well-characterized murine model of prostate cancer was used to investigate the regulation of hypoxia-inducible factor 1α (HIF1A/HIF1α) in CSCs and a basal stem cell subpopulation (Lin(-)/Sca-1(+)/CD49f(+)) was identified, in primary prostate tumors of mice, with elevated HIF1α expression. To further analyze the consequences of hypoxic upregulation on stem cell proliferation and HIF1α signaling, CSC subpopulations from murine TRAMP-C1 cells (Sca-1(+)/CD49f(+)) as well as from a human prostate cancer cell line (CD44(+)/CD49f(+)) were isolated and characterized. HIF1α levels and HIF target gene expression were elevated in hypoxic CSC-like cells, and upregulation of AKT occurred through a mechanism involving an mTOR/S6K/IRS-1 feedback loop. Interestingly, resistance of prostate CSCs to selective mTOR inhibitors was observed because of HIF1α upregulation. Thus, prostate CSCs show a hypoxic deactivation of a feedback inhibition of AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway through HIF1α is critical for CSC quiescence and maintenance by attenuating CSC metabolism and growth via mTOR and promoting survival by AKT signaling. We also propose that prostate CSCs can exhibit primary drug resistance to selective mTOR inhibitors. This work contributes to a deeper understanding of hypoxic regulatory mechanisms in CSCs and will help devise novel therapies against prostate cancer.

Highlights of This Issue

High-grade serous ovarian cancer (HGSOC) remains one of the most lethal cancer types despite aggressive standard treatment. HGSOC is characterized by widespread recurrent regions of copy number gain and loss. Luo and colleagues performed genomic analyses and interrogated a diverse number of genes that are recurrently amplified in HGSOC and are essential for cancer proliferation and survival in ovarian cancer cell lines. One of the genes, FGF receptor substrate 2 (FRS2), encodes an adaptor protein and induces transformation in immortalized human cell lines. These results implicate FRS2 as an oncogene and a potential therapeutic target in a subset of HGSOC that harbors FRS2 amplifications.Telomere maintenance is essential for oncogenic transformation of human cells through the bypass of senescence, known to be an important tumor-suppressive mechanism. In addition to the three activities known to regulate telomere function (telomerase, shelterin, and the CST complex), a fourth complex was found to interact with these, the POLA/primase complex. Diotti and colleagues demonstrate that the polymerase-α (p180) enzyme and its regulatory subunit (p68) interact with telomeric complexes and are important for telomere function. Therefore specific DNA replication components play a role at telomeres and can be seen as relevant targets against tumor cell growth.Neuroblastoma is a common solid tumor in children, and high-risk patients often present with genomic alterations suggesting the importance of DNA repair fidelity. Therefore, it is critical to identify components of the alternative NHEJ pathway as promising new therapeutic targets. Newman and colleagues identify a potential mechanistic explanation for the frequent chromosomal rearrangements found in neuroblastoma tumors. Importantly, clinical tumor specimens with high mRNA expression of alternative NHEJ pathway components have worse survival outcomes than those with low expression. These findings are of interest to both basic scientists in the DNA repair community and to clinical oncologists.HIF1α is a master regulator of adaptive responses to hypoxia, and its activation leads to metabolic reprogramming, angiogenesis, and tumor progression. Its role in hypoxic signaling in cancer stem cells (CSC) is not well known. Marhold and colleagues show that elevated HIF1α in a prostate CSC subpopulation leads to deregulated PI3K/AKT/mTOR. This suggests that CSCs rely on HIF1α-mediated inhibition of mTOR, which promotes CSC quiescence and survival in the hypoxic tumor microenvironment. Downregulation of HIF1α consequently attenuates this effect and increases proliferation of CSCs. This study also implicates primary resistance of CSCs to selective mTOR inhibition and provides a rationale for the use of dual PI3K/mTOR inhibitors.

Abstract 3047: Hypoxia leads to deregulation of PI3K/AKT/mTOR signaling in prostate cancer stem cells

Abstract Tumor initiating subpopulations of cancer cells, also known as cancer progenitor or cancer stem cells (CSCs) were recently identified and characterized in prostate cancer. Emerging evidence suggests that hypoxia-inducible factor 1 alpha (HIF1A) contributes to CSC maintenance and influences the PI3K/AKT/mTOR axis, a pathway which is frequently altered in prostate cancer. In our study, we assessed the effects of hypoxia on PI3K/AKT/mTOR signaling in a subset of basal prostate cancer stem cells in a murine model of prostate cancer. First, we successfully isolated Lin-/Sca-1+/CD49f+ basal stem cell subpopulations from primary prostate tumors of TRAMP (transgenic adenocarcinoma of mouse prostate) mice. We observed a consistent decrease in S6 phosphorylation within the CSC subpopulation, in agreement with an mTOR pathway inhibition. However, AKT phosphorylation in the CSC subpopulation was simultaneously elevated, thus prompting further investigations. To analyze the molecular pathways leading to this deregulation, we used a cell line model and isolated Sca-1+/CD49f+ CSC-like subpopulations from the murine prostate cancer cell line TRAMP-C1 and CD44+/CD49f+ CSC-like subpopulations from the androgen independent human prostate cancer cell line DU145. We used sphere formation assays and expression of Notch 1 and Oct 3/4 to confirm the stem and progenitor cell properties of the sorted subpopulations. We could confirm the decrease in mTOR/S6 signaling and identify the upregulation of AKT through IRS-1 mediated feedback loop in prostate CSC-like cells under hypoxic conditions. This AKT/mTOR deregulation was promptly reversed in normoxia, suggestive of the involvement of hypoxia inducible factors. We found elevated HIF1A protein levels as well as increased target gene expression in CSC subpopulations in vitro and in vivo. Further, lentiviral knockdown of HIF1A restored AKT/mTOR activity in CSC-like cells. We could show that CSC-like prostate cancer cells with elevated expression of basal stem cell markers, such as CD44, Sca-1 or CD49f, show a hypoxic deactivation of feedback inhibition on AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway via HIF1A may be critical for cancer stem cell quiescence and maintenance, slowing down cancer stem cell metabolism and growth via mTOR and promoting their survival via AKT signaling. Citation Format: Maximilian Marhold, Erwin Tomasich, Zuzana Pernicová, Radek Fedr, Karel Soucek, Andreas Spittler, Michael Krainer, Peter Horak. Hypoxia leads to deregulation of PI3K/AKT/mTOR signaling in prostate cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2014-3047

Stem Cell Quiescence

AbstractAdult stem cells are maintained in a quiescent state but are able to exit quiescence and rapidly expand and differentiate in response to stress. The quiescent state appears to be necessary for preserving the self-renewal of stem cells and is a critical factor in the resistance of cancer stem cells (CSCs) to chemotherapy and targeted therapies. Limited knowledge about quiescence mechanisms has prevented significant advances in targeting of drug-resistant quiescent CSCs populations in the clinic. Thus, an improved understanding of the molecular mechanisms of quiescence in adult stem cells is critical for the development of molecularly targeted therapies against quiescent CSCs in different cancers. Recent studies have provided a better understanding of the intrinsic and extrinsic regulatory mechanisms that control stem cell quiescence. It is now appreciated that the p53 gene plays a critical role in regulating stem cell quiescence. Other intrinsic regulatory mechanisms include the FoxO, HIF-1α, and NFATc1 transcription factors and signaling through ATM and mTOR. Extrinsic microenvironmental regulatory mechanisms include angiopoietin-1, TGF-β, bone morphogenic protein, thrombopoietin, N-cadherin, and integrin adhesion receptors; Wnt/β-catenin signaling; and osteopontin. In this article, we review current advances in understanding normal stem cell quiescence, their significance for CSC quiescence and drug resistance, and the potential clinical applications of these findings. Clin Cancer Res; 17(15); 4936–41. ©2011 AACR.

Mechanistic analysis of a novel, telomerase-specific oncolytic adenovirus targeting human gastric cancer stem cells.

e13626 Background: Cancer stem cells (CSCs) are established firmly to play a key role in tumor formation, maintenance, and resistance to the conventional therapies. This resistance is reported to depend on quiescence of CSCs, and controlling cell cycle of CSCs is indispensable to eliminate CSCs. We constructed a telomerase-specific oncolytic adenovirus (OBP-301 [Telomelysin]), in which the hTERT promoter drives expression of E1 genes. OBP-301 causes selective replication and lysis of a variety of human cancer cells. A phase I study has been completed in the US to confirm the safety and pharmacokinetics of OBP-301 in patients with advanced solid tumors. Since telomerase activity is one of the stem-cell properties, OBP-301 may be capable of targeting human CSCs. Here, we examined the molecular mechanism of OBP-301-mediated cytotoxicity on CD133+ CSCs isolated from human gastric cancer cells. Methods: Radioresistant MKN45 and MKN7 human gastric cancer cells were obtained by sequential irradiation, and sorted for CD133+ and CD133− cells by flow cytometry. The CD133+ cells were confirmed for the characteristics of CSCs. We compared antitumor effects of chemotherapeutic agents, ionizing radiation, and OBP-301 on CD133+ and CD133-cells by XTT analysis. Western blotting was performed to determine the protein expression essential for the maintenance of stemness. Results: Although the fraction of CD133+ cells was 1-2% in untreated cells, radiation increased the CD133+ population to 18-20%. The CD133+ cells only have asymmetric division ability, sphere formation ability, and tumorigenicity. The cell viability assay demonstrated that CD133+ cells were less sensitive to the chemotherapeutic agents and ionizing radiation compared to CD133- cells, whereas OBP-301 could efficiently kill both CD133+ and CD133- cells. As CD133+ cells showed a higher telomerase activity, OBP-301 could replicate more efficiently in CD133+ cells. OBP-301 mediated cell cycle progression on quiescent CD133+ cells, while protein expression associated with the growth and stemness was reduced. Conclusions: Our data indicate that telomerase-specific OBP-301 might be a promising anticancer treatment against human gastric CSCs. No significant financial relationships to disclose.