Cancer stem cells in cholangiocarcinoma: emerging roles as therapeutic targets

Covering the Cover

Cancer Stem Cell–Directed Therapies: Recent Data From the Laboratory and Clinic

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer.

Many cancers, including breast cancer are maintained by a subpopulation of cells that display stem cell properties, mediate metastasis and contribute to treatment resistance. These cancer stem cells are regulated by the interaction of cell intrinsic pathways as well as extracellular signals originating from the tumor microenvironment. Developing tumors recruit a diverse collection of cells that make up the tumor microenvironment and through iterative interactions both the tumor cell and its microenvironment co-evolve. The tumor microenvironment is composed of multiple cell types including mesenchymal cells, fibroblasts, endothelial cells and immune cells. We have utilized in vitro and mouse xenograft models to examine the interaction between breast cancer stem cells and cells within the microenvironment. We previously demonstrated that bone marrow derived mesenchymal stem cells like tumor stem cells are hierarchically organized and are recruited to the growing sites of breast cancers via cytokines such as interlukin-6 (IL-6). Mesenchymal stem cells interact with cancer stem cells forming “niches” which influences the growth and differentiation of both cellular compartments. Both IL-8 and IL-6 play seminal roles in the regulation of breast cancer stem cells. IL-6 also plays an important role in mediating resistance of breast cancer stem cells to a variety of therapeutic agents. These include development of resistance of HER2 overexpressing breast cancers to trastuzumab-targeted therapy. Although inactivation of the PTEN gene has been implicated in the development of resistance to HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance have remained elusive. We have found that development of trastuzumab resistance in PTEN deleted cells is mediated by activation of an IL-6 inflammatory loop leading to expansion of the cancer stem cell population. Interestingly, long term trastuzumab treatment generates highly enriched cancer stem cells with an epithelial mesenchymal (EMT) phenotype which secrete over 100-fold more IL-6 than parental cells. The IL-6 receptor antibody tociluzumab was able to interrupt this inflammatory loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenografts. These studies demonstrate that trastuzumab resistance may be mediated by an IL-6 inflammatory loop and suggests that blocking this loop represents a novel strategy to overcome trastuzumab resistance. Another inflammatory cytokine intimately involved in breast cancer stem cell regulation is IL-8. The IL-8 receptor CXCR1 is selectively over expressed in breast cancer stem cell populations. Recombinant IL-8 stimulates breast cancer self-renewal and a small molecule CXCR1 inhibitor, reparaxin is able to block this signaling pathway. Reparaxin is able to target breast cancer stem cells in mouse xenograft models reducing tumor growth and metastasis. Based on these preclinical models a clinical trial utilizing reparaxin in combination with chemotherapy for the treatment of women with advanced breast cancers is currently being planned. Together, these studies suggest that strategies aimed at blocking the interaction of breast cancer stem cells with the microenvironment may prove an effective strategy to target this important cell population.

Methylation of Cancer-Stem-Cell-Associated Wnt Target Genes Predicts Poor Prognosis in Colorectal Cancer Patients

Mitochondrial oxidative metabolism contributes to a cancer stem cell phenotype in cholangiocarcinoma

Moving Forward in Human Mammary Stem Cell Biology and Breast Cancer Prognostication Using ALDH1

Drug resistance, radioresistance and metastasis are the biggest hurdle in effective cancer treatment. Recent studies suggest that these problems might be caused by the presence of cancer stem cells (CSCs). CSCs are a small group of cells which can self-renew, remain undifferentiated, resist to cancer drugs and radiotherapy. It can be predicted that selective killing of CSCs may lead to tutor regression and may be a new therapeutic solution. Naturally occurring flavonoids are weakly active in inhibiting ABC transporter leading to drug accumulation in cancer cells. We have been working on the design, synthesis and characterisation of novel flavonoid dimers that can be used to reverse drug resistance in cancer cells. We recently discovered that a new class of triazole-linked flavonoid dimers 13_9 and 15_2 were effective in reversing drug resistance in CD44+CD24-/low MCF7 breast cancer stem cells at an effective concentration of less than 32 nM. Our studies suggested that a novel mechanism was involved which is independent of ABC transporters. To determine the mechanism behind, we have designed and synthesised a photoactivatable derivative of 13_9 (13_9X2) to identify the protein target(s). 13_9X2 contains a photoactivating functional group which can capture the target protein by forming a covalent bond under UV irradiation and a clickable functional group which can be used for pull-down and further identification using mass spectrometry. One candidate target is peroxiredoxin 1 (PRDX-1) which is a reactive oxygen species (ROS) scavenger. We hypothesise that CSCs are resistant to doxorubicin-induced ROS damage by overexpressing PRDX-1. By binding and disrupting PRDX-1, 13_9 and 15_2 reduce ROS scavenging ability of PRDX-1, resulting in the reversal of drug resistance in CSCs. This work provides new insights into the mechanisms of cell signalling regulation of cancer cell resistance in breast CSCs and provides a better understanding of how flavonoid dimers reverse drug resistance in CSCs. Additionally, this work contributes to the development of new anticancer drugs which can sensitize CSCs to current chemotherapy. Citation Format: Gege Sun, Iris L. Wong, Jiahua Cui, Zhen Liu, Bill T. Chan, Larry M. Chow. Investigate the mechanism of actions of flavonoid dimers in reversing doxorubicin resistance in breast cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3795.

Cholangiocarcinoma (CCAs) may be defined as tumors that derived from the biliary tree with the differentiation in the biliary epithelial cells. This tumor is malignant, extremely aggressive with a poor prognosis. It can be treated surgically and its pathogenesis is poorly understood. The tumor microenvironment (TME) is a very important factor in the regulation of tumor angiogenesis, invasion, and metastasis. Besides cancer stem cells (CSCs) can modulate tumor growth, stroma formation, and migratory capability. The initial stage of tumorigenesis is characterized by genetic mutations and epigenetic alterations due to intrinsic factors which lead to the generation of oncogenes thus inducing tumorigenesis. CSCs may result from precancerous stem cells, cell de-differentiation, normal stem cells, or an epithelial-mesenchymal transition (EMT). CSCs have been found in the cancer niche, and EMT may occur early within the tumor microenvironment. Previous studies have demonstrated evidence of cholangiocarcinoma stem cells (CD133, CD24, EpCAM, CD44, and others) and the presence of these markers has been associated with malignant potential. The interaction between TME and cholangiocarcinoma stem cells via signaling mediators may create an environment that accommodates tumor growth, yielding resistance to cytotoxic insults (chemotherarapeutic). While progress has been made in the understanding of the mechanisms, the interactions in the tumorigenic process still remain a major challenge. Our review, addresses recent concepts of TME-CSCs interaction and will emphasize the importance of early detection with the use of novel diagnostic mechanisms such as CCA-CSC biomarkers and the importance of tumor stroma to define new treatments. J. Cell. Physiol. 231: 768-776, 2016. © 2015 Wiley Periodicals, Inc.

The most promising therapeutic option is cancer stem cells (CSCs). CSCs, leaders of a cancer hierarchy, possess stem cell-like traits such as self-rehabilitation and the cognition to differentiate abnormally. CSCs do not have to be uncommon in tumors; cancer and non-cancer stem cells can change correctable phenotypic modification, and the phenotype of CSCs can differ significantly between the patients. The function of CSC metabolism and the mechanisms underlying CSC metabolic adaptability have become a key focus of modern cancer research. CSC resistance can develop as a result of radiotherapy and chemotherapy, as well as following chemotherapy secession. Neurodegenerative diseases are involved in continuous damage of neurons structure and/or function which denotes multiple degrees of paralysis and declined cognition and sensation. Globally, millions of people were infected by Alzheimer’s disease (AD) which is a complicated, irretrievable, forceful neurodegenerative infirmity. Commonly, the populace over 65 years may acquire this senile disease with characteristic features of destruction, mental function minimization which comprises loss of memory, cognitive injury, and finally declined life eminence of patients. Asymmetrical expression of proteins specifically Beta-amyloid “plaques” and Tau “Tangles” in the brain is measured as pathogenic properties of AD. Worldwide Alzheimer dementia (AD) pervasiveness has been calculated as elevated as 25 million, and it has been forecasted that AD will be twofold every 20 years till 2040. Mutually developed and developing nations had a remarkable collision on Alzheimer’s disease had a remarkable collision on infected individuals, caretakers, and civilization. Older age and genetic susceptibility are the major etiological factors for Alzheimer’s dementia. Existing treatment protocols facilitate recuperate from symptoms, and there is a deficiency in permanent recovery from the disease. Cell therapy strategies have been considered as an authoritative utensil for treating Alzheimer’s disease patients. Stem cell therapy also entitled regenerative cell therapy has been considered an unquestionably influential pioneering tactic in treating the disease of neurodegenerative for the past 20 years. Stem cells have the competence of recuperating damaged neuronal tissues through substituting the injured vanished neuronal cells with differentiated cells providing an advantageous atmosphere that promotes regeneration and safeguarding existing healthy neurons and glial cells from imminent injury. Therefore, in this chapter, we have elaborately discussed different stem cell types, neurodegenerative disease management through stem cell-based therapies, and current advancements in stem cell rehabilitation. However, improved sympathetic and additional investigation in stem cell methodologies may lead to the sensible and successful handling of ailments of neurodegenerative.KeywordsCancer stem cell therapyAlzheimer’s disease (AD)DementiaBeta-amyloidNeurodegenerative disordersStem cell therapy

The most promising therapeutic option is cancer stem cells (CSCs). CSCs, leaders of a cancer hierarchy, possess stem cell-like traits such as self-rehabilitation and the cognition to differentiate abnormally. CSCs do not have to be uncommon in tumors; cancer and non-cancer stem cells can change correctable phenotypic modification, and the phenotype of CSCs can differ significantly between the patients. The function of CSC metabolism and the mechanisms underlying CSC metabolic adaptability have become a key focus of modern cancer research. CSC resistance can develop as a result of radiotherapy and chemotherapy, as well as following chemotherapy secession. Neurodegenerative diseases are involved in continuous damage of neurons structure and/or function which denotes multiple degrees of paralysis and declined cognition and sensation. Globally, millions of people were infected by Alzheimer’s disease (AD) which is a complicated, irretrievable, forceful neurodegenerative infirmity. Commonly, the populace over 65 years may acquire this senile disease with characteristic features of destruction, mental function minimization which comprises loss of memory, cognitive injury, and finally declined life eminence of patients. Asymmetrical expression of proteins specifically Beta-amyloid “plaques” and Tau “Tangles” in the brain is measured as pathogenic properties of AD. Worldwide Alzheimer dementia (AD) pervasiveness has been calculated as elevated as 25 million, and it has been forecasted that AD will be twofold every 20 years till 2040. Mutually developed and developing nations had a remarkable collision on Alzheimer’s disease had a remarkable collision on infected individuals, caretakers, and civilization. Older age and genetic susceptibility are the major etiological factors for Alzheimer’s dementia. Existing treatment protocols facilitate recuperate from symptoms, and there is a deficiency in permanent recovery from the disease. Cell therapy strategies have been considered as an authoritative utensil for treating Alzheimer’s disease patients. Stem cell therapy also entitled regenerative cell therapy has been considered an unquestionably influential pioneering tactic in treating the disease of neurodegenerative for the past 20 years. Stem cells have the competence of recuperating damaged neuronal tissues through substituting the injured vanished neuronal cells with differentiated cells providing an advantageous atmosphere that promotes regeneration and safeguarding existing healthy neurons and glial cells from imminent injury. Therefore, in this chapter, we have elaborately discussed different stem cell types, neurodegenerative disease management through stem cell-based therapies, and current advancements in stem cell rehabilitation. However, improved sympathetic and additional investigation in stem cell methodologies may lead to the sensible and successful handling of ailments of neurodegenerative.KeywordsCancer stem cell therapyAlzheimer’s disease (AD)DementiaBeta-amyloidNeurodegenerative disordersStem cell therapy

Cholangiocarcinoma (CCA) is the second most common type of liver cancer, and is highly aggressive with very poor prognosis. CCA is classified into intrahepatic cholangiocarcinoma (iCCA) and extra-hepatic cholangiocarcinoma (eCCA), which is further stratified into perihilar (pCCA) and distal (dCCA). Cancer stem cells (CSCs) are a subpopulation of cancer cells capable of tumor initiation and malignant growth, and are also responsible for chemoresistance. Thus, CSCs play an important role in CCA carcinogenesis. Surface markers such as CD133, CD24, CD44, EpCAM, Sox2, CD49f, and CD117 are important for identifying and isolating CCA CSCs. CSCs are present in the tumor microenvironment (TME), termed ‘CSC niche’, where cellular components and soluble factors interact to promote tumor initiation. Epithelial-to-mesenchymal transition (EMT) is another important mechanism underlying carcinogenesis, involved in the invasiveness, metastasis and chemoresistance of cancer. It has been demonstrated that EMT plays a critical role in generating CSCs. Therapies targeting the surface markers and signaling pathways of CCA CSCs, proteins involved in TME, and immune checkpoint proteins are currently under investigation. Therefore, this review focuses on recent studies on the roles of CSCs in CCA; the possible therapeutic strategies targeting CSCs of CCA are also discussed.

Inflammatory processes and cancer stem cells (CSCs) are increasingly recognized as factors in the development of tumors. Emerging evidence indicates that CSCs are associated with cancer properties such as metastasis, treatment resistance, and disease recurrence. However, the precise interaction between CSCs and the immune microenvironment remains unexplored. Although evasion of the immune system by CSCs has been extensively studied, new research demonstrates that CSCs can also control and even profit from the immune response. This review provides an overview of the reciprocal interplay between CSCs and tumor-infiltrating immune cells, collecting pertinent data about how CSCs stimulate leukocyte reprogramming, resulting in pro-tumor immune cells that promote metastasis, chemoresistance, tumorigenicity, and even a rise in the number of CSCs. Tumor-associated macrophages, neutrophils, Th17 and regulatory T cells, mesenchymal stem cells, and cancer-associated fibroblasts, as well as the signaling pathways involved in these pro-tumor activities, are among the immune cells studied. Although cytotoxic leukocytes have the potential to eliminate CSCs, immune evasion mechanisms in CSCs and their clinical implications are also known. We intended to compile experimental findings that provide direct evidence of interactions between CSCs and the immune system and CSCs and the inflammatory milieu. In addition, we aimed to summarize key concepts in order to comprehend the cross-talk between CSCs and the tumor microenvironment as a crucial process for the effective design of anti-CSC therapies.

Malignant tumors contain heterogeneous populations of cells in various states of proliferation and differentiation. The presence of cancer stem or initiating cells is a well-established concept wherein quiescent and poorly differentiated cells within a tumor mass contribute to drug resistance, and under permissive conditions, are responsible for tumor recurrence and metastasis. A number of studies have identified molecular markers that are characteristic of tissue-specific cancer stem cells (CSCs). Isolation of CSCs has enabled studies on the metabolic status of CSCs. As metabolic plasticity is a hallmark of cancer cell adaptation, the intricacies of CSC metabolism and their phenotypic behavior are critical areas of research. Unlike normal stem cells, which rely heavily on oxidative phosphorylation (OXPHOS) as their primary source of energy, or cancer cells, which are primarily glycolytic, CSCs demonstrate a unique metabolic flexibility. CSCs can switch between OXPHOS and glycolysis in the presence of oxygen to maintain homeostasis and, thereby, promote tumor growth. Here, we review key factors that impact CSC metabolic phenotype including heterogeneity of CSCs across different histologic tumor types, tissue-specific variations, tumor microenvironment, and CSC niche. Furthermore, we discuss how targeting key players of glycolytic and mitochondrial pathways has shown promising results in cancer eradication and attenuation of disease recurrence in preclinical models. In addition, we highlight studies on other potential therapeutic targets including complex interactions within the microenvironment and cellular communications in the CSC niche to interfere with CSC growth, resistance, and metastasis.

Most cancer patients experience disease progression despite one or multiple lines of treatment with cytotoxic chemotherapy. Evidence from human trials and in vivo experiments on patient-derived xenograft (PDX) tumor lines indicates that cancer stem cells (CSCs) can survive chemotherapy treatment. However, the mechanism of chemotherapy resistance in CSCs is not fully elucidated. To identify new mechanisms of the drug-resistance in colorectal cancer, we used gene-expression microarrays to examine the gene expression profile of CSCs and non-CSCs in four human PDX models of colorectal cancer, both before-and-after treatment with cytotoxic chemotherapy. The gene-expression data were analyzed using Independent Component Analysis (ICA) followed by Transcription Factor Target Genes (TFTG) enrichment analysis, which identified the transcription factor E2F4 as a candidate regulator of chemotherapy resistance in colorectal CSCs. E2F4 was predominantly expressed in the CSC-enriched cell population (CD44+/CD166+ human colorectal cancer cells). E2F4 knock-down sensitizes cancer cells to chemotherapy in three different PDX tumors as well as HT29 cells. Co-immunoprecipitation experiments showed that E2F4 forms a complex with p107 and DP-1, which also contributed to the chemotherapy resistance. Simultaneous knock-down of both E2F4 and p107 enhanced chemotherapy sensitivity more than E2F4 knock-down alone. In normal intestinal crypt cells E2F4 was dispensable for chemotherapy resistance. ChIP-seq and RNA-seq experiments revealed that LDHA is a key downstream target of E2F4. LDHA knock-down sensitizes cancer cells to chemotherapy. High LDHA expression in tumor tissues was an independent poor prognostic factor in colorectal cancer patients (n=466), as well as in non-small cell lung cancer (n=1,926) and breast cancer (n=3,554). Thus, the E2F4/p107-LDHA pathway represents a novel therapeutic target to overcome chemotherapy resistance in CSCs. Citation Format: Junichi Matsubara, Yong F. Li, Piero Dalerba, Debashis Sahoo, Taichi Isobe, Russ B. Altman, Michael F. Clarke. E2F4/p107 complex regulates chemotherapy resistance in human colorectal cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 81. doi:10.1158/1538-7445.AM2017-81