The roles of cancer stem cell (CSC)-derived exosomal non-coding RNAs in cancer progression

Comparison of hematopoietic cancer stem cells with normal stem cells leads to discovery of novel differentially expressed SSRs

Finding and Killing the CRABs of Pancreatic Cancer

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

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

1942P IL6 pre-treatment promotes chemosensitivity by eliminating quiescent cancer stem cells

Continuously increasing evidence has shown that most primary tumours contain subpopulations of cells with stem-like characteristics and their existence is associated with resistance to therapy and formation of metastases at distant sites. This is further supported by the identification of several common key pathways and genes between cancer and normal stem cells involved mainly in proliferation, differentiation and de-differentiation steps. Cancer stem cells (CSCs) can self-renew as well as give rise to non-CSC progeny. CSCs may exist within the tumour since the early stages of cancer or develop during cancer progression through the developmental process of epithelial-to-mesenchymal transition (EMT). Cells with stem-like features have been recently identified within the circulating tumour cells (CTCs) that are responsible for the generation of metastases. Dissemination from the primary tumour can occur in the early stages of tumour development or later during tumour progression. Here, we review current knowledge concerning CSCs’ development, metastatic potential, implications for therapy and mechanisms that govern these processes, including molecular pathways and the role of microenvironment.

Simple SummaryExosomes are a class of extracellular vesicles released by cancer cells that play important roles in cancer progression and therapy resistance. Using RNA sequencing in plasma exosomes from patients with metastatic colorectal cancer (mCRC), we investigated exosomal RNA expression profiles between initial recurrence and cancer progression after metastasectomy. The differential expression level of the exosome RNA was enriched in samples from patients with metastases to the liver, multiple metastatic sites and large tumor burden. In terms of liver metastasis, the interferon-α response gene set was enriched in a tumor burden of ≥1 cm3, and CXCL10, CXCL11 and SAMD 9 were highly expressed in the exosomal RNA which was validated using GSEA. High CXCL10 expression was associated with shorter progression-free survival (PFS), but CXCL11 and SAMD 9 were not correlated with PFS. Exosomal CXCL10 RNA could be a novel biomarker for liver metastasis from mCRC and a potential target for the prevention and treatment of these patients.This study aimed to identify novel biomarkers for metastatic colorectal cancer progression using exosomal RNA expression profiling. The exosomal RNA expression profiles of 54 patients with mCRC were investigated. Exosomal RNA profiling was performed at the time of relapse immediately before metastasectomy and cancer recurrence or progression after metastasectomy. The up- and down-regulated RNA expression profiles were screened and analyzed using H-cluster, principle component analysis and gene ontology. The tissue expression profile of the liver metastases was compared with the GSE 41258 set using GSEA tools. We identified two distinctive biological process gene sets (IFNA and PCDB families) related to metastatic progression. The interferon-α response gene set was enriched, especially when the tumor volume was ≥1 cm3. CXCL10, CXCL11 and SAMD 9 mRNA were highly expressed in the plasma exosome samples of patients with mCRC to the liver. Furthermore, high expression of CXCL10 but not CXCL11 or SAMD9 was associated with a poor prognosis and shorter progression-free survival. Conclusions: Cancer-derived exosomal CXCL10 may be a novel biomarker for liver metastasis of mCRC and a potential target for the prevention and treatment of mCRC with liver metastasis.

We have used hTERT (the catalytic component of telomerase) to immortalize a variety of human cell types (sometimes in combination with Cdk4 to bypass cell culture stress). Cell types immortalized include skin keratinocytes and fibroblasts, muscle satellite cells, breast epithelial and stromal cells, corneal epithelial cells and fibroblasts (keratocytes), and human colonic epithelial cells. In addition, we have immortalized human bronchial epithelial cells (HBEC) and have determined that these cells can terminally differentiate into both central and peripheral lung cell types. These immortalized HBECs have been used to study the molecular pathogenesis of lung cancer by stable “knock down” of TP53 and also by over-expression of C-myc and mutant K-rasV12. When these cells containing multiple genetic alterations are introduced into immunosuppressed mice, the experimentally transformed cells make tumors that represent several distinct histological types. This suggests that these immortalized and transformed HBECs have bronchiolar-alveolar stem-like characteristics that can differentiate into multiple lineages. Stem cells are defined by both their ability to make more stem cells (self renewal) and their ability to produce cells that can differentiate. Experimentally immortalized human bronchial epithelial cells fulfill this definition of normal stem cells by continuous self renewal and by retaining the capability of differentiating into several cell types. Since experimentally transformed cells make lung tumors representing several major lineages, this is also an indication that the HBECs are derived from a multi-potent lung stem cell. Similar to normal stem cells, cancer (initiating) stem cells also have the ability to self-renew as well as undergo differentiation to give rise to phenotypically diverse types of cancer cells. There is mounting evidence that these rare cancer stem cells may be multidrug resistant and responsible for tumor relapse and metastasis. Targeted cancer therapeutic approaches seek to identify pathways that are more tumor specific, resulting in fewer side-effects and that may produce long-term durable responses. Telomerase is a novel cancer therapeutic target since it is activated in the vast majority of human cancers and telomeres of almost all human tumor cells are maintained at short but stable lengths. In addition, telomerase is not expressed or is expressed at levels that do not fully maintain telomeres in normal tissues, and telomeres are generally longer in normal stem cells compared to cancer cells. This potentially provides a therapeutic advantage for targeting telomerase over approaches that affect both normal and cancer cells equally. We have previously reported that telomerase positive cancer cells that are experimentally induced to undergo quiescence, down regulate telomerase. As part of our anti-telomerase therapeutics program, we have addressed the following questions: Are putative cancer stem cell populations quiescent and do they have short or long telomeres? While the molecular characteristics of cancer stem cells are not completely defined and subject to some controversies, we have isolated and examined cells expressing these cancer stem cell putative markers reported for breast, brain, prostate, pancreas, and lung cancer. In each case we have observed that purified cancer stem cell populations are positive for telomerase activity, indicating they are not quiescent. In addition, a telomerase inhibitor currently being tested in clinical trials robustly inhibits the activity of telomerase in these sorted sub-populations of putative cancer stem cells as well as the mass population of cancer cells. Finally, we have observed that cancer stem cells have short telomeres in comparison to normal stem cells. These findings support the idea that there may be a therapeutic window of opportunity to target cancer stem cells by inhibiting telomerase, thus driving telomeres progressively shorter leading to cancer stem cell death, potentially without irreversible damage to normal stem cells. Cancer remains a major cause of death in spite of substantial progress towards understanding the molecular basis of many types of cancers. The discovery of new drugs is a high priority, and telomerase inhibitors have the potential to act by a novel mechanism that will provide new options for cancer therapy. A review of ongoing anti-telomerase clinical trials will be presented. In summary, telomerase inhibitors might not only directly limit or stop the growth of human tumors including cancer stem cells, but might also act in an additive or synergistic fashion with existing therapies to amplify their effectiveness. Citation Format: Jerry W. Shay. Role of telomerase in normal and neoplastic stem cells [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY33-03

Chapter 13 - Regulation of cancer stemness, cell signaling, reactive oxygen species, and microRNAs in cancer stem cells

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Prostate cancer stem cells (CSC) are implicated in tumor initiation, cancer progression, metastasis, and the development of therapeutic-resistant disease. It is well known that the bulk of prostate cancer cells express androgen receptor (AR) and that androgens are required for prostate cancer growth, progression, and emergence of castration-resistant disease. In contrast, the small subpopulation of self-renewing CSCs exhibits an AR-negative (AR-) signature. The mechanisms underlying the absence of AR are unknown. Using CSC-like cell models isolated from clinical biopsy tissues, we identify the E3 ligase MDM2 as a key regulator of prostate CSC integrity. First, unlike what has been reported for the bulk of AR+ tumor cells where MDM2 regulates the temporal expression of AR during transcriptional activity, MDM2 in CSCs promoted the constant ubiquitination and degradation of AR, resulting in sustained loss of total AR protein. Second, MDM2 promoted CSC self-renewal, the expression of stem cell factors, and CSC proliferation. Loss of MDM2 reversed these processes and induced expression of full-length AR (and not AR variants), terminal differentiation into luminal cells, and cell death. Selectively blocking MDM2-mediated activity in combination with androgen/AR-targeted therapy may offer a novel strategy for eliminating AR- CSCs in addition to the bulk of AR+ prostate cancer cells, decreasing metastatic tumor burden and inhibiting the emergence of therapeutic resistance.Significance: These findings provide a novel mechanistic aspect of prostate cancer cell stemness that advances our understanding of the diverse transcriptional activity that bypasses AR in contributing to therapeutic resistance, tumor progression, and metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/6/1124/F1.large.jpg.

<div>Abstract<p>Prostate cancer stem cells (CSC) are implicated in tumor initiation, cancer progression, metastasis, and the development of therapeutic-resistant disease. It is well known that the bulk of prostate cancer cells express androgen receptor (AR) and that androgens are required for prostate cancer growth, progression, and emergence of castration-resistant disease. In contrast, the small subpopulation of self-renewing CSCs exhibits an AR-negative (AR<sup>−</sup>) signature. The mechanisms underlying the absence of AR are unknown. Using CSC-like cell models isolated from clinical biopsy tissues, we identify the E3 ligase MDM2 as a key regulator of prostate CSC integrity. First, unlike what has been reported for the bulk of AR<sup>+</sup> tumor cells where MDM2 regulates the temporal expression of AR during transcriptional activity, MDM2 in CSCs promoted the constant ubiquitination and degradation of AR, resulting in sustained loss of total AR protein. Second, MDM2 promoted CSC self-renewal, the expression of stem cell factors, and CSC proliferation. Loss of MDM2 reversed these processes and induced expression of full-length AR (and not AR variants), terminal differentiation into luminal cells, and cell death. Selectively blocking MDM2-mediated activity in combination with androgen/AR-targeted therapy may offer a novel strategy for eliminating AR<sup>−</sup> CSCs in addition to the bulk of AR<sup>+</sup> prostate cancer cells, decreasing metastatic tumor burden and inhibiting the emergence of therapeutic resistance.</p><p><b>Significance:</b> These findings provide a novel mechanistic aspect of prostate cancer cell stemness that advances our understanding of the diverse transcriptional activity that bypasses AR in contributing to therapeutic resistance, tumor progression, and metastasis.</p></div>

<div>Abstract<p>Prostate cancer stem cells (CSC) are implicated in tumor initiation, cancer progression, metastasis, and the development of therapeutic-resistant disease. It is well known that the bulk of prostate cancer cells express androgen receptor (AR) and that androgens are required for prostate cancer growth, progression, and emergence of castration-resistant disease. In contrast, the small subpopulation of self-renewing CSCs exhibits an AR-negative (AR<sup>−</sup>) signature. The mechanisms underlying the absence of AR are unknown. Using CSC-like cell models isolated from clinical biopsy tissues, we identify the E3 ligase MDM2 as a key regulator of prostate CSC integrity. First, unlike what has been reported for the bulk of AR<sup>+</sup> tumor cells where MDM2 regulates the temporal expression of AR during transcriptional activity, MDM2 in CSCs promoted the constant ubiquitination and degradation of AR, resulting in sustained loss of total AR protein. Second, MDM2 promoted CSC self-renewal, the expression of stem cell factors, and CSC proliferation. Loss of MDM2 reversed these processes and induced expression of full-length AR (and not AR variants), terminal differentiation into luminal cells, and cell death. Selectively blocking MDM2-mediated activity in combination with androgen/AR-targeted therapy may offer a novel strategy for eliminating AR<sup>−</sup> CSCs in addition to the bulk of AR<sup>+</sup> prostate cancer cells, decreasing metastatic tumor burden and inhibiting the emergence of therapeutic resistance.</p><p><b>Significance:</b> These findings provide a novel mechanistic aspect of prostate cancer cell stemness that advances our understanding of the diverse transcriptional activity that bypasses AR in contributing to therapeutic resistance, tumor progression, and metastasis.</p></div>

Simple SummaryCancer stem cells (CSCs) represent a distinct cancer subpopulation that can influence the tumour microenvironment, in addition to cancer progression and relapse. A multitude of factors including CSC properties, long noncoding RNAs (lncRNAs), and autophagy play pivotal roles in maintaining CSCs. We discuss the methods of detection of CSCs and how our knowledge of regulatory and cellular processes, and their interaction with the microenvironment, may lead to more effective targeting of these cells. Autophagy and lncRNAs can regulate several cellular functions, thereby promoting stemness factors and CSC properties, hence understanding this triangle and its associated signalling networks can lead to enhanced therapy response, while paving the way for the development of novel therapeutic approaches. Cancer stem cells (CSCs) possess properties such as self-renewal, resistance to apoptotic cues, quiescence, and DNA-damage repair capacity. Moreover, CSCs strongly influence the tumour microenvironment (TME) and may account for cancer progression, recurrence, and relapse. CSCs represent a distinct subpopulation in tumours and the detection, characterisation, and understanding of the regulatory landscape and cellular processes that govern their maintenance may pave the way to improving prognosis, selective targeted therapy, and therapy outcomes. In this review, we have discussed the characteristics of CSCs identified in various cancer types and the role of autophagy and long noncoding RNAs (lncRNAs) in maintaining the homeostasis of CSCs. Further, we have discussed methods to detect CSCs and strategies for treatment and relapse, taking into account the requirement to inhibit CSC growth and survival within the complex backdrop of cellular processes, microenvironmental interactions, and regulatory networks associated with cancer. Finally, we critique the computationally reinforced triangle of factors inclusive of CSC properties, the process of autophagy, and lncRNA and their associated networks with respect to hypoxia, epithelial-to-mesenchymal transition (EMT), and signalling pathways.

The concept of the cancer stem cell (CSC) is neither new nor without controversy. It is important to study these cells since they are often hypothesized to be androgen insensitive and resistant to chemotherapeutics. Therefore, finding reliable markers for identifying and targeting these cells has become increasingly attractive. The hyaluronic acid receptor, CD44, is associated with a wide variety of cell types, including CSCs. CD44 has many different isoforms which can be categorized as belonging to one of two groups; one expressing different combinations of 10 variant extracellular exons (CD44v), and the other only expressing standard exons found in all isoforms, but none of the 10 variant exons (CD44s). CD44s is typically associated with the mesenchymal stem cell niche, while the various CD44v forms are related to hematopoietic stem cells and increased cell adhesion. However, both CD44s and CD44v have been linked to the CSC niche and cancer progression. Similar to CD44, the aldehyde dehydrogenases (ALDH) superfamily expression is also associated with CSCs and cancer progression. Previously, our laboratory demonstrated that epithelial cancer cells undergo an EMT upon exposure to M2 macrophages. In order to obtain a purely epithelial PCa population, our laboratory isolated a single cell clone of PC3 that had high E-cadherin expression, denoted PC3-Epi. PC3-Epi cells were then incubated with M2 macrophages, which caused a stable EMT to occur after only a few days in culture, denoted PC3-EMT. Our finding demonstrate that these PC3 clones exhibit numerous CSC characteristics, such as high ALDH activity, CD44 expression and cell plasticity. FACS analysis of CD44 isoform expression show distinct CSC subpopulations within these PC3-Epi and PC3-EMT cell types. These subpopulations showed the ability to recapitulate the presorted parental population after being returned to culture. This data suggests not only that the PC3 cell line displays numerous CSC characteristics, but also that distinct CSC subpopulations may exist. Note: This abstract was not presented at the meeting. Citation Format: James R. Hernandez, Steven M. Mooney, Gonzalo Torga, James E. Verdone, Kenneth J. Pienta. CD44 isoform expression illuminates multiple CSC subpopulations in PC3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1394. doi:10.1158/1538-7445.AM2015-1394