activate a complex response, that includes the stimulation of an “emergency” biochemical and biological programs. Under these conditions, cancercells survive by entering into a transient quiescent state, which is reversed when appropriate environmental conditions are sufficient to support cell proliferation and ultimately metastasis . As such, the effectiveness of anti-angiogenic agents could be improved by using combination strategies aimed at inhibiting both cancer- and cancerstem-likecells. Based on our current observations, we suggest the combined use of Doxycycline, with angiogenesis inhibitors, such as Bevacizumab (Avastin). This new proposed combination therapy would effectively block both: i) blood vessel formation and ii) CSC propagation, ultimately making anti-angiogenic therapy more effective.
Proteomic analysis of human breast cancerstem- likecells also revealed the significant over-expression of several mitoribosomal proteins, such as MRPL45 and MRPL17, and 6 other proteins associated with mitochondrial biogenesis (HSPA9, TIMM8A, GFM1, HSPD1 [a.k.a., HSP60], TSFM, TUFM) . Importantly, functional inhibition of mitochondrial biogenesis, using the off-target effects of certain bacteriostatic antibiotics, effectively ablated the propagation of CSCs, in 12 cell lines representing 8 different tumor types (breast, DCIS, prostate, ovarian, pancreatic, lung, melanoma and glioblastoma) [3, 5]. Virtually identical results were also obtained with bonafide OXPHOS inhibitors (pyrvinium pamoate and atovaquone), providing additional complementary evidence that functional mitochondria are required for the propagation of CSCs [3, 16]. Taken together, these preliminary studies provide the necessary evidence that the development of novel mitoribosome inhibitors might be a beneficial approach for the more effective treatment of cancer patients.
targeting mitochondrial complex V in breast cancercells. To test this hypothesis, we determined its metabolic effects on MCF7 cells, using the Seahorse XF-e96, to measure mitochondrial function and glycolysis. Our results demonstrate that bedaquiline dramatically inhibits oxygen consumption and ATP production in the low micromolar range. In addition, bedaquiline treatment also reduced aerobic glycolysis (a.k.a., the Warburg effect). In this context, bedaquiline reduced mitochondrial membrane potential, but increased mitochondrial mass and overall ROS production. Most importantly, bedaquiline potently inhibited the propagation of CSCs, with an IC-50 of 1 μM, as measured using the mammosphere assay. Remarkably, bedaquiline treatment increased mitochondrial respiratory function, ATP production, and glycolysis in normal human fibroblasts. Thus, these results provide clear evidence that bedaquiline specifically inhibits mitochondrial respiration in MCF7 cancercells, but not in normal human fibroblasts. As such, our new findings suggest that bedaquiline could be used to target the mitochondrial F0F1-ATPase/ATP synthase in CSCs.
Hepatocellular carcinoma (HCC) is one of the most malignant tumors in Chinese people and offers poor prognosis. Tumor tissue, like normal tissue, is hierarchically differentiated. Thus, minor tumor cell populations able to differentiate, such as stemcells, sustain tumor self-renewal and proliferation. The fact that liver cancerstemcells (CSCs) with different surface markers appear heterogeneous with respect to oncogenesis and drug resistance indicates that subpopulations of surface markers preserve the hierarchical potential of differentiation during proliferation, deterioration and relapse. The epithelial to mesenchymal transition (EMT) is correlated to tumor malignancy and aggression, and hepatocytes bearing EMT have obvious hierarchical differentiation potential with respect to signaling pathways such as transforming growth factor β, Wnt/β-catenin and microRNA. Therefore, it may be more effective for early diagnosis to monitor HCC recurrence using peripherally circulating CSCs, and these may also offer potential for HCC immunotherapy or for targeting HCC treatment using these markers. Thus, we reviewed the generation, hierarchical differentiation and clinical application of hepatic CSCs.
A wealth of experimental data suggest that qui- escence is a vital feature of CSCs , and quiescence is considered to be a resistance mechanism in re- sponse to some chemotherapeutics . In chronic myeloid leukemia (CML), exposure of patient-derived cells to imatinib resulted in the enrichment of a qui- escent CD34+ population ; similar results were observed in an independent study by Jorgensen et al. . Moreover, quiescent patient-derived CD34+ CML cells were sensitized to imatinib in vitro by cell cycle induction using granulocyte colony stimulating factor . In colon cancer, 5Fu-resistant cells express a typical CSC-like phenotype and enter into a re- versible quiescent G0 state on re-exposure to higher 5Fu concentrations. Consistent with these findings, we found that residual GC cells contained a larger fraction of slowly growing cells that accumulated in the G0 phase of the cell cycle after 5Fu chemotherapy. The growth curve also revealed that the 5Fu-resistant cells proliferated at a slower rate than their parental counterparts. In addition, the percentage of quiescent SGC7901-FR cells increased gradually following 5Fu re-exposure. Because 5Fu activity is dependent on cell cycle progression , remaining in a quiescent state may be a possible mechanism of 5Fu resistance in GC cells.
Cancerstemcells (CSCs) are considered to be the major source of tumor recurrence after radiation and chemotherapy, and the literature suggests that targeting CSCs is one of the promising approaches to overcome treatment resistance of cancers.[19-21] Additionally, stem cell niches maintaining CSCs are also considered to be important targets to treat glioblastoma, and currently, two types of stem cell niches are reported: perivascular niche and hypoxic niche.[4, 23] In the present study, the expression of nestin, a marker of glioma stem cell-like phenotype, was found to be decreased in the six tumors resected under neoadjuvant Bev treatment as compared with control glioblastomas. However, numerous nestin- positive tumor cells remained after Bev treatment, with relative concentration around vessels (Figure. 6A, 6B, 6C). These observation as well as vascular co-option of the tumor cells noted in cases 3, 5, and 6 may suggest that improvement of a low -oxygen environment by Bev treatment could make it difficult to maintain a hypoxic niche. In contrast, the perivascular niche is still likely present around the apparently “normalized” vasculature under antiangiogenic therapy, and the other therapeutic approach or combination therapy may be necessary to eradicate perivascular CSCs.
Multiple myeloma (MM) is a hematological malignancy, which remains incurable because most patients eventually relapse or become refractory to current treatments. Due to heterogeneity within the cancer cell microenvironment, cancer cell populations employ a dynamic survival strategy to chemotherapeutic treatments, which frequently results in a rapid acquisition of therapy resistance. Besides resistance-conferring genetic alterations within a tumor cell population selected during drug treatment, recent findings also reveal non-mutational mechanisms of drug resistance, involving a small population of “ cancerstemcells ” (CSCs) which are intrinsically more refractory to the effects of a variety of anticancer drugs. Other studies have implicated epigenetic mechanisms in reversible drug tolerance to protect the population from eradication by potentially lethal exposures, suggesting that acquired drug resistance does not necessarily require a stable heritable genetic alteration. Clonal evolution of MM cells and the bone marrow microenvironment changes contribute to drug resistance. MM-CSCs may not be a static population and survive as phenotypically and functionally different cell types via the transition between stem-like and non-stem-like states in local microenvironments, as observed in other types of cancers. Targeting MM-CSCs is clinically relevant, and different approaches have been suggested to target molecular, metabolic and epigenetic signatures, and the self- renewal signaling characteristic of MM CSC-likecells. Here, we summarize epigenetic strategies to reverse drug resistance in heterogeneous multiple myeloma.
In a recent report, Fan, Chen and colleagues provided additional evidence of a role for ACAT1 as an oncogene, using pre-clinical models [9,10]. Moreover, they screened a chemical library containing mainly FDA- approved drugs and identified arecoline, a natural product as a potential ACAT1 inhibitor [9,10]. Arecoline is a nicotinic acid-based alkaloid found within the areca nut, which is the fruit of the areca palm tree (Areca catechu) . In certain Asian countries, arecoline is administered by chewing the areca nut, together with the betel leaf (like chewing tobacco), as it behaves as a natural CNS stimulant . Importantly, this natural product, arecoline, showed anti-tumor activity, further validating that drugs targeting ACAT1 might be valuable as anti-cancer agents [9,10]. However, the authors did not assess its capacity to target CSCs. As arecoline is very small molecule (shown in Figure 10B for comparison), it would need to be modified significantly by medicinal chemistry to increase its potency.
Moreover, different subpopulations of CSCs exhibit dif- ferent metabolic patterns. Recent publications imply the existence of an epithelial-like (mesenchymal to epithelial transition (MET)) and a mesenchymal-like (epithelial to mesenchymal transition (EMT)) CSC phenotype, states that might be interconvertible [67, 68]. In breast cancer, MET CSCs are characterized by high ALDH activity and enhanced proliferative capacity, whereas EMT CSCs are identified by the expression of the CD44 hi /CS24 – surface markers and a slow-cycling, quiescent state . Mesenchymal-like EMT CSCs seem to favour glycolysis, and have a marked reduction in oxygen consumption, decreased mitochondrial mass and membrane potential, lower ROS production and higher antioxidant capacity compared with the epithelial-like fraction of CSCs [36, 67]. Indeed, CD44 acts as a metabolic modulator, by activating glycolysis under hypoxia. CD44 ablation reduces glycolysis and the antioxidant response, and moves the energy production to the mitochondria, with an increase in ROS . Conversely, a proteomics study revealed that the ALDH-expressing population of CSCs express more glycolytic enzymes than the CD44 hi /CS24 – CSCs . Finally, a recent study shows that highly metastatic mur- ine cancercells enhance both glycolysis and OXPHOS pathways compared with cells with the same genetic background that lack metastatic potential . Another report links metabolic plasticity to the acquisition of therapy resistance by showing that although most CSCs have limited metabolic malleability and predominantly rely on OXPHOS, a subpopulation of metformin-resistant CSCs is able to acquire a more adaptable intermediate glycolytic/respiratory phenotype. The metabolic phenotype of CSCs thus appears to be heterogeneous with distinct metabolic programmes activated in different subpopula- tions of cancercells (Fig. 2).
In such a peculiar microenvironment where inflamma- tion is responsible for both normal liver homeostasis and function, and for liver pathology, several immune cells as well as non-hematopoietic cells have shown to correlate with HCC progression. Among them, the fol- lowing are worth to mention: tumor associated macro- phages (TAMs), hepatic stellate cells (HSCs), cancer- associated fibroblasts (CAFs), neutrophils, cancerstem- likecells (CSLCs) and regulatory T cells (Tregs) (Fig. 1).
Cancerstemcells (CSCs) are cancercells with stem cell-like abilities. This type of cancer cell has captured the attention of cancer clinicians and may be one of the key factors associated with their difficulty in treating cancers. Ever since CSCs were reported in leukaemia two decades ago, they have been attracting a lot of interest; however, many details are still under debate (Simard and Engels, 2010). CSCs are also called cancer-initiating cells because when injected into immunodeficient mice, they can generate tumours (Charafe-Jauffret et al., 2009). Numerous studies suggest that the eradication of CSCs could be among the most important strategies for developing successful cancer treatments because they impact upon cancer self- renew, cancer metastasis, drug resistance, and are responsible for tumour recurrence (Liu et al., 2015, Kai et al., 2010, Boyle and Kochetkova, 2014, Charafe-Jauffret et al., 2009). Under most conditions CSCs are quiescent, but they can be triggered to induce self-renewal activities and to reproduce progenitor cells (Chen et al., 2016). Clearly, the existence of CSCs is a major problem in treating cancers. Researchers have recently shown that if anti-cancer drugs kill only non-CSCs, CSCs will rapidly induce further cancer growth. Thus, by targeting CSCs, the issues of cancer metastasis and cancer recurrence might be addressed, and patients’ lives might be extended. The discovery of CSCs has provided an insight into why cancer is sometimes very difficult to treat and eliminate. Cancer treatment regimens need to be devised to overcome technical challenges.
Fig. 1. Three signal pathways contribute to stemness properties of gastric cancerstem-likecells: Wnt/β-catenin signal pathway, Notch signal pathway, and Hedgehog signal pathway. (A) Wnt/β-catenin signal pathway: Wnt binds to its receptor-Frizzled to activate Dsh protein. The activated Dsh protein en- hances the phosphorylation of GSK3β (a component of the cytoplasmic complex that promotes phosphorylation of β-catenin and its degradation), which inhibits the ability of GSK3β, further causing the accumulation of free and unphosphorylated β-catenin in the cytoplasm that is then translocated to the nucleus. In the nucleus, β-catenin binds to TCF/LEF to promote downstream target genes expression. (B) Notch signal pathway: Ligand binding-induced Notch activation causes γ-secretase (including Presenilin and Nicastrin) to cleave Notch COOH-terminal fragment to release NICD into the cytoplasm. Then, NICD translocates to the nucleus to interact with SKIP and CSL, which lead to SMRT/HDACs dissociation, further converting CSL to a transcription- al activator to initiate downstream gene expression. (C) Hedgehog signal pathway: Ptc-induced inhibition of Smo is reversed by Hh binding with Ptc, lead- ing to the release of the complex of GLI (GLI/SUFU/SKT36) from microtubules, with GLI protein entering the nucleus to transcriptionally activate down- stream target genes.
Abstract: In recent years, many studies have shown that some types of tumors are characterized by the presence of cells with stem-like characteristics, called cancerstemcells (CSCs). These are considered cells that initiate the tumor and are probably responsible for tumor recurrence. CSCs have the capacity for self-renewal, the potential to give rise to one or more cell types within the tumor, and the ability to drive, in a continuous manner, the proliferation of malignant cells. The failure of current cancer therapies can be attributed to the relative ineffectiveness of drugs against CSCs, which remain viable while retaining their full ability to reproduce the tumor. The development of new strategies is currently hampered by the lack of reliable mark- ers to identify CSCs. One promising surface marker of CSCs in head and neck cancer is the CD44 molecule, which has been shown in preliminary studies to have high specificity, although there are discrepant data because its prognostic value may depend on the specific tumor location. More rigorous studies are needed to investigate the usefulness of CD44 expression in head and neck tumors for possible clinical applicability.
. Candidate RNA editing loci were selected from this dataset to validate an RNA editing fingerprint of CML pro- gression using a site-specific RNA editing detection assay. Eight LSC-specific loci (Table 1) were selected on the basis of greatest average fold-change in RNA editing frequency among significantly different sites (p < 0.005) . These sites were located within transcripts of the ubiquitin ligase hu- man homolog of mouse double minute 2 (MDM2); the cyti- dine deaminase apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3D (APOBEC3D); antizyme inhibi- tor 1 (AZIN1); signal recognition particle 9 kDa (SRP9); spli- cing factor 3b, subunit 3 (SF3B3); abl-interactor 1 (ABI1); lysosomal trafficking regulator (LYST); and MDM4. Analysis of editing rates in individual samples from our RNA-seq dataset  showed increased RNA editing of MDM2, APO- BEC3D, and AZIN1 in BC CML LSC compared with CP progenitors (Figure 1B-D). Notably, site-specific RNA edit- ing of AZIN1 – a regulator of tumor growth  – causes exon recoding resulting in enhanced protein stability and is associated with aggressive hepatocellular carcinoma . The majority of BC CML LSC samples displayed AZIN1 RNA editing, while RNA editing at this site was virtually undetect- able in CP CML progenitor cells (Figure 1D). Consistent with these findings, Sanger sequencing of PCR products amplified with high-fidelity DNA polymerase confirmed in- creased APOBEC3D and AZIN1 RNA editing in BC versus CP CML progenitors (Additional file 3: Figure S1A-D).
Hyper-activation of cyclins, especially the G1/S adminis- trator cyclin D1 may favor tumor development by indu- cing unscheduled cell division in progenitor cells . The abundance of cyclin D1 has a significant relation- ship with neoplastic progression of NSCLC [90,91]. In epithelial ovary cancercells, stem-like CD24- cells or spher- oids highly expressed cyclin D1, Bmi-1, and vimentin with reduced expression of E-cadherin, while non stem-like CD24+ or parental cells showed the opposite expression. Furthermore, cyclin D1-targeted small interfering RNA re- sulted in decreased vimentin expression in spheroids, ac- companying with reduced cell viability and migration . In cervical squamous carcinoma, most invasive tumour cells expressed cyclin D1 and showed a reduction in E- cadherin and beta-catenin staining . Epidermoid A431 cells expressing SIP1 along with exogenous cyclin D1 were highly invasive, indicating that SIP1-regulated invasion is independent of attenuation of G1/S progression . Con- versely, knock down Notch-3 reduced cyclin D1 with re- duced proliferation and enhanced apoptosis . Given the importance of Notch and cyclin D1 in tumorigenesis, it is not surprising that Notch pathway would participate in regulation of cyclin D1-mediated EMT. Activation of Notch signaling during neural induction in the ES cells led to sig- nificantly enhanced cell proliferation, accompanied by Notch-mediated activation of cyclin D1 expression. A re- duction of cyclin-D1-expressing cells was observed in the developing CNS of Notch signaling-deficient mouse em- bryos. Moreover, expression of a dominant negative form of cyclin D1 in the ES cells abrogated the Notch-induced proliferative ability, and, conversely, a constitutively active form of cyclin D1 mimicked the effect of Notch on cell pro- liferation . Gifitinib-acquired resistant lung cancercells displayed EMT phenotype. Molecular analyses confirmed that activation of Notch-1 and its target genes, which in- duced expression of mesenchymal marker vimentin, snail and hes-1, and decreased epithelial marker E-cadherin. The elevated expression of Notch-1 could directly suppress p21 Waf1/Cipl accompanied with the increasing of cyclin D1, giving rise to the EMT phenotype. Meanwhile, knockdown of Notch-1 decreased cyclinD1 expression and reversed EMT. These findings suggest that the aggressive behavior caused by cyclinD1 might be driven by the mis-expression of Notch pathway in NSCLC, which finally down regulates p21 Waf1/Cipl and promotes the EMT phenotype .
Cells were harvested into radioimmunoprecipitation assay lysate buffer containing freshly added 1% phenylmethyl- sulfonyl fluoride. Protein was subjected to 10% SDS– PAGE and then transferred to 0.45 µ m transfer membranes (Millipore, Billerica, MA, USA). Membranes were blocked at room temperature for 1 hour in Tris-buffered saline with Tween-20 containing 5% nonfat milk, and then incubated with rabbit anti-cyclin D1 monoclonal antibody (1:5000), anti-N-cadherin (1:10,000) (Epitomics Inc, Burlingame, CA, USA), anti-vimentin (1:700; Abcam, Cambridge, UK), and mouse anti- β -actin monoclonal antibody (1:2000) (Sigma- Aldrich) at 4 ° C overnight. After incubation with appropriated secondary antibodies, goat-anti-mouse IgG (1:1000) or goat- anti-rabbit IgG (1:1000) (Sigma-Aldrich), at room tempera- ture for 1 hour, bands were visualized by Chemiluminescence Detection Kit for HRP (Biological Industries, Kibbutz Beit Table 1 DnA oligonucleotides sequence used in synthesis of
NANOG is capable of maintaining embryonic stem cell pluripotency independently of the LIF-STAT3 pathway, which OCT4 is incapable of doing [68, 73]. NANOG exerts many functions through its tran- scriptional regulatory activities. NANOG regulates the cell cycle and proliferation by directly binding the cyclin D1 promoter [75, 76]. In prostate cancer cell lines, the induction of NANOG causes the upre- gulations of CD133 and ALDH1 . NANOG alone is sufficient to induce SLUG transcription . Furthermore, NANOG is capable of inducing CSC- like properties in primary p53-deficient mature mouse astrocytes; however, astrocytes with intact p53 could not be induced . Some functional re- dundancy and cooperation between NANOG and STAT3 have been reported. The two form a complex in head and neck squamous cell carcinoma cells , and microarray analysis showed that NANOG also regulated 14 out of the 22 STAT3 target genes involved in the maintenance of an undifferentiated state .
RhoC in tumor phenotypes and molecular pathways The role of RhoC in carcinoma progression has been ex- tensively clarified by several research groups over the years. The first report, which suggested that RhoC con- tributed to progression of cancer, was by Suwa et al. in the year 1998. This group investigated changes in the ex- pression levels of the Rho family of genes—RhoA, B and C in pancreatic ductal carcinoma. It was discovered that the expression of the RhoC gene was significantly higher in metastatic tumors than in primary tumors, whereas RhoA and RhoB did not show significant changes in ex- pression under these conditions. Also, increased RhoC expression significantly correlated with poor prognosis of patients, unlike RhoA and RhoB, which showed no such correlation . Following this study, several other groups reported the role of RhoC in numerous other cancers, including those of the breasts, skin, ovaries, liver and head and neck, among several others [28–33]. The increased expression of RhoC is therefore positively correlated to poor prognosis. However, activation of the molecule is necessary to enable its downstream effects. MyoGEF, a molecule responsible for activation of RhoA and RhoC was found to regulate both polarity and inva- sive phenotypes of MDA-MB-231 (an invasive breast cancer cell line) . On the contrary, p190RhoGAP which converts GTP-bound Rho to the inactive GDP- bound form, is associated with reduced proliferation, mi- gration and invasion in breast and pancreatic cancer models, thus acting as the antithesis of MyoGEF in this context [35, 36]. The role of RhoC as a transforming oncogene was postulated by van Golen et al. This group demonstrated that the stable transfectants of human mammary epithelial cells over-expressing RhoC not only gained tumorigenic properties but were also highly inva- sive . In the year 2013, Xie et al. showed that stable transfection of the RhoC expression vector into a normal hepatocyte cell line, imparted tumor phenotypes like proliferation, anchorage-independent growth, migration, invasion, increased expression of matrix metallopro- teases like MMP2 and MMP9, and elevated levels of Vascular Endothelial Growth Factor (VEGF), further cementing RhoC’s role as an oncogene . Addition- ally, RhoC was found to have a positive association with dedifferentiation and the phosphorylated form of
FBXO32, which is also known as a muscle-specific F-box protein, is expressed largely in skeletal muscle cells and cardiomyocytes [105, 106]. It has been docu- mented that FBXO32 plays a crucial role in regulating muscle homeostasis by targeting multiple substrates such as calcineurin , eIF-3 , MyoD , MAPK-1 , and IκBα . Emerging evidence has identified FBXO32 as a tumor suppressor because it in- duces apoptosis . In support of this evidence, FBXO32 dysregulation promotes the EMT process in urothelial carcinoma after acquisition of platinum resist- ance. This was verified by accumulation of MyoD pro- teins and the subsequent increase in the expression of the mesenchymal markers Snail and vimentin and the reduction in the expression of the epithelial molecule E-cadherin . However, interestingly, Sahu et al. re- ported conversely that FBXO32 directly targeted CtBP1 for its ubiquitination and enhanced its nuclear retention, which generated a suitable microenvironment for EMT progression and cancer metastasis in human breast can- cer cells . Moreover, in the same study, the expres- sion of FBXO32 was elevated in breast cancercells with invasive properties (MDA-MB-231 cells), and depletion of FBXO32 in a xenograft mouse model suppressed tumor growth and metastasis .
In the study of Zhang et al., they explore the role of Wnt/β-catenin signaling in maintaining the cancerstemcells (CSCs) in lung cancer, the results show that Wnt signal pathway plays an vital role in maintaining highly resistant CSCs and regulating cell cycle, apoptosis and metas- tasis in the development and progression of lung cancer . What’s more, several genes related with the Wnt signaling pathway have been explored the relationship with lung can- cer. Li et al. have found that Wnt3a, a key gene in Wnt signaling pathway, can accelerate cell invasion and the growth of anchorage-indepen- dent and promote the metastasis ability of NSCLC through regulating the expression of Notch3 in Notch signaling pathway . Coscio et al. indicate that the Wnt signaling pathway may play a crucial role in tumorigenesis and the progression of cancer and study 441 single nucleotide polymorphisms (SNPs) from 54 genes in the Wnt signaling pathway in NSCLC patients, the final results show that genetic variants contribute to the clinical outcomes for patients with the early-stage NSCLC . However, in previous studies, they mostly fo- cus on the function of single gene. As we all known, the occurrence of diseases are influ- enced by many factors in complex human body. Therefore, in our study, we explored the rele- vance of 4 polymorphisms from two genes in the Wnt signaling pathway with NSCLC suscep- tibility and the environment factor was also considered. The homozygous mutant geno- types of rs2240307, rs2240308 in AXIN2 gene significantly increased the risk of NSCLC and MMP7 rs11568818 polymorphism was also considered to be associated with the increas- ed susceptibility to NSCLC. What’s more, the haplotype A-G-G of rs2240307, rs4791169, rs2240308 in a block of AXIN2 might be a pro- tective factor for against the occurrence of NSCLC in Chinese Han population. In addition, we found that there were the interaction be- tween AXIN2, MMP7 polymorphisms and envi- ronment factor.