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Intratumoral heterogeneity is tightly associated with the failure of anticancer treatment modalities including conventional chemotherapy, radiation therapy, and molecularly targeted therapy. Such heterogeneity is generated in an evolutionary manner not only as a result of genetic alterations but also by the presence of cancer stem cells (CSCs). CSCs are proposed to exist at the top of a tumor cell hierarchy and are undifferentiated tumor cells that manifest enhanced tumorigenic and metastatic potential, self-renewal capacity, and therapeutic resistance. Properties that contribute to the robustness of CSCs include the abilities to withstand redox stress, to rapidly repair damaged DNA, to adapt to a hyperinflammatory or hyponutritious tumor microenvironment, and to expel anticancer drugs by the action of ATP-binding cassette transporters as well as plasticity with regard to the transition between dormant CSC and transit-amplifying progenitor cell phenotypes. In addition, CSCs manifest the phenomenon of metabolic reprogramming, which is essential for maintenance of their self-renewal potential and their ability to adapt to changes in the tumor microenvironment. Elucidation of the molecular underpinnings of these biological features of CSCs is key to the development of novel anticancer therapies. In this review, we highlight the pathological relevance of CSCs in terms of their hallmarks and identification, the properties of their niche—both in primary tumors and at potential sites of metastasis—and their resistance to oxidative stress dependent on system xc (−)., Highlights • Intratumoral heterogeneity driven by CSCs is responsible for therapeutic resistance. • CTCs survive in the distant organs and achieve colonization, causing metastasis. • E/M hybrid cancer cells due to partial EMT exhibit the highest metastatic potential. • The CSC niche regulates stemness in metastatic disease as well as in primary tumor. • Activation of system xc(-) by CD44 variant in CSCs is a promising therapeutic target.

The concept of a "cancer stem cell" has evolved over the past decades, and research on cancer stem cell biology has entered into a stage of remarkable progress. Cancer stem cells are a major determining factor contributing to the establishment of phenotypic and functional intratumoral heterogeneity in synchronization with their surrounding "cancer stem cell niches." They serve as the driving force for cancer initiation, metastasis, and therapeutic resistance in various types of malignancies. In verity, reciprocal interplay between ovarian cancer stem cells and their niches involves a complex but ingeniously orchestrated tumor microenvironment within the intraperitoneal milieu and especially contribute to chemotherapy resistance in patients with advanced ovarian cancer. Herein, we review the principles of our current understanding of the biological features of ovarian cancer stem cells, focusing mainly on the precise mechanisms underlying acquired chemotherapy resistance. Furthermore, we highlight the specific roles of various cancer-associated stromal and immune cells in creating possible cancer stem cell niches that regulate ovarian cancer stemness.

In 2018, Schneider and Zouboulis analysed the available tools for studying sebaceous gland pathophysiology in vitro. Since then, the interest in this field remains unbroken, as demonstrated by recent reviews on sebaceous gland physiology, endocrinology and neurobiology, the role of sebaceous glands beyond acne, and several original works on different areas of sebaceous gland function, including sebaceous lipogenesis. Landmark developments in the first part of the 30-year modelling research dedicated to the sebaceous gland, which is considered by several scientists as the brain of the skin, were the short-term culture of human sebaceous glands, the culture of human sebaceous gland cells and the development of immortalized sebaceous gland cell lines exhibiting characteristics of normal sebocytes. On the other hand, current developments represent the establishment of sebaceous gland spheroids, the 3D-SeboSkin model of viable skin explants ex vivo, the combination of culture-expanded epidermal stem cells of mice and adult humans to form de novo hair follicles and sebaceous glands, when they are transplanted into excisional wounds in mice, and 3D-printed scaffolds coated with decellularized matrix of adipose-derived mesenchymal stromal cells and SZ95 sebocytes. These novel tools may become useful platforms for better understanding of cellular and molecular mechanisms governing sebocyte biology and sebaceous gland homeostasis, such as the changes in sebum synthesis and composition, the infundibular differentiation and the influence of the innate immunity and the cutaneous microbiome and for identifying potential therapeutic targets of skin diseases affecting the sebaceous glands.

Neoplastic epithelial cells coexist in carcinomas with various non-neoplastic stromal cells, together creating the tumor microenvironment. There is a growing interest in the cross-talk between tumor cells and stromal fibroblasts referred to as carcinoma-associated fibroblasts (CAFs), which are frequently present in human carcinomas. CAF populations extracted from different human carcinomas have been shown to possess the ability to influence the hallmarks of cancer. Indeed, several mechanisms underlying CAF-promoted tumorigenesis are elucidated. Activated fibroblasts in CAFs are characterized as alpha-smooth muscle actin-positive myofibroblasts and actin-negative fibroblasts, both of which are competent to support tumor growth and progression. There are, however, heterogeneous CAF populations presumably due to the diverse sources of their progenitors in the tumor-associated stroma. Thus, molecular markers allowing identification of bona fide CAF populations with tumor-promoting traits remain under investigation. CAFs and myofibroblasts in wound healing and fibrosis share biological properties and support epithelial cell growth, not only by remodeling the extracellular matrix, but also by producing numerous growth factors and inflammatory cytokines. Notably, accumulating evidence strongly suggests that anti-fibrosis agents suppress tumor development and progression. In this review, we highlight important tumor-promoting roles of CAFs based on their analogies with wound-derived myofibroblasts and discuss the potential therapeutic strategy targeting CAFs.

I read with great interest the excellent work of Talukdar et al. (1), in which the authors demonstrate that melanoma differentiation associated gene-9 (MDA-9)/Syntenin (syndecan binding protein; SDCBP) regulates protective autophagy in glioblastoma stem cells (GSCs) through two cascades: First, the complex composed of MDA-9/Syntenin and focal adhesion kinase (FAK) increases phosphorylated Bcl-2 via PKCα, and, second, EGFR activated by the complex of MDA-9/Syntenin and FAK induces autophagy-related molecules such as Atg5, LC-3, and Lamp1 (1). Anoikis resistance is one of the hallmarks of cancer, which is essential for circulating tumor cells to persistently survive in the bloodstream and to exhibit the colonization/proliferation in the premetastatic niche (the favorable microenvironment for undifferentiated tumor-initiating cells to potentially form the distant metastatic … [↵][1]1Email: medical21go{at}yahoo.co.jp. [1]: #xref-corresp-1-1

I read with great interest the work of Qin et al. (1), in which the authors reveal that acyclic retinoid (ACR) selectively eliminates cancer stem cells (CSCs) of hepatocellular carcinoma (HCC), which highly express MYCN with enhanced Wnt/β-catenin signal pathway. There exist several molecules that are highly expressed in HCC-CSCs, such as CD90, CD133, EpCAM, GPC3, etc. (2). Among them, MYCN is not only a promising biomarker of HCC-CSCs but also a novel therapeutic target of ACR in preventing de novo HCC recurrence (1). Although NMYC gene amplification is frequently recognized in neuroendocrine tumors characterized by small-cell lung cancer and neuroblastoma (3), the restricted expression of … [↵][1]1Email: medical21go{at}yahoo.co.jp. [1]: #xref-corresp-1-1

Cancer stem-like cells exhibit the multi-functional roles to survive and persist for a long period in the minimal residual disease after the conventional anti-cancer treatments. Cancer stem-like cells of solid malignant tumors which highly express CD44v8-10, the variant isoform of CD44 generated by alternative splicing, has a resistance to redox stress by the robust production of glutathione mediated by ESRP1-CD44v-xCT (cystine/glutamate antiporter) axis. It has been reported that CD44v and c-Myc tend to show the inversed expression pattern at the invasive front of the aggressive tumors. Given that the accumulation of reactive oxygen species triggers the activation of Wnt/β-catenin signal pathway, it is hypothesized that CD44v causes the negative feedback machinery in the regulation of c-Myc expression via the attenuated ROS-induced Wnt signal pathway. To address the fundamental question whether and how both proliferative and quiescent cancer stem-like cells heterogeneously exist at the invasive/metastatic edge, researchers need to investigate into the E3-ubiquitin ligase activity essential for c-Myc degradation. CSCs heterogeneity at the invasive/metastatic front is expected to demonstrate the dynamic tumor evolution with the selective pressure of anti-cancer treatments. Furthermore, the novel molecular targeting therapeutic strategies would be established to disrupt the finely-regulated c-Myc expression in the heterogeneous CSC population in combination with the typical drug-repositioning with xCT inhibitor.

Introduction Epithelial cell adhesion molecule (EpCAM) is expressed in tumors with an epithelial cell of origin, in a heterogeneous manner. Prostate cancer stem-like cells highly express EpCAM. However, little is known about how EpCAM is involved in the ability of cells to adapt to micro-environmental changes in available growth factors, which is one of the essential biological phenotypes of cancer stem-like cells (CSCs). Methods EpCAM-high and EpCAM-low subpopulations of cells were established from the prostate cancer cell line PC-3. Signal transductions in response to serum starvation, and on the exposure to EGF ligand or the specific inhibitor were analyzed in terms. Furthermore, we analyzed the expression level of amino acid transporters which contribute to the activation of mTOR signal between the two subgroups. Results EpCAM-high and EpCAM-low PC-3 subpopulations showed markedly different responses to serum starvation. EpCAM expression was positively correlated with activation of the mTOR and epithelial growth factor receptor (EGFR) signaling pathways. Furthermore, AMP-activated protein kinase (AMPK) was gradually de-activated in EpCAM-low PC-3 cells in the absence of serum. Conclusions EpCAM regulates the AMPK signaling pathway, essential for the response to growth factors characterized by EGF. LAT1, the amino acid transporter stabilized at the cellular membrane by EpCAM, is likely to be responsible for the difference in the susceptibility to EGF between EpCAM-high and EpCAM-low PC-3 cells.

We hypothesized that sorafenib plus transarterial chemoembolization (TACE) would confer survival benefits over sorafenib alone for advanced hepatocellular carcinoma (aHCC). We investigated this while using the population-based All-Cancer Dataset to assemble a cohort (n = 3674, median age, 60, 83% men) of patients receiving sorafenib for aHCC (Child-Pugh A) with macro-vascular invasion or nodal/distant metastases. The patients were classified into the sorafenib-TACE group (n = 426) or the propensity score-matched sorafenib-alone group (n = 1686). All of the participants were followed up until death or the end of the study. Time-dependent Cox model and the Mantel&ndash, Byar test were used for survival analysis. During the median follow-ups of 221 and 133 days for the sorafenib-TACE and sorafenib-alone groups, 164 (39%) and 916 (54%) deaths occurred, respectively, the corresponding median overall survivals (OS) were 381 and 204 days, respectively (hazard ratio, HR: 0.74, 95% confidence interval, CI, 0.63&ndash, 0.88, p = 0.021). The one-year and six-month OS were 53.5% and 80.3% in the sorafenib-TACE group and 32.4% and 54.4% in the sorafenib-alone group, respectively. The major complications were comparable between the two groups. The addition of TACE to sorafenib improves survival, with a 26% reduction in mortality. These findings provide strong real-world evidence that supports this combination strategy for eligible Child-Pugh A aHCC patients.

Apoptosis plays a pivotal role in development and tissue homeostasis in multicellular organisms. Clustering of Bak proteins on the mitochondrial outer membrane is responsible for the induction of apoptosis by evoking a release of pro-apoptotic proteins from mitochondria into cytosol. However, how the protein cluster permeabilizes the mitochondrial membrane remains unclear because elucidation of the cluster characteristics such as size and protein density has been hampered by the diffraction-limited resolution of light microscopy. Here, we describe an approach to quantitatively characterize Bak clusters in situ based on single molecule localization. We showed that Bak proteins form densely packed clusters at the nanoscale on mitochondria during apoptosis. Quantitative analysis based on the localization of each Bak protein revealed that the density of Bak protein is uniform among clusters although the cluster size is highly heterogeneous. Our approach provides unprecedented information on the size and protein density of Bak clusters possibly critical for the permeabilization and is applicable for the analysis of different cluster formations.

New treatment strategies for platinum-resistant recurrent ovarian cancer are needed. Programmed cell death-1 (PD-1) signaling is a new target of antitumor therapy that involves immune reactivation; however, its antitumor effect on subsequent chemotherapy is unclear. Two representative cases are described here.

Tumor tissue is composed of cancer cells and surrounding stromal cells with diverse genetic/epigenetic backgrounds, a situation known as intra-tumoral heterogeneity. Cancer cells are surrounded by a totally different microenvironment than that of normal cells; consequently, tumor cells must exhibit rapidly adaptive responses to hypoxia and hypo-nutrient conditions. This phenomenon of changes of tumor cellular bioenergetics, called “metabolic reprogramming”, has been recognized as one of 10 hallmarks of cancer. Metabolic reprogramming is required for both malignant transformation and tumor development, including invasion and metastasis. Although the Warburg effect has been widely accepted as a common feature of metabolic reprogramming, accumulating evidence has revealed that tumor cells depend on mitochondrial metabolism as well as aerobic glycolysis. Remarkably, cancer-associated fibroblasts in tumor stroma tend to activate both glycolysis and autophagy in contrast to neighboring cancer cells, which leads to a reverse Warburg effect. Heterogeneity of monocarboxylate transporter expression reflects cellular metabolic heterogeneity with respect to the production and uptake of lactate. In tumor tissue, metabolic heterogeneity induces metabolic symbiosis, which is responsible for adaptation to drastic changes in the nutrient microenvironment resulting from chemotherapy. In addition, metabolic heterogeneity is responsible for the failure to induce the same therapeutic effect against cancer cells as a whole. In particular, cancer stem cells exhibit several biological features responsible for resistance to conventional anti-tumor therapies. Consequently, cancer stem cells tend to form minimal residual disease after chemotherapy and exhibit metastatic potential with additional metabolic reprogramming. This type of altered metabolic reprogramming leads to adaptive/acquired resistance to anti-tumor therapy. Collectively, complex and dynamic metabolic reprogramming should be regarded as a reflection of the “robustness” of tumor cells against unfavorable conditions. This review focuses on the concept of metabolic reprogramming in heterogeneous tumor tissue, and further emphasizes the importance of developing novel therapeutic strategies based on drug repositioning.

Cancer stem cells (CSCs) are undifferentiated cancer cells with a high tumorigenic activity, the ability to undergo self‐renewal, and a multilineage differentiation potential. Cancer stem cells are responsible for the development of tumor cell heterogeneity, a key feature for resistance to anticancer treatments including conventional chemotherapy, radiation therapy, and molecularly targeted therapy. Furthermore, minimal residual disease, the major cause of cancer recurrence and metastasis, is enriched in CSCs. Cancer stem cells also possess the property of “robustness”, which encompasses several characteristics including a slow cell cycle, the ability to detoxify or mediate the efflux of cytotoxic agents, resistance to oxidative stress, and a rapid response to DNA damage, all of which contribute to the development of therapeutic resistance. The identification of mechanisms underlying such characteristics and the development of novel approaches to target them will be required for the therapeutic elimination of CSCs and the complete eradication of tumors. In this review, we focus on two prospective therapeutic approaches that target CSCs with the aim of disrupting their quiescence or redox defense capability.

Aquaporin-7 (AQP7) is expressed in adipose tissue, permeated by water and glycerol, and is involved in lipid metabolism. AQP7-null mice develop obesity, insulin resistance, and adipocyte hypertrophy. Here, we show that AQP7 is expressed in adipocyte plasma membranes, and is re-localized to intracellular membranes in response to catecholamine in mouse white adipose tissue. We found that internalization of AQP7 was induced by PKA activation and comparative gene identification 58 (CGI-58). This relocation was confirmed by functional studies in 3T3-L1 adipocytes. Collectively, these results suggest that AQP7 makes several contributions to adipocyte metabolism, in both cortical and intracellular membranes.

It has recently been reported that molecular targeting drugs fail to overcome the “oncogene/oncogenic signaladdiction” of cancer cells. Tumor tissue is composed of heterogeneous cancer cells, so that the therapeutic response is difficult to predict. Recent advance in cancer research has strongly suggests that cancer stem cells contribute to the formation and maintenance of the heterogeneous cellular society in the tumor tissue. After all, this heterogeneity is the major cause of the acquired resistance to anti-tumor therapies. In this commentary, we would like to briefly explain the promising therapeutic targets of CD44 variant isoform and EpCAM, the “functional” cancer stem cell markers.

Cancer stem-like cells express high amount of CD44 variant8-10 which protects cancer cells from redox stress. We have demonstrated by immunohistochemical analysis and Western blotting, and reverse-transcription polymerase chain reaction, that CD44 variant8-10 and c-Myc tend to show the inversed expression manner in gastric cancer cells. That is attributable to the oxidative stress-induced canonical Wnt activation, and furthermore, the up-regulation of the downstream molecules, one of which is oncogenic c-Myc, is not easily to occur in CD44 variant-positive cancer cells. We have also found out that CD44v8-10 expression is associated with the turn-over of the c-Myc with the experiments using gastric cancer cell lines. This cannot be simply explained by the model of oxidative stress-induced Wnt activation. CD44v8-10-positive cancer cells are enriched at the invasive front. Tumor tissue at the invasive area is considered to be composed of heterogeneous cellular population; dormant cancer stem-like cells with CD44v8-10 (high)/ Fbw7 (high)/ c-Myc (low) and proliferative cancer stem-like cells with CD44v8-10 (high)/ Fbw7 (low)/ c-Myc (high).

In cancer metastasis, various environmental stressors attack the disseminating cells. The successful colonization of cancer cells in secondary sites therefore requires the ability of the cells to avoid the consequences of such exposure to the stressors. Here we show that orthotopic transplantation of a CD44 variant isoform-expressing (CD44v(+)) subpopulation of 4T1 breast cancer cells, but not that of a CD44v(-) subpopulation, in mice results in efficient lung metastasis accompanied by expansion of stem-like cancer cells. Such metastasis is dependent on the activity of the cystine transporter xCT, and the stability of this protein is controlled by CD44v. We find that epithelial splicing regulatory protein 1 regulates the expression of CD44v, and knockdown of epithelial splicing regulatory protein 1 in CD44v(+) cells results in an isoform switch from CD44v to CD44 standard (CD44s), leading to reduced cell surface expression of xCT and suppression of lung colonization. The epithelial splicing regulatory protein 1-CD44v-xCT axis is thus a potential therapeutic target for the prevention of metastasis.

Cancer stem cells (CSCs) , which are subset of tumor cells resistant to radiation and chemotherapy, are associated with malignant characteristics of tumor and possess both self-renewal ability and pluripotency for tumor formation. In the process of generating non-CSCs from CSCs, gene mutations and epigenetic changes are induced in those cells, resulting in composition of tumor tissue with heterogeneous cell population. CSCs have been recognized as the source of metastatic foci. Epithelial-mesenchymal transition (EMT) is a change in cellular phenotype characterized by the loss of cell-to-cell adhesions and the gain of migratory behaviors,which has been shown to be a critical factor for initiating cancer invasion and metastasis. However, some recent studies suggest that EMT is not essential requirement for tumor invasion and metastasis. Herein, we discuss the biological significance of CSCs and EMT in tumor invasion and metastasis.

Introduction: Recent studies have shown that intra-tumor heterogeneity is the main cause of therapeutic resistance in cancer. Tumor tissue is composed of heterogeneous cellular populations with a hierarchical organization governed by stem/progenitor-like cells, also referred to as cancer stem-like cells (CSCs). Epithelial cell adhesion molecule (EpCAM), also known as CD326, is over-expressed ubiquitously in CSCs that originate from epithelial lesions. Prostate cancer stem-like cells highly express EpCAM. However, little is known about how EpCAM is involved in the ability of cells to adapt to micro-environmental changes in available growth factors, which is one of the essential biological phenotypes of CSCs. Methods: EpCAM-high and EpCAM-low subpopulations of cells were established from the prostate cancer cell line PC-3. Signal transduction in response to serum starvation, signaling pathways essential for tumorigenic potential in vivo, and the machinery of serum depletion-induced dormancy and plasticity were analyzed by immunoblot, quantitative RT-PCR, flow cytometry, and immunohistochemical staining. Results: EpCAM-high and EpCAM-low PC-3 subpopulations showed markedly different responses to serum starvation. EpCAM expression was positively correlated with activation of the mammalian target of rapamycin (mTOR) and epithelial growth factor receptor (EGFR) signaling pathways. Contrary to accepted wisdom, AMP-activated protein kinase (AMPK) was gradually de-activated in EpCAM-low PC-3 cells in the absence of serum. In addition, the Spearman's rank correlation between EpCAM and LAT1 in parental, EpCAM-high, and EpCAM-low PC-3 cells was very high (0.859), which implied that the expression of EpCAM correlates with the potential of leucine uptake. Both EpCAM-high and EpCAM-low PC-3 cells exhibited tumorigenic potential, which depended on mTOR and canonical Wnt signaling, respectively. Finally, serum starvation promoted cell cycle arrest and the acquisition of a quiescent phenotype. It has been well established that there are two major mechanisms that promote G0 entry; the SCF (Fbw7)-c-Myc axis and the Skp2-p27 axis. EpCAM-high PC-3 cancer cells entered the G0 phase via the SCF (Fbw7)-c-Myc axis, while EpCAM-low cells did so via the Skp2-p27 axis. EGF-induced plasticity in EpCAM-high PC-3 cells but not in EpCAM-low PC-3 cells implies that EGFR signaling is likely to be involved in cell cycle re-entry in EpCAM-high PC-3 cells. Discussion: EpCAM regulates the AMPK signaling pathway, which is essential for the response to growth factors, characterized by EGF, in the tumor microenvironment. Furthermore, EpCAM enhances the ability of cells to enter the G0 phase under long-term serum starvation and promotes cell cycle re-entry from this dormancy upon exposure to growth factors. Taken together, these data suggest that “stemness,” including sensitivity to growth factors and plasticity between dormancy and proliferation, is maintained and regulated by EpCAM. Significance: The phenotypes induced by EpCAM expression are mainly responsible for the persistence of minimal residual disease (MRD) and the latent relapse of prostate cancers. Therefore, EpCAM may be a promising therapeutic target to block the ability of prostate cancer CSCs to adapt to the lack of growth factors in the tumor microenvironment. References: EpCAM expression in the prostate cancer makes the difference in the response to growth factors (Biochemical and Biophysical Research Communications, 2013, in press) EpCAM functions as a sensor that allows prostate cancer cells to become quiescent in response to lack of growth factors (In preparation) Citation Format: Go J. Yoshida, Hideyuki Saya. EpCAM functions as a sensor that allows prostate cancer cells to become quiescent in response to lack of growth factors.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B06. doi:10.1158/1538-7445.CHTME14-B06

Cancer metastasis is established by the seeding and successful colonization of stem-like cancer cells which show tumor-initiating ability at distant organs. CD44 is a cell surface marker protein which is highly expressed in stem-like cells of several human cancers including breast cancer. CD44 exists in various isoforms produced through alternative splicing of mRNA. Increased expression of CD44 variant isoform (CD44v) has been shown to correlate with metastatic potential and aggressive clinical behavior of various human cancers. We recently showed that CD44v contributes to reactive oxygen species (ROS)-defense in cancer cells by enhancing cystine uptake by the cystine transporter xCT. However, the functional relevance of CD44v and xCT in the metastatic spread of stem-like cancer cells has remained elusive. In the present study, we show that metastatic breast cancer 4T1 cells consist of CD44v+ and CD44v- cells. The orthotopic transplantation of CD44v+ population but not that of CD44v-, in mice resulted in efficient lung metastasis. We found that CD44v expressing metastatic cancer cells highly express xCT and thereby maintain high reduced glutathione (GSH) level. A specific xCT inhibitor sulfasalazine suppressed lung metastatsis of CD44v+ cells. Alternative splicing of CD44mRNA in such metastatic cancer cells is regulated by Epithelial Splicing Related Protein1 (ESRP1) and short hairpin RNA (shRNA) targeting ESRP1 switched isoform from CD44v to CD44 standard (CD44s), leading to the suppression of lung metastasis. Furthermore, we found that ESRP1 expression is regulated epigenetically through histone modification by performing chromatin immunoprecipitation (ChIP) sequencing analysis of the ESRP1 locus in CD44v+ and CD44v- cells. These findings establish a novel role of ESRP1-regulated alternative splicing program in the regulation of antioxidant status of stem-like cancer cells which is crucial for the metastatic ability. Citation Format: Kenji Tsuchihashi, Osamu Nagano, Toshifumi Yae, Takatsugu Ishimoto, Takeshi Motohara, Momoko Yoshikawa, Go J Yoshida, Takeyuki Wada, Takashi Masuko, Kaoru Mogushi, Hiroshi Tanaka, Tsuyoshi Osawa, Yasuharu Kanki, Takashi Minami, Hiroyuki Aburatani, Mitsuyo Ohmura, Akiko Kubo, Makoto Suematsu, Kazuhisa Takahashi, Eishi Baba, Koichi Akashi, Hideyuki Saya. ESRP1 regulated alternative splicing of CD44mRNA enhances lung colonization of metastatic cancer cell. [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 4967. doi:10.1158/1538-7445.AM2014-4967

Metastasis of tumor cells is a multi-step phenomenon including dissociation from primary site, intravasation, long-term circulation in the blood, extravasation, and colonization in the pre-metastatic niche in the distant organs. It is quite important that cancer cells are always exposed to redox stress throughout this muti-step metastatic phenomenon. This is why metastasis occurs only when cancer cells are refractory to the high level of reactive oxygen species (ROS) in the tumor microenvironment. CD44, one of the cancer stem cell (CSC) markers, makes diversity in isofroms due to the alternative splicing via epithelial splicing regulatory protein1 (ESRP1) or transcription factor grainyhead-like 2 (Grhl2). In the clinical perspective, a high expression level of ESRP1 or Grhl-2 in the tumor cells is generally associated with a poor prognosis in breast cancer patients. In addition, epigenetic modifications including DNA and histone methylation at metastasis-related genes have been also considered as a cause of cancer metastasis. This is why the depletion of CD44variant isoform (CD44v) is not necessarily accompanied by epithelial-mesenchymal transition (EMT), given that EMT has widely been recognized to expand the CSC population. Besides, CD44 is not only a surface marker of CSCs but also a functional molecule; for instance, the interaction between CD44v6 and c-Met, the receptor of hepatocyte growth factor (HGF), activates the Ras-dependent survival signal of tumor cells. We have recently identified that CD44 variant 8-10 (CD44v9) stabilizes the localization of xCT glutamate-cystine transporter at the cellular membrane, thereby increasing the intracellular level of reduced glutathione (GSH). In fact, enrichment of CD44v9-positive tumor cells is frequently recognized in the metastatic foci and recalcitrant cases. The expression level of CD44v9 is positively correlated with the Akt/PI3K/mTOR signal activity of metastatic breast tumor cells. Akt/PI3K/mTOR signal is related to cholesterol synthesis and to adaptation to the hypo-nutrition, or hypoxia. In the case of metastasis of breast cancer cells to the lungs, tumor-entrained neutrophils (TEN) accumulate in the lung to inhibit lung metastasis through the increased redox stress in the NADPH-dependent manner. Thus, the presence of xCT transporter promotes the GSH synthesis and the protection from tumor stroma-derived ROS, which plays a fundamental role in the adaptive resistance to conventional cancer therapy for the metastasis. We have also revealed that ESRP1-dependent alternative splicing of CD44 mRNA increases both the intracellular GSH level and the metastatic potential of the breast cancer cells. We have demonstrated that depletion of ESRP1 or Grhl2 in metastatic breast cancer cells adversely affects the metastatic potential, since they fail to express CD44v9. Notably, even the tumor cells which are lack of CD44v9 are able to form the metastasis to lungs, but the ability of survival and proliferation in the microenvironment rich in redox stress are considerably lower. Akt/PI3K/mTOR activity is significantly suppressed compared with the aggressive metastatic foci mainly composed of CD44v9-positive tumor cells. Therapies targeted to alternative splicing of CD44 as well as those that target CD44v-mediated stabilization of xCT might be expected to imapir the adaptive resistance to ROS in the metastatic stem-like cancer cells and thereby rendering them susceptible to currently available cancer treatments. Considering the high dependency to lipid synthesis In the survival and proliferation of metastatic breast cancer cells, HMG-CoA reductase inhibitors contribute to the synergistic inhibition of the metastatic exacerbation. Citation Format: Go J. Yoshida, Kenji Tsuchihashi, Toshifumi Yae, Osamu Nagano, Hideyuki Saya. ESRP1-CD44v-xCT axis as a potential therapeutic target for the metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C89.

EpCAM (Epithelial cell adhesion molecule: CD326) is a type I transmembrane glycoprotein that functions as an epithelial-specific cell-adhesion molecule. This molecule has long been recognized as one of the cancer stem cell markers, but the detailed function of EpCAM is still elusive. EpCAM is expressed in tumor cells in the vast majority of patients with epithelial cancers, such as ovarian, gastric, colorectal, pancreatic, breast, lung, and endometrial tumors. EpCAM-positive carcinomas maintain EpCAM expression even in the metastatic conditions, including dissemination into the peritoneal cavity. In some carcinomas, highly expressed EpCAM is reportedly associated with a poor prognosis due to the repeated relapse and latent metastasis. In the clinical perspective, catumaxomab has two different antigen-binding specificities: one for EpCAM on tumor cells and one for the CD3 antigen on T-cells. Since this drug is effective for advanced ovarian cancers with malignant ascites, the molecular mechanism underlying the significantlly clinical development should be further investigated. Recent study using the proteomics technology has strongly suggested that EpCAM expression promotes the active transport of amino acids into tumor cells by forming the complex with CD147, CD98hc (heavy chain) and other amino acid transporters such as LAT1, LAT2, and xCT. Besides, a novel MCT (monocarboxylate transporter)-CD147-CD98hc-LAT1 (L-type amino acid transporter 1) complex has previously been reported to promote the proliferation and metabolism of epithelial cells via the activation of AMPK. Therefore, we firstly performed the immunocytochemical and immunoblot analyzes to identify what kinds of amino acid transporters co-localize to EpCAM in HCT-116 and SKOV-III cells. Secondly, we investigated the relationship between EpCAM expression and the cell proliferation and ATP synthesis. While EpCAM-positive cancer cells have the robust potential for proliferation, EpCAM-negative cancer cells have the robust potential for ATP production. The treatment with rapamycin, one of the immune-suppressing drugs, reduced the activity of mTOR(mammalian target of rapamycin)/Akt pathway in cancer cells. The phenotypes of EpCAM-positive cells, such as the dependency to glycolysis and the proliferative ability, were attenuated by the treatment with rapamycin. These results strongly suggest that mTOR/Akt signal is the downstream pathway of EpCAM expression. Taken together, EpCAM is thought of as not only a cancer stem cell marker but also a functional molecule to maintain stemness for the following reasons.EpCAM-positive cancer cells tend to more depend on glycolysis than EpCAM-negative cancer cells.EpCAM expression activates mTOR/Akt signal.EpCAM molecule functions as a sensor to the nutrient in the tumor microenvironment, thereby adapting to starvation by acquiring dormancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A55.

Accumulating evidence suggests that the phenomenon of epithelial-mesenchymal-transition (EMT) plays a fundamental role in the tumor development. Several research articles have been published from Journal of Experimental and Clinical Cancer Research (JECCR) which have investigated into the molecular machineries underlying the importance of EMT for hepatic cancer. Given those recent publications by JECCR, this commentary focuses on the pathological significance of EMT for liver tumor.

Background Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome that is commonly associated with a germline mutation in the tumor suppressor gene p53. Loss of p53 results in increased expression of CD44, a cancer stem cell (CSC) marker, which is involved in the scavenging of reactive oxygen species (ROS). Here, we report a change in the expression of a CD44 variant isoform (CD44v8-10) in an 8-year-old female LFS patient with osteosarcoma and atypical liver cancer after chemotherapy. Case presentation The patient visited a clinic with a chief complaint of chronic pain in a bruise on her right knee. Magnetic resonance imaging (MRI) raised the possibility of a bone malignancy. Biochemical testing also revealed significantly elevated levels of AFP, which strongly suggested the existence of a primary malignancy in the liver. MRI imaging showed the simultaneous development of osteosarcoma and liver cancer, both of which were confirmed upon biopsy. Combined therapy with surgical resection after chemotherapy was successful in this patient. Regardless of the absence of a familial history of hereditary cancer, a germline mutation in p53 was identified (a missense mutation defined as c.722 C>T, p.Ser241Phe). To better understand the cancer progression and response to treatment, immunohistochemical (IHC) analysis of biopsy specimens obtained before and after chemotherapy was performed using a specific antibody against CD44v8-10. Conclusion This case demonstrates the ectopic up-regulation of CD44v8-10 in a biopsy sample obtained after cytotoxic chemotherapy, which confers high levels of oxidative stress on cancer cells. Because the alternative splicing of CD44 is tightly regulated epigenetically, it is possible that micro-environmental stress resulting from chemotherapy caused the ectopic induction of CD44v8-10 in vivo.

The 2016 Nobel Prize in Physiology or Medicine was awarded to the researcher that discovered autophagy, which is an evolutionally conserved catabolic process which degrades cytoplasmic constituents and organelles in the lysosome. Autophagy plays a crucial role in both normal tissue homeostasis and tumor development and is necessary for cancer cells to adapt efficiently to an unfavorable tumor microenvironment characterized by hypo-nutrient conditions. This protein degradation process leads to amino acid recycling, which provides sufficient amino acid substrates for cellular survival and proliferation. Autophagy is constitutively activated in cancer cells due to the deregulation of PI3K/Akt/mTOR signaling pathway, which enables them to adapt to hypo-nutrient microenvironment and exhibit the robust proliferation at the pre-metastatic niche. That is why just the activation of autophagy with mTOR inhibitor often fails in vain. In contrast, disturbance of autophagy–lysosome flux leads to endoplasmic reticulum (ER) stress and an unfolded protein response (UPR), which finally leads to increased apoptotic cell death in the tumor tissue. Accumulating evidence suggests that autophagy has a close relationship with programmed cell death, while uncontrolled autophagy itself often induces autophagic cell death in tumor cells. Autophagic cell death was originally defined as cell death accompanied by large-scale autophagic vacuolization of the cytoplasm. However, autophagy is a “double-edged sword” for cancer cells as it can either promote or suppress the survival and proliferation in the tumor microenvironment. Furthermore, several studies of drug re-positioning suggest that “conventional” agents used to treat diseases other than cancer can have antitumor therapeutic effects by activating/suppressing autophagy. Because of ever increasing failure rates and high cost associated with anticancer drug development, this therapeutic development strategy has attracted increasing attention because the safety profiles of these medicines are well known. Antimalarial agents such as artemisinin and disease-modifying antirheumatic drug (DMARD) are the typical examples of drug re-positioning which affect the autophagy regulation for the therapeutic use. This review article focuses on recent advances in some of the novel therapeutic strategies that target autophagy with a view to treating/preventing malignant neoplasms.