Flubendazole elicits anti‐metastatic effects in triple‐negative breast cancer via STAT3 inhibition

Tumor metastasis remains the cause of 90% of cancer‐related deaths. Cancer stem cells (CSC) are thought to be responsible for the aggressive and metastatic nature of triple‐negative breast cancers (TNBC), and new therapeutic strategies are being devised to target them. Flubendazole (FLU) is a widely used anthelmintic agent that also exhibits anticancer activity in several cancer types. The aim of this study was to characterize the mechanism of action of FLU on breast cancer stem cell (BCSC)‐like properties and metastasis in TNBC. FLU treatment caused a significant induction of apoptosis, accompanied by G2/M phase accumulation, caspase‐3/‐7 activation and the dysregulation of STAT3 activation in TNBC cells. The latter phenomenon was associated with impairment of cancer stem‐like traits, concomitant with a reduction in the CD24^low/CD44^high, CD24^high/CD49f^high subpopulation, ALDH1 activity and mammosphere formation. The BCSC‐enriched populations exhibited enhanced metastasis with higher STAT3 activation, while FLU administration inhibited tumor growth, angiogenesis and lung and liver metastasis, coinciding with decreased MMP‐2 and MMP‐9 levels in circulating blood. FLU kills not only rapid proliferating tumor cells but also effectively eradicates BCSC‐like cells in vitro and in vivo. Our findings warrant further investigation of FLU as a treatment for metastatic TNBC.     

PD‐1 blockade enhances the antitumor efficacy of GM‐CSF surface‐modified bladder cancer stem cells vaccine

Eliminating cancer stem cells (CSCs) is a key issue in eradicating tumor. The streptavidin–granulocyte‐macrophage‐colony stimulating factor (SA–GM‐CSF) surface‐modified bladder CSCs vaccine previously developed using our protein–anchor technology could effectively induce specific immune response for eliminating CSCs. However, program death receptor‐1 (PD‐1)/program death ligand 1 (PD‐L1) signaling in tumor microenvironment results in tumor‐adaptive immune resistance. Although the CSCs vaccine could increase the number of CD8⁺T cells, a part of these CD8⁺T cells expressed PD‐1. Moreover, the CSCs vaccine upregulated the PD‐L1 expression of tumor cells, resulting in immune resistance. Adding PD‐1 blockade to the CSCs vaccine therapy increased the population of CD4⁺, CD8⁺ and CD8⁺IFN‐γ⁺ but not CD4⁺ Foxp3⁺T cells and induced the highest production of IFN‐γ. PD‐1 blockade could effectively enhance the functions of tumor‐specific T lymphocytes generated by the CSCs vaccine. This combination therapy improved the cure rate among mice and effectively protected the mice against a second CSCs cell challenge, but not a RM‐1 cell challenge. These results indicate that PD‐1 blockade combined with the GM‐CSF‐modified CSCs vaccine effectively induced a strong and specific antitumor immune response against bladder cancer.     

Hypoxia promotes the phenotypic change of aldehyde dehydrogenase activity of breast cancer stem cells

Stable breast cancer cell (BCC) lines are valuable tools for the identification of breast cancer stem cell (BCSC) phenotypes that develop in response to several stimuli as well as for studying the basic mechanisms associated with the initiation and maintenance of BCSCs. However, the characteristics of individual, BCC‐derived BCSCs varies and these cells show distinct phenotypes depending on the different BCSC markers used for their isolation. Aldehyde dehydrogenase (ALDH) activity is just such a recognized biomarker of BCSCs with a CD44⁺/CD24^? phenotype. We isolated BCSCs with high ALDH activity (CD44⁺/CD24^?/Aldefluor^pos) from a primary culture of human breast cancer tissue and observed that the cells had stem cell properties compared to BCSCs with no ALDH activity (CD44⁺/CD24^?/Aldefluor^neg). Moreover, we found Aldefluor^pos BCSCs had a greater hypoxic response and subsequent induction of HIF‐1α expression compared to the Aldefluor^neg BCSCs. We also found that knocking down HIF‐1α, but not HIF‐2α, in Aldefluor^pos BCSCs led to a significant reduction of the stem cell properties through a decrease in the mRNA levels of genes associated with the epithelial‐mesenchymal transition. Indeed, HIF‐1α overexpression in Aldefluor^neg BCSCs led to Slug and Snail mRNA increase and the associated repression of E‐cadherin and increase in Vimentin. Of note, prolonged hypoxic stimulation promoted the phenotypic changes of Aldefluor^neg BCSCs including ALDH activity, tumorigenesis and metastasis, suggesting that hypoxia in the tumor environment may influence BCSC fate and breast cancer clinical outcomes.     

Targeting highly expressed extracellular HSP90 in breast cancer stem cells inhibits tumor growth in vitro and in vivo

Breast cancer stem cells (BCSC) have been identified in breast carcinoma as CD44⁺/CD24^?/low cells, which display tumorigenic activity and have the ability to self-renew, differentiate and metastasize. Previous studies showed that extracellular HSP90 (eHSP90) participates in the invasion and metastatic processes of various cancers including breast cancer. Here, we show for the first time that eHSP90 is over-expressed in mammosphere cultures that are derived from the MDA-MB-231, MDA-MB-453 and MCF-7 breast cancer cell lines. These mammospheres are highly enriched in cells of the CD44⁺/CD24^?/low BCSC phenotype and additionally show high expression of the BCSC markers CD49f and Sox2. Thus our results indicate that eHSP90 represents a potential novel BCSC marker. Moreover, we present evidence that eHSP90 is functionally involved in BCSC activity in vitro and in vivo. Selective neutralization of eHSP90, using the monoclonal antibody mAb 4C5, has the capacity to inhibit stem cell activity in vitro because the formation of mammosphere-derived colonies is dramatically reduced in its presence. In vivo, the treatment of mice with mAb4C5 using a prophylactic protocol, significantly inhibited the primary growth of MDA-MB-231 and mammosphere-derived tumors. More importantly, administration of this antibody in a therapeutic protocol caused a statistically significant regression of established tumors derived from MDA-MB-231 originating mammospheres. Tumor regression was even greater when mAb 4C5 was administered in combination with paclitaxel. Overall, our findings implicate eHSP90 as a potential novel BCSC biomarker. Moreover they show that eHSP90 participates in BCSC-derived primary tumor growth. Finally, we provide additional support for the possible therapeutic value of mAb4C5 in the treatment of breast cancer.     

Isolation and characterization of tumorigenic extrahepatic cholangiocarcinoma cells with stem cell‐like properties

Recent studies suggest that the ability to form and grow tumors specifically resides in a small cell population called cancer stem cells (CSCs). These studies were conducted mainly on various human cancers; however, isolation and characterization of stem cells from cholangiocarcinoma have not been attempted. The molecular markers CD24, CD44, CD34, and epithelial cell adhesion molecule (EpCAM) are widely used, individually or in combination, to characterize some types of CSCs. In this study, we used these markers to identify a subpopulation of cells in extrahepatic cholangiocarcinoma (ECC) with cancer stem/progenitor cell‐like properties. We found that CD24⁺CD44⁺EpCAM^high cells (0.39–2.27%) were present in human ECC tissues. The expression of a CD24⁺CD44⁺EpCAM^high subpopulation was consistent with primary cancers and could be duplicated during serial in vivo passaging in NOD/SCID mice. CD24⁺CD44⁺EpCAM^high cells isolated from 3 cholangiocarcinoma xenografts showed high tumorigenic potential compared with CD24^?CD44^?EpCAM^low/? cells. These tumorigenic ECC cells exhibited the stem cell properties of self‐renewal and ability to produce heterogeneous progeny. We report the identification of a CSC population in ECC characterized by CD24, CD44 and EpCAM phenotypes. Our findings could provide new insight into the tumorigenesis of cholangiocarcinoma and offer a potential target for anti‐cancer therapy.     

Cancer stem cells in Epstein‐Barr virus‐associated gastric carcinoma

Cancer stem cells (CSCs) play a decisive role in the development and progression of cancer. To investigate CSCs in Epstein‐Barr virus (EBV)‐associated carcinoma (EBVaGC), we screened previously reported stem cell markers of gastric cancer in EBV‐infected gastric cancer cell lines (TMK1 and NUGC3) and identified CD44v6v9 double positive cells as candidate CSCs. CD44v6/v9^+/+ cells were sorted from EBVaGC cell line (SNU719) cells and EBV‐infected TMK1 cells and these cell populations showed high spheroid‐forming ability and tumor formation in SCID mice compared with the respective CD44v6/v9^?/? cells. Sphere‐forming ability was dependent on the nuclear factor‐κB (NF‐κB) signaling pathway, which was confirmed by decrease of sphere formation ability under BAY 11‐7082. Small interfering RNA knockdown of latent membrane protein 2A (LMP2A), one of the latent gene products of EBV infection, decreased spheroid formation in SNU719 cells. Transfection of the LMP2A gene increased the sphere‐forming ability of TMK1 cells, which was mediated through NF‐κB signaling. Together, these results indicate that CD44v6v9^+/+ cells are CSCs in EBVaGC that are maintained through the LMP2A/NF‐κB pathway. Future studies should investigate CD44v6/v9^+/+ cells in normal and neoplastic gastric epithelium to prevent and treat this specific subtype of gastric cancer infected with EBV.     

Predictive factors of the tumor immunological microenvironment for long‐term follow‐up in early stage breast cancer

The aim of this research was to investigate the correlation of immunologic factors in the tumor environment of breast cancer, using immunohistological staining to evaluate the expression of programmed death 1/programmed death ligand 1 (PD‐1/PD‐L1), phosphatase and tensin homolog (PTEN), tumor infiltrating lymphocytes (TILs), and macrophages, and to analyze the association between the immunologic factors and clinical outcome for patients with early stage breast cancer (EBC). A total of 97 EBC patients who underwent standard surgery were investigated. Expression of PD‐1/PD‐L1 and PTEN and the density of CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages were evaluated by immunohistochemical analysis. The association between the immunologic factors and clinical outcome was statistically analyzed. The density of CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages and non‐expression of PTEN was significantly higher in cases of triple negative breast cancer. CD8⁺ TIL density and CD8⁺/PD‐L1⁺ expression were predictive factors for disease‐free survival and overall survival (OS). Human epidermal growth factor 2 (HER2)‐positive patients with PTEN expression and luminal/HER2‐negative patients without PD‐L1 expression had significantly longer OS compared to patients without PTEN expression (P = 0.049) and with PD‐L1 expression (P = 0.036), respectively. Furthermore, patients with PD‐L1⁺/CD8⁺ expression had worse median progression‐free survival (P = 0.022) and median OS (P = 0.037) compared with patients without PD‐L1⁺/CD8⁺ expression. The CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages were shown to infiltrate the tumor area of EBC. In particular, triple negative breast cancer had a higher rate of TIL infiltration within the tumor environment. Expression of PTEN and lack of PD‐L1 expression were associated with favorable survival in HER2‐positive and luminal/HER2‐negative EBC patients, respectively. The PD‐L1 expression combined with CD8⁺ density was significantly associated with an aggressive clinical outcome.     

Cancer stem/progenitor cells are highly enriched in CD133+CD44+ population in hepatocellular carcinoma

Both our previous study and other reports have suggested that CD133, originally classified as a hematopoietic stem cell marker, could be used for enrichment of cancer stem cells (CSCs) in human hepatocellular carcinoma (HCC). It was also noted that not all of CD133⁺ cells were representative of CSCs. Further identification and characterization of CSCs or tumor‐initiating cells in HCC are necessary to better understand hepatocarcinogenesis. In present study, we demonstrated that CSC phenotype could be precisely defined by co‐expression of CD133 and CD44 cell surface markers. CD133⁺CD44⁺ HCC cells showed stem cell properties, including extensive proliferation, self‐renewal, and differentiation into the bulk of cancer cells. In vivo xenograft experiments revealed that, actually, the highly tumorigenic capacity of CD133⁺ cells as previously described was primarily attributed to CD133⁺CD44⁺ cell subpopulation, instead of their CD133⁺CD44^? counterparts. Moreover, cells double‐positive for CD133 and CD44 exhibited preferential expression of some stem cell‐associated genes and were more resistant to chemotherapeutic agents due to the upregulation of ATP‐binding cassette (ABC) superfamily transporters, including ABCB1, ABCC1, and ABCG2, further supporting these cells as HCC cell origin. Our findings suggest that CD133⁺CD44⁺ cells might represent true cancer stem/progenitor cells in HCC, which could allow a better understanding of HCC initiation and progression, as well as establish a precise target for the development of more effective therapies.     

Amurensin G enhances the susceptibility to tumor necrosis factor‐related apoptosis‐inducing ligand‐mediated cytotoxicity of cancer stem‐like cells of HCT‐15 cells

Cancer stem cells (CSCs) are resistant to radiotherapy and chemotherapy and play a significant role in cancer recurrence. Design of better treatment strategies that can eliminate or otherwise control CSC populations in tumors is necessary. In this study, the sensitivity to tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐induced cytotoxicity and the effect of amurensin G, a novel sirtuin 1 (SIRT1) inhibitor, were examined using the CSC‐enriched fraction of HCT‐15 human colon cancer cells. Cancer stem cell‐enriched HCT‐15 colony cells were paradoxically less sensitive to doxorubicin, and more sensitive to TRAIL‐induced cytotoxicity, than their parental cells. Also, CD44⁺ HCT‐15 cells were more susceptible to TRAIL‐mediated cytotoxicity than CD44^? HCT‐15 cells, possibly due to increased levels of death receptors DR4 and DR5 as well as c‐Myc, and decreased levels of c‐FLIP_L/S in CD44⁺ cells compared with CD44^? HCT‐15 cells. The combination effect of amurensin G on TRAIL‐mediated cytotoxicity was much more apparent in CD44⁺ cells than in CD44^? HCT‐15 cells, and this was associated with more prominent downregulation of c‐FLIP_L/S in CD44⁺ cells than in CD44^? HCT‐15 cells. These results indicate that HCT‐15 colony or CD44⁺ cells, which may have CSC properties, are more sensitive to TRAIL than parental or CD44^? HCT‐15 cells. Amurensin G may be effective in eliminating colon CSCs and be applicable to potentiate the sensitivity of colon CSCs to TRAIL.     

CD44+ slow‐cycling tumor cell expansion is triggered by cooperative actions of Wnt and prostaglandin E2 in gastric tumorigenesis

(Cancer Sci 2010; 101: 673–678) Similar to normal tissue stem cells, cancer stem cells (CSCs) are thought to be quiescent or slow‐cycling and, thereby, insensitive to chemo‐ and radiotherapies. CD44, a cell surface component that interacts with the extracellular matrix, has been found to be highly expressed in CSCs of several solid tumors. However, the relevancy between CD44⁺ cells and slow‐cycling cells and the underlying mechanisms for the emergence of CD44⁺ CSCs during tumorigenesis have not been elucidated. Here we show that a gastric gland residing at the squamo‐columnar junction (SCJ) in normal mouse stomach contains CD44⁺ stem cell‐like slow‐cycling cells and that this characteristic CD44⁺ gland was expanded by prostaglandin E2 (PGE₂) and Wnt signaling in K19‐Wnt1/C2mE mouse, a genetic mouse model for gastric tumorigenesis. The analysis of three transgenic mouse lines, K19‐Wnt1, K19‐C2mE and K19‐Wnt1/C2mE, revealed that the expansion of CD44⁺ SCJ cells is triggered by PGE₂‐mediated signaling and is prominently enhanced by the addition of Wnt activation. Furthermore, each expanded CD44⁺ gland in gastric tumor of K19‐Wnt1/C2mE mouse contains a few BrdU label‐retaining quiescent or slow‐cycling cells, suggesting that the CD44⁺ SCJ cells in normal mouse are candidates for the cell‐of‐origin of gastric CSCs. These observations suggest that PGE₂‐mediated inflammatory signaling and Wnt signaling cooperatively trigger the expansion of CD44⁺ slow‐cycling stem‐like cells in SCJ, leading to development of lethal gastric tumors in mice.     

Characterization of a subpopulation of colon cancer cells with stem cell‐like properties

The biology of the normal colonic mucosa suggests that colon cancer originates from normal colon stem cells. CD44 cancer stem cells have been identified in breast and prostate cancer, and we therefore examined whether CD44 similarly identified colon cancer stem cells. Initial assays found CD44^hi colon tumor cells to have enhanced soft agar colony‐forming ability. Subsequently, CD44^hi cells isolated from 4 primary colon adenocarcinoma xenografts were found to be highly tumorigenic in immune deficient mice. CD44^hi cells consistently formed tumors with 1,000 cells, and in multiple experiments, as few as 10 and 100 CD44^hi cells formed tumors in 7/10 and 21/28 mice, respectively. In contrast, CD44^? colon tumor cells were either nontumorigenic or 10–50‐fold less tumorigenic. CD44^hi cells could be serially passaged up to 4 times in vivo, suggesting self‐renewal capacity, and formed tumors that recapitulated the heterogeneity of the original patient tumor. CD44^hi cells were significantly enriched for nuclear activated β‐catenin, a key element in normal stem/progenitor cells and in early colon tumor progression. Bromodeoxyuridine (BrdU) labeling studies indicated that CD44^hi cells divide slowly relative to the CD44^? cells, suggesting their tumorigenicity is not simply due to faster proliferation. Aldehyde dehydrogenase (ALDH) sort further increased the tumorigenicity of CD44^hi cells from 2/2 patient tumors, but CD133 tumor cells in our hands did not have increased tumorigenicity. Our observations indicate that CD44 is a marker of stem‐like cells in colon cancer, and support the use of additional markers to further purify colon cancer stem cells. © 2008 Wiley‐Liss, Inc.     

GPER-induced signaling is essential for the survival of breast cancer stem cells

G protein-coupled estrogen receptor-1 (GPER), a member of the G protein-coupled receptor (GPCR) superfamily, mediates estrogen-induced proliferation of normal and malignant breast epithelial cells. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here we showed greater expression of GPER in BCSCs than non-BCSCs of three patient-derived xenografts of ER⁻/PR⁺ breast cancers. GPER silencing reduced stemness features of BCSCs as reflected by reduced mammosphere forming capacity in vitro, and tumor growth in vivo with decreased BCSC populations. Comparative phosphoproteomics revealed greater GPER-mediated PKA/BAD signaling in BCSCs. Activation of GPER by its ligands, including tamoxifen (TMX), induced phosphorylation of PKA and BAD-Ser118 to sustain BCSC characteristics. Transfection with a dominant-negative mutant BAD (Ser118Ala) led to reduced cell survival. Taken together, GPER and its downstream signaling play a key role in maintaining the stemness of BCSCs, suggesting that GPER is a potential therapeutic target for eradicating BCSCs.     

miR-155 increases stemness and decitabine resistance in triple-negative breast cancer cells by inhibiting TSPAN5

Effective therapeutic targets for triple-negative breast cancer (TNBC), a special type of breast cancer (BC) with rapid metastasis and poor prognosis, are lacking, especially for patients with chemotherapy resistance. Decitabine (DCA) is a Food and Drug Administration-approved DNA methyltransferase inhibitor that has been proven effective for the treatment of tumors. However, its antitumor effect in cancer cells is limited by multidrug resistance. Cancer stem cells (CSCs), which are thought to act as seeds during tumor formation, regulate tumorigenesis, metastasis, and drug resistance through complex signaling. Our previous study found that miR-155 is upregulated in BC, but whether and how miR-155 regulates DCA resistance is unclear. In this study, we demonstrated that miR-155 was upregulated in CD24⁻CD44⁺ BC stem cells (BCSCs). In addition, the overexpression of miR-155 increased the number of CD24⁻CD44⁺ CSCs, DCA resistance and tumor clone formation in MDA-231 and BT-549 BC cells, and knockdown of miR-155 inhibited DCA resistance and stemness in BCSCs in vitro. Moreover, miR-155 induced stemness and DCA resistance by inhibiting the direct target gene tetraspanin-5 (TSPAN5). We further confirmed that overexpression of TSPAN5 abrogated the effect of miR-155 in promoting stemness and DCA resistance in BC cells. Our data show that miR-155 increases stemness and DCA resistance in BC cells by targeting TSPAN5. These data provide a therapeutic strategy and mechanistic basis for future possible clinical applications targeting the miR-155/TSPAN5 signaling axis in the treatment of TNBC.     

Glucose metabolism‐targeted therapy and withaferin A are effective for epidermal growth factor receptor tyrosine kinase inhibitor‐induced drug‐tolerant persisters

In pathway‐targeted cancer drug therapies, the relatively rapid emergence of drug‐tolerant persisters (DTPs) substantially limits the overall therapeutic benefit. However, little is known about the roles of DTPs in drug resistance. In this study, we investigated the features of epidermal growth factor receptor–tyrosine kinase inhibitor‐induced DTPs and explored a new treatment strategy to overcome the emergence of these DTPs. We used two EGFR‐mutated lung adenocarcinoma cell lines, PC9 and II‐18. They were treated with 2 μM gefitinib for 6, 12, or 24 days or 6 months. We analyzed the mRNA expression of the stem cell‐related markers by quantitative RT‐PCR and the expression of the cellular senescence‐associated proteins. Then we sorted DTPs according to the expression pattern of CD133 and analyzed the features of sorted cells. Finally, we tried to ablate DTPs by glucose metabolism targeting therapies and a stem‐like cell targeting drug, withaferin A. Drug‐tolerant persisters were composed of at least two types of cells, one with the properties of cancer stem‐like cells (CSCs) and the other with the properties of therapy‐induced senescent (TIS) cells. The CD133^high cell population had CSC properties and the CD133^low cell population had TIS properties. The CD133^low cell population containing TIS cells showed a senescence‐associated secretory phenotype that supported the emergence of the CD133^high cell population containing CSCs. Glucose metabolism inhibitors effectively eliminated the CD133^low cell population. Withaferin A effectively eliminated the CD133^high cell population. The combination of phloretin and withaferin A effectively suppressed gefitinib‐resistant tumor growth.     

CD44 variant 6 is correlated with peritoneal dissemination and poor prognosis in patients with advanced epithelial ovarian cancer

Cancer stem cells (CSCs) drive tumor initiation and metastasis in several types of human cancer. However, the contribution of ovarian CSCs to peritoneal metastasis remains unresolved. The cell adhesion molecule CD44 has been identified as a major marker for CSCs in solid tumors, including epithelial ovarian cancer. CD44 exists as a standard form (CD44s) and also as numerous variant isoforms (CD44v) generated by alternative mRNA splicing. Here we show that disseminated ovarian tumors in the pelvic peritoneum contain highly enriched CD44v6‐positive cancer cells, which drive tumor metastasis and are responsible for tumor resistance to chemotherapy. Clinically, an increased number of CD44v6‐positive cancer cells in primary tumors was associated with a shortened overall survival in stage III–IV ovarian cancer patients. Furthermore, a subpopulation of CD44v6‐positive cancer cells manifested the ability to initiate tumor metastasis in the pelvic peritoneum in an in vivo mouse model, suggesting that CD44v6‐positive cells show the potential to serve as metastasis‐initiating cells. Thus, the peritoneal disseminated metastasis of epithelial ovarian cancer is initiated by the CD44v6‐positive subpopulation, and CD44v6 expression is a biomarker for the clinical outcome of advanced ovarian cancer patients. Given that a distinct subpopulation of CD44v6‐positive cancer cells plays a critical role in peritoneal metastasis, definitive treatment should target this subpopulation of CD44v6‐positive cells in epithelial ovarian cancer.     

Combination therapy targeting both cancer stem-like cells and bulk tumor cells for improved efficacy of breast cancer treatment

Many types of tumors are organized in a hierarchy of heterogeneous cell populations. The cancer stem-like cells (CSCs) hypothesis suggests that tumor development and metastasis are driven by a minority population of cells, which are responsible for tumor initiation, growth and recurrences. The inability to efficiently eliminate CSCs during chemotherapy, together with CSCs being highly tumorigenic and invasive, may result in treatment failure due to cancer relapse and metastases. CSCs are emerging as a promising target for the development of translational cancer therapies. Ideal panacea for cancer would kill all malignant cells, including CSCs and bulk tumor cells. Since both chemotherapy and CSCs-specific therapy are insufficient to cure cancer, we propose combination therapy with CSCs-targeted agents and chemotherapeutics for improved breast cancer treatment. We generated in vitro mammosphere of 2 breast cancer cell lines, and demonstrated ability of mammospheres to grow and enrich cancer cells with stem-like properties, including self-renewal, multilineage differentiation and enrichment of cells expressing breast cancer stem-like cell biomarkers CD44⁺/CD24^?/low. The formation of mammospheres was significantly inhibited by salinomycin, validating its pharmacological role against the cancer stem-like cells. In contrast, paclitaxel showed a minimal effect on the proliferation and growth of breast cancer stem-like cells. While combination therapies of salinomycin with conventional chemotherapy (paclitaxel or lipodox) showed a potential to improve tumor cell killing, different subtypes of breast cancer cells showed different patterns in response to the combination therapies. While optimization of combination therapy is warranted, the design of combination therapy should consider phenotypic attributes of breast cancer types.     

Identification of NY‐BR‐1‐specific CD4+ T cell epitopes using HLA‐transgenic mice

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer‐related deaths among women. The differentiation antigen NY‐BR‐1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen‐specific CD4⁺ effector T cells, NY‐BR‐1 was screened for the presence of HLA‐restricted CD4⁺ T cell epitopes that could be included in immunological treatment approaches. Upon NY‐BR‐1‐specific DNA immunization of HLA‐transgenic mice and functional ex vivo analysis, a panel of NY‐BR‐1‐derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY‐BR‐1‐specific, HLA‐DRB1*0301– or HLA‐DRB1*0401‐restricted CD4⁺ T cell lines from splenocytes of peptide immunized HLA‐transgenic mice. Notably, all four CD4⁺ T cell lines recognized human HLA‐DR‐matched dendritic cells (DC) pulsed with lysates of NY‐BR‐1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4⁺ T cells specific for all four CD4⁺ T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4⁺ T cell responses against the new epitopes are not deleted nor inactivated by self‐tolerance mechanisms. Our results present the first NY‐BR‐1‐specific HLA‐DRB1*0301– and HLA‐DRB1*0401‐restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer.     

PGE2/EP4 antagonism enhances tumor chemosensitivity by inducing extracellular vesicle‐mediated clearance of cancer stem cells

Cells expressing mesenchymal/basal phenotypes in tumors have been associated with stem cell properties. Cancer stem cells (CSCs) are often resistant to conventional chemotherapy. We explored overcoming mesenchymal CSC resistance to chemotherapeutic agents. Our goal was to reduce CSC numbers in vivo, in conjunction with chemotherapy, to reduce tumor burden. Analysis of clinical samples demonstrated that COX‐2/PGE₂/EP₄ signaling is elevated in basal‐like and chemoresistant breast carcinoma and is correlated with survival and relapse of breast cancer. EP₄ antagonism elicts a striking shift of breast cancer cells from a mesenchymal/CSC state to a more epithelial non‐CSC state. The transition was mediated by EP₄ antagonist‐induced extracellular vesicles [(EVs)/exosomes] which removed CSC markers, mesenchymal markers, integrins, and drug efflux transporters from the CSCs. In addition, EP₄ antagonism‐induced CSC EVs/exosomes can convert tumor epithelial/non‐CSCs to mesenchymal/CSCs able to give rise to tumors and to promote tumor cell dissemination. Because of its ability to induce a CSC‐to‐non‐CSC transition, EP₄ antagonist treatment in vivo reduced the numbers of CSCs within tumors and increased tumor chemosensitivity. EP₄ antagonist treatment enhances tumor response to chemotherapy by reducing the numbers of chemotherapy‐resistant CSCs available to repopulate the tumor. EP₄ antagonism can collaborate with conventional chemotherapy to reduce tumor burden.