Inhibition of breast cancer growth via miR-7 suppressing ALDH1A3 activity concomitant with decreasing breast cancer stem cell subpopulation

Breast cancer patients with high expression of aldehyde dehydrogenases (ALDHs) cell population have higher tolerability to chemotherapy since the cells posses a characteristic of breast cancer stem cells (BCSCs) that are resistant to conventional chemotherapy. In this study, we found that the ALDH-positive cells were higher in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in both BT549 and MDA-MB-231 cell lines but microRNA-7 (miR-7) level was lower in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in MDA-MB-231 cells. Moreover, miR-7 overexpression in MDA-MB-231 cells decreased ALDH1A3 activity by miR-7 directly binding to the 3′-untranslated region of ALDH1A3; while the ALDH1A3 expression was downregulated in MDA-MB-231 cells, the expressions of CD44 and Epithelium Specific Antigen (ESA) were reduced along with decreasing the BCSC subpopulation. Significantly, enforced expression of miR-7 in CD44⁺CD24⁻ESA⁺BCSC markedly inhibited the BCSC-driven xenograft growth in mice by decreasing an expression of ALDH1A3. Collectively, the findings demonstrate the miR-7 inhibits breast cancer growth via suppressing ALDH1A3 activity concomitant with decreasing BCSC subpopulation. This approach may be considered for an investigation on clinical treatment of breast cancers.     

Comparative stemness and differentiation of luminal and basal breast cancer stem cell type under glutamine‐deprivation

Glutamine (gln) metabolism has emerged as a cancer therapeutic target in past few years, however, the effect of gln-deprivation of bCSCs remains elusive in breast cancer. In this study, effect of glutamine on stemness and differentiation potential of bCSCs isolated from MCF-7 and MDAMB-231 were studied. We have shown that bCSCs differentiate into CD24⁺ epithelial population under gln-deprivation and demonstrated increased expression of epithelial markers such as e-cadherin, claudin-1 and decreased expression of mesenchymal protein n-cadherin. MCF-7-bCSCs showed a decrease in EpCAM^high population whereas MDAMB-231-bCSCs increased CD44^high population in response to gln-deprivation. The expression of intracellular stem cell markers such sox-2, oct-4 and nanog showed a drastic decrease in gene expression under gln-deprived MDAMB-231-bCSCs. Finally, localization of β-catenin in MCF-7 and MDAMB-231 cells showed its accumulation in cytosol or perinuclear space reducing its efficiency to transcribe downstream genes. Conclusively, our study demonstrated that gln-deprivation induces differentiation of bCSCs into epithelial subtypes and also reduces stemness of bCSCs mediated by reduced nuclear localization of β-catenin. It also suggests that basal and luminal bCSCs respond differentially towards changes in extracellular and intracellular gln. This study could significantly affect the gln targeting regimen of breast cancer therapeutics.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-020-00603-1.     

Autophagy regulates cisplatin‐induced stemness and chemoresistance via the upregulation of CD44, ABCB1 and ADAM17 in oral squamous cell carcinoma

Objective

We inspected the relevance of CD44, ABCB1 and ADAM17 in OSCC stemness and deciphered the role of autophagy/mitophagy in regulating stemness and chemoresistance.

Material and methods

A retrospective analysis of CD44, ABCB1 and ADAM17 with respect to the various clinico‐pathological factors and their correlation was analysed in sixty OSCC samples. Furthermore, the stemness and chemoresistance were studied in resistant oral cancer cells using sphere formation assay, flow cytometry and florescence microscopy. The role of autophagy/mitophagy was investigated by transient transfection of siATG14, GFP‐LC3, tF‐LC3, mKeima‐Red‐Mito7 and Western blot analysis of autophagic and mitochondrial proteins.

Results

In OSCC, high CD44, ABCB1 and ADAM17 expressions were correlated with higher tumour grades and poor differentiation and show significant correlation in their co‐expression. In vitro and OSCC tissue double labelling confirmed that CD44⁺ cells co‐expresses ABCB1 and ADAM17. Further, cisplatin (CDDP)‐resistant FaDu cells displayed stem‐like features and higher CD44, ABCB1 and ADAM17 expression. Higher autophagic flux and mitophagy were observed in resistant FaDu cells as compared to parental cells, and inhibition of autophagy led to the decrease in stemness, restoration of mitochondrial proteins and reduced expression of CD44, ABCB1 and ADAM17.

Conclusion

The CD44⁺/ABCB1⁺/ADAM17⁺ expression in OSCC is associated with stemness and chemoresistance. Further, this study highlights the involvement of mitophagy in chemoresistance and autophagic regulation of stemness in OSCC.     

Wnt/β-catenin Signaling Inhibitors suppress the Tumor-initiating properties of a CD44+CD133+ subpopulation of Caco-2 cells

Tumor-initiating cells or cancer stem cells are a subset of cancer cells that have tumorigenic potential in human cancer. Although several markers have been proposed to distinguish tumor-initiating cells from colorectal cancer cells, little is known about how this subpopulation contributes to tumorigenesis. Here, we characterized a tumor-initiating cell subpopulation from Caco-2 colorectal cancer cells. Based on the findings that Caco-2 cell subpopulations express different cell surface markers, we were able to discriminate three main fractions, CD44^-CD133^-, CD44^-CD133⁺, and CD44⁺CD133⁺ subsets, and characterized their biochemical and tumorigenic properties. Our results show that CD44⁺CD133⁺ cells possessed an unusual capacity to proliferate and could form tumors when transplanted into NSG mice. Additionally, primary tumors grown from CD44⁺CD133⁺ Caco-2 cells contained mixed populations of CD44⁺CD133⁺ and non-CD44⁺CD133⁺ Caco-2 cells, indicating that the full phenotypic heterogeneity of the parental Caco-2 cells was re-created. Notably, only the CD44⁺CD133⁺ subset of Caco-2-derived primary tumors had tumorigenic potential in NSG mice, and the tumor growth of CD44⁺CD133⁺ cells was faster in secondary xenografts than in primary transplants. Gene expression analysis revealed that the Wnt/β-catenin pathway was over-activated in CD44⁺CD133⁺ cells, and the growth and tumorigenic potential of this subpopulation were significantly suppressed by small-molecule Wnt/β-catenin signaling inhibitors. Our findings suggest that the CD44⁺CD133⁺ subpopulation from Caco-2 cells was highly enriched in tumorigenic cells and will be useful for investigating the mechanisms leading to human colorectal cancer development.     

Effect of Melatonin in Epithelial Mesenchymal Transition Markers and Invasive Properties of Breast Cancer Stem Cells of Canine and Human Cell Lines

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44⁺/CD24^low/- marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44⁺/CD24^low/- positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.     

DLG5 suppresses breast cancer stem cell‐like characteristics to restore tamoxifen sensitivity by inhibiting TAZ expression

Tamoxifen (TAM) is a primary drug for treatment of estrogen receptor positive breast cancer. However, TAM resistance remains a serious threat to breast cancer patients and may be attributed to increased stemness of breast cancer. Here, we show that discs large homolog 5 (DLG5) expression is down‐regulated in TAM‐resistant breast cancer and cells. DLG5 silencing decreased the sensitivity to TAM and increased the frequency and stemness of CD44⁺/CD24^? breast cancer stem cells (BCSCs) and TAZ, a transducer of the Hippo pathway, expression in MCF7 cells while DLG5 overexpression had opposite effects. TAZ silencing restored the sensitivity to TAM and reduced the frequency and stemness in TAM‐resistant breast cancer cells. Taken together, our data indicate that down‐regulated DLG5 expression increases the stemness of breast cancer cells by enhancing TAZ expression, contributing to TAM resistance in breast cancer.     

Krüppel‐like factor 4 regulates stemness and mesenchymal properties of colorectal cancer stem cells through the TGF‐β1/Smad/snail pathway

Krüppel‐like factor 4 (KLF4) was closely associated with epithelial‐mesenchymal transition and stemness in colorectal cancer stem cells (CSCs)‐enriched spheroid cells. Nonetheless, the underlying molecular mechanism is unclear. This study showed that KLF4 overexpression was accompanied with stemness and mesenchymal features in Lgr5⁺CD44⁺EpCAM⁺ colorectal CSCs. KLF4 knockdown suppressed stemness, mesenchymal features and activation of the TGF‐β1 pathway, whereas enforced KLF4 overexpression activated TGF‐β1, phosphorylation of Smad 2/3 and Snail expression, and restored stemness and mesenchymal phenotypes. Furthermore, TGF‐β1 pathway inhibition invalidated KLF4‐facilitated stemness and mesenchymal features without affecting KLF4 expression. The data from the current study are the first to demonstrate that KLF4 maintains stemness and mesenchymal properties through the TGF‐β1/Smad/Snail pathway in Lgr5⁺CD44⁺EpCAM⁺ colorectal CSCs.     

miR‐200c suppresses stemness and increases cellular sensitivity to trastuzumab in HER2+ breast cancer

Resistance to trastuzumab remains a major obstacle in HER2‐overexpressing breast cancer treatment. miR‐200c is important for many functions in cancer stem cells (CSCs), including tumour recurrence, metastasis and resistance. We hypothesized that miR‐200c contributes to trastuzumab resistance and stemness maintenance in HER2‐overexpressing breast cancer. In this study, we used HER2‐positive SKBR3, HER2‐negative MCF‐7, and their CD44⁺CD24^? phenotype mammospheres SKBR3‐S and MCF‐7‐S to verify. Our results demonstrated that miR‐200c was weakly expressed in breast cancer cell lines and cell line stem cells. Overexpression of miR‐200c resulted in a significant reduction in the number of tumour spheres formed and the population of CD44⁺CD24^? phenotype mammospheres in SKBR3‐S. Combining miR‐200c with trastuzumab can significantly reduce proliferation and increase apoptosis of SKBR3 and SKBR3‐S. Overexpression of miR‐200c also eliminated its downstream target genes. These genes were highly expressed and positively related in breast cancer patients. Overexpression of miR‐200c also improved the malignant progression of SKBR3‐S and SKBR3 in vivo. miR‐200c plays an important role in the maintenance of the CSC‐like phenotype and increases drug sensitivity to trastuzumab in HER2+ cells and stem cells.     

Curcumin Improves the Tumoricidal Effect of Mitomycin C by Suppressing ABCG2 Expression in Stem Cell-Like Breast Cancer Cells

Cancer cells with stem cell–like properties contribute to the development of resistance to chemotherapy and eventually to tumor relapses. The current study investigated the potential of curcumin to reduce breast cancer stem cell (BCSC) population for sensitizing breast cancer cells to mitomycin C (MMC) both in vitro and in vivo. Curcumin improved the sensitivity of paclitaxel, cisplatin, and doxorubicin in breast cancer cell lines MCF-7 and MDA-MB-231, as shown by the more than 2-fold decrease in the half-maximal inhibitory concentration of these chemotherapeutic agents. In addition, curcumin sensitized the BCSCs of MCF-7 and MDA-MB-231 to MMC by 5- and 15-fold, respectively. The BCSCs could not grow to the fifth generation in the presence of curcumin and MMC. MMC or curcumin alone only marginally reduced the BCSC population in the mammospheres; however, together, they reduced the BCSC population in CD44⁺CD24^−/low cells by more than 75% (29.34% to 6.86%). Curcumin sensitized BCSCs through a reduction in the expression of ATP-binding cassette (ABC) transporters ABCG2 and ABCC1. We demonstrated that fumitremorgin C, a selective ABCG2 inhibitor, reduced BCSC survival to a similar degree as curcumin did. Curcumin sensitized breast cancer cells to chemotherapeutic drugs by reducing the BCSC population mainly through a reduction in the expression of ABCG2.     

Adipose-derived stem cells and cancer cells fuse to generate cancer stem cell-like cells with increased tumorigenicity

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells isolated from adipose tissue and have the ability to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Despite their great therapeutic potentials, previous studies showed that ADSCs could enhance the proliferation and metastatic potential of breast cancer cells (BCCs). In this study, we found that ADSCs fused with BCCs spontaneously, while breast cancer stem cell (CSC) markers CD44⁺CD24^-/lowEpCAM⁺ were enriched in this fusion population. We further assessed the fusion hybrid by multicolor DNA FISH and mouse xenograft assays. Only single nucleus was observed in the fusion hybrid, confirming that it was a synkaryon. In vivo mouse xenograft assay indicated that the tumorigenic potential of the fusion hybrid was significantly higher than that of the parent tumorigenic triple-negative BCC line MDA-MB-231. We had compared the fusion efficiency between two BCC lines, the CD44-rich MDA-MB-231 and the CD44-poor MCF-7, with ADSCs. Interestingly, we found that the fusion efficiency was much higher between MDA-MB-231 and ADSCs, suggesting that a potential mechanism of cell fusion may lie in the dissimilarity between these two cell lines. The cell fusion efficiency was hampered by knocking down the CD44. Altogether, our findings suggest that CD44-mediated cell fusion could be a potential mechanism for generating CSCs.     

A preliminary study of the role of extracellular -5′- nucleotidase in breast cancer stem cells and epithelial-mesenchymal transition

Tumor stem cell theory may well explain a variety of malignant behaviors of tumors. Cells undergoing epithelial-mesenchymal transition (EMT) share many characteristics with tumor stem cells. Our previous studies showed that extracellular -5'- nucleotidase (CD73), one of the important surface markers of mesenchymal stem cells, may promote growth and metastasis of breast cancer cells both in vivo and in vitro. In this study, we assessed breast cancer stem cell (BCSC) markers [acetaldehyde dehydrogenase (ALDH)⁺ and CD44⁺CD24^?] in various breast cancer cell lines with flow cytometry after overexpression (by lentivirus infection) or suppression (by siRNA interference) of CD73. We measured CD73 expression in breast cancer mammospheres with real-time PCR and western blots. Finally, we examined the expression of CD73 and EMT markers in different breast cancer cell lines, as well as in mammary cells (MCF10A) that underwent EMT induced by transforming growth factor beta (TGF-β). We found that CD73 positively correlated with ALDH⁺ or CD44⁺CD24^? subsets of breast cancer cells. CD73 was expressed more in breast cancer mammospheres than in adherent cells. CD73 and mesenchymal marker expression was higher in breast cancer cells with more malignant features, while CD73 was lower in low malignant breast cancer cells with higher epithelial markers. Furthermore, CD73 expression increased during the process of TGF-β-induced EMT. Our results indicate that CD73 may play an important role in BCSCs.     

CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549

Cancer stem cells (CSCs) are subpopulations of tumor cells that are responsible for tumor initiation, maintenance and metastasis. Recent studies suggested that lung cancer arises from CSCs. In this study, the expression of potential CSC markers in cell line A549 was evaluated. We applied flow cytometry to assess the expression of putative stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), CD24, CD44, CD133 and ABCG2. Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) Aria II and characterized using their clonogenic and sphere-forming capacity. A549 cells expressed the CSC markers CD44 and CD24 at 68.16% and 54.46%, respectively. The expression of the putative CSC marker ALDH1 was 4.20%, whereas the expression of ABCG2 and CD133 was 0.93%. Double-positive CD44/133 populations were rare. CD44⁺/24⁺ and CD44⁺/CD24^?/low subpopulations respectively exhibited 64% and 27.92% expression. The colony-forming potentials in the CD44⁺/CD24⁺ and CD44⁺/CD24^?/low subpopulations were 84.37 ± 2.86% and 90 ± 3.06%, respectively, while the parental A549 cells yielded 56.65 ± 2.33% using the colony-formation assay. Both isolated subpopulations formed spheres in serumfree medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). CD44 and CD24 cannot be considered potential markers for isolating lung CSCs in cell line A549, but further investigation using in vivo assays is required.     

Endothelial Cells Provide a Notch-Dependent Pro-Tumoral Niche for Enhancing Breast Cancer Survival,Stemness and Pro-Metastatic Properties

Treating metastasis has been challenging due to tumors complexity and heterogeneity. This complexity is partly related to the crosstalk between tumor and its microenvironment. Endothelial cells -the building blocks of tumor vasculature- have been shown to have additional roles in cancer progression than angiogenesis and supplying oxygen and nutrients. Here, we show an alternative role for endothelial cells in supporting breast cancer growth and spreading independent of their vascular functions. Using endothelial cells and breast cancer cell lines MDA-MB231 and MCF-7, we developed co-culture systems to study the influence of tumor endothelium on breast tumor development by both in vitro and in vivo approaches. Our results demonstrated that endothelial cells conferred survival advantage to tumor cells under complete starvation and enriched the CD44^HighCD24^Low/- stem cell population in tumor cells. Moreover, endothelial cells enhanced the pro-metastatic potential of breast cancer cells. The in vitro and in vivo results concordantly confirmed a role for endothelial Jagged1 to promote breast tumor through notch activation. Here, we propose a role for endothelial cells in enhancing breast cancer progression, stemness, and pro-metastatic traits through a perfusion-independent manner. Our findings may be beneficial in developing novel therapeutic approaches.     

Melatonin regulates cancer migration and stemness and enhances the anti-tumour effect of cisplatin

Melatonin, a lipophilic hormone released from the pineal gland, has oncostatic effects on various types of cancers. However, its cancer treatment potential needs to be improved by deciphering its corresponding mechanisms of action and optimising therapeutic strategy. In the present study, melatonin inhibited gastric cancer cell migration and soft agar colony formation. Magnetic-activated cell sorting was applied to isolate CD133⁺ cancer stem cells. Gene expression analysis showed that melatonin lowered the upregulation of LC3-II expression in CD133⁺ cells compared to CD133⁻ cells. Several long non-coding RNAs and many components in the canonical Wnt signalling pathway were altered in melatonin-treated cells. In addition, knockdown of long non-coding RNA H19 enhanced the expression of pro-apoptotic genes, Bax and Bak, induced by melatonin treatment. Combinatorial treatment with melatonin and cisplatin was investigated to improve the applicability of melatonin as an anticancer therapy. Combinatorial treatment increased the apoptosis rate and induced G0/G1 cell cycle arrest. Melatonin can regulate migration and stemness in gastric cancer cells by modifying many signalling pathways. Combinatorial treatment with melatonin and cisplatin has the potential to improve the therapeutic efficacy of both.     

Requirement of monocytes and T-helper cells during development of tumor cell cytotoxicity in targeted T cells

In cocultures of human plancental alkaline phosphatase(PLAP)-positive MO₄ tumor cells and human peripheral blood mononuclear cells (PBMC), also containing a heteroconjugate (7E8-OKT3) synthesized between the anti-PLAP monoclonal antibody 7E8 and the anti-CD3 antibody OKT3, and supplemented with low levels of recombinant interleukin-2 (rIL-2), T cells are progressively activated, resulting in tumor cell lysis. To unravel the contribution of PBMC subsets to the generation of this targetable cytotoxicity, PBMC subsets were studied after their isolation by cell sorting, either from fresh PBMC or from PBMC peractivated with OKTe3 and rIL-2. Whereas no targetable cytotoxicity was found in Fc-receptor-bearing CD3-cells, tumor cells were lysed by CD3⁺ T cells (mostly CD8⁺) isolated from pre-activated PBMC. When isolated from fresh PBMC, neither the CD8⁺ T cell subset, nor the total CD3⁺ T cell population developed significant targetable cytotoxicity, even in the presence of rIL-2. Thus, additional cell types are essential for the CD8⁺ T cell activation. Indeed. CD4⁺ T cells isolated from pre-activated but not from fresh PBMC were capable of eliciting cytotoxicity in fresh CD8⁺ T cells. The non-targeted monocytes were found to be the activators of the CD4⁺ T cells. In summary, targeting T cells to the surface of a tumor cell is not sufficientper se to achieve activation and lysis. The progressive tumor cell lysis by targeted T cells seems to be initiated by non-targeted monocytes activating CD4⁺ T cells, these cells in turn promoting CD8⁺ T cell activation, necessary for the development of cytotoxicity.