Markers of tumor-initiating cells predict chemoresistance in breast cancer

Purpose

Evidence is lacking whether the number of breast tumor-initiating cells (BT-ICs) directly correlates with the sensitivity of breast tumors to chemotherapy. Here, we evaluated the association between proportion of BT-ICs and chemoresistance of the tumors.

Methods

Immunohistochemical staining(IHC) was used to examine the expression of aldehyde dehydrogenase 1 (ALDH1) and proliferating cell nuclear antigen, and TUNEL was used to detect the apoptosis index. The significance of various variables in patient survival was analyzed using a Cox proportional hazards model. The percentage of BT-ICs in breast cancer cell lines and primary breast tumors was determined by ALDH1 enzymatic assay, CD44⁺/CD24⁻ phenotype and mammosphere formation assay.

Results

ALDH1 expression determined by IHC in primary breast cancers was associated with poor clinical response to neoadjuvant chemotherapy and reduced survival in breast cancer patients. Breast tumors that contained higher proportion of BT-ICs with CD44⁺/CD24⁻ phenotype, ALDH1 enzymatic activity and sphere forming capacity were more resistant to neoadjuvant chemotherapy. Chemoresistant cell lines AdrR/MCF-7 and SK-3rd, had increased number of cells with sphere forming capacity, CD44⁺/CD24⁻ phenotype and side-population. Regardless the proportion of T-ICs, FACS-sorted CD44⁺/CD24⁻ cells that derived from primary tumors or breast cancer lines were about 10–60 fold more resistant to chemotherapy relative to the non- CD44⁺/CD24⁻ cells and their parental cells. Furthermore, our data demonstrated that MDR1 (multidrug resistance 1) and ABCG2 (ATP-binding cassette sub-family G member 2) were upregulated in CD44⁺/CD24⁻ cells. Treatment with lapatinib or salinomycin reduced the proportion of BT-ICs by nearly 50 fold, and thus enhanced the sensitivity of breast cancer cells to chemotherapy by around 30 fold.

Conclusions

These data suggest that the proportion of BT-ICs is associated with chemotherapeutic resistance of breast cancer. It highlights the importance of targeting T-ICs, rather than eliminating the bulk of rapidly dividing and terminally differentiated cells, in novel anti-cancer strategies.     

Autotaxin delays apoptosis induced by carboplatin in ovarian cancer cells

Drug resistance remains a barrier to the effective long term treatment of ovarian cancer. We have established an RNAi-based screen to identify genes which confer resistance to carboplatin or paclitaxel. To validate the screen we showed that siRNA interfering with the apoptosis regulators FLIP and Bcl-X_L conferred sensitivity to paclitaxel and carboplatin respectively. The expression of 90 genes which have previously been shown to be over-expressed in drug-resistant ovarian cancer was inhibited using siRNA and the impact on sensitivity to carboplatin and paclitaxel was assessed. ENPP2 was identified as a candidate gene causing drug resistance. ENPP2 encodes autotaxin, a phospholipase involved in the synthesis of the survival factor lysophosphatidic acid. siRNA directed to ENPP2 resulted in earlier apoptosis following treatment with carboplatin. 2-carbacyclic phosphatidic acid (ccPA 16:1), a small molecule inhibitor of autotaxin, also accelerated apoptosis induced by carboplatin. Stable ectopic expression of autotaxin in OVCAR-3 cells led to a delay in apoptosis. When serum was withdrawn to remove exogenous LPA, ccPA caused a pronounced potentiation of apoptosis induced by carboplatin in cells expressing autotaxin. These results indicate that autotaxin delays apoptosis induced by carboplatin in ovarian cancer cells.     

Resveratrol chemosensitizes breast cancer cells to melphalan by cell cycle arrest

Melphalan (MEL) is a chemotherapeutic agent used in breast cancer therapy; however, MEL's side effects limit its clinical applications. In the last 20 years, resveratrol (RSV), a polyphenol found in grape skins, has been proposed to reduce the risk of cancer development. The aim of this study was to investigate whether RSV would be able to enhance the antitumor effects of MEL in MCF-7 and MDA-MB-231 cells. RSV potentiated the cytotoxic effects of MEL in human breast cancer cells. This finding was related to the ability of RSV to sensitize MCF-7 cells to MEL-induced apoptosis. The sensitization by RSV involved the enhancement of p53 levels, the decrease of procaspase 8 and the activation of caspases 7 and 9. Another proposed mechanism for the chemosensitization effect of MCF-7 cells to MEL by RSV was the cell cycle arrest in the S phase. The treatment with RSV or MEL increased the levels of p-Chk2. The increase became pronounced in the combined treatments of the compounds. The expression of cyclin A was decreased by treatment with RSV and by the combination of RSV with MEL. While the levels of cyclin dependent kinase 2 (CDK2) remained unchanged by treatments, its active form (Thr(160) -phosphorylated CDK2) was decreased by treatment with RSV and by the combination of RSV with MEL. The activity of CDK7, kinase that phosphorylates CDK2 at Thr(160), was inhibited by RSV and by the combination of RSV with MEL. These results indicate that RSV could be used as an adjuvant agent during breast cancer therapy with MEL.     

Genistein-induced mitotic arrest of gastric cancer cells by downregulating KIF20A, a proteomics study

Genistein exerts its anticarcinogenic effects by inducing G2/M arrest and apoptosis of cancer cells. However, the precise molecular mechanism of action of genistein has not been completely elucidated. In this study, we used quantitative proteomics to identify the genistein-induced protein alterations in gastric cancer cells and investigate the molecular mechanism responsible for the anti-cancer actions of genistein. Total 86 proteins were identified to be regulated by genistein, most of which were clustered into the regulation of cell division and G2/M transition, consistent with the anti-cancer effect of genistein. Many proteins including kinesin family proteins, TPX2, CDCA8, and CIT were identified for the first time to be regulated by genistein. Interestingly, five kinesin family proteins including KIF11, KIF20A, KIF22, KIF23, and CENPF were found to be simultaneously downregulated by genistein. Significantly decreased KIF20A was selected for further functional studies. The silencing of KIF20A inhibited cell viability and induced G2/M arrest, similar to the effects of genistein treatment in gastric cancer. And the silencing of KIF20A also increased cancer cell sensitivity to genistein inhibition, whereas overexpression of KIF20A markedly attenuated genistein-induced cell viability inhibition and G2/M arrest. These observations suggested that KIF20A played an important role in anti-cancer actions of genistein, and thus may be a potential molecular target for drug intervention of gastric cancer.     

Checkpoint kinase 1 regulates diallyl trisulfide-induced mitotic arrest in human prostate cancer cells

We have shown previously that diallyl trisulfide (DATS), a constituent of processed garlic, inhibits proliferation of PC-3 and DU145 human prostate cancer cells by causing G(2)-M phase cell cycle arrest in association with inhibition of cyclin-dependent kinase 1 activity and hyperphosphorylation of Cdc25C at Ser(216). Here, we report that DATS-treated PC-3 and DU145 cells are also arrested in mitosis as judged by microscopy following staining with anti-alpha-tubulin antibody and 4',6-diamidino-2-phenylindole and flow cytometric analysis of Ser(10) phosphorylation of histone H3. The DATS treatment caused activation of checkpoint kinase 1 and checkpoint kinase 2, which are intermediaries of DNA damage checkpoints and implicated in Ser(216) phosphorylation of Cdc25C. The diallyl trisulfide-induced Ser(216) phosphorylation of Cdc25C as well as mitotic arrest were significantly attenuated by knockdown of check-point kinase 1 protein in both PC-3 and DU145 cells. On the other hand, depletion of checkpoint kinase 2 protein did not have any appreciable effect on G(2) or M phase arrest or Cdc25C phosphorylation caused by diallyl trisulfide. The lack of a role of checkpoint kinase 2 in diallyl trisulfide-induced phosphorylation of Cdc25C or G(2)-M phase cell cycle arrest was confirmed using HCT-15 cells stably transfected with phosphorylation-deficient mutant (T68A mutant) of checkpoint kinase 2. In conclusion, the results of the present study suggest existence of a checkpoint kinase 1-dependent mechanism for diallyl trisulfide-induced mitotic arrest in human prostate cancer cells.     

Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and -sensitive cancer cells

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.     

Psychological stress induces chemoresistance in breast cancer by upregulating mdr1

Psychological distress reduces the efficacy of chemotherapy in breast cancer patients. The mechanism may be related to the altered neuronal or hormonal secretions during stress. Here, we reported that adrenaline, a hormone mediating the biological activities of stress, upregulates mdr1 gene expression in MCF-7 breast cancer cells via alpha(2)-adrenergic receptors in a dose-dependent manner. Mdr1 upregulation can be specifically inhibited by pretreatment with mdr1-siRNA. Consequently, adrenergic stimulation enhances the pump function of P-glycoprotein and confers resistance of MCF-7 cells to paclitaxel. In vivo, restraint stress increases mdr1 gene expression in the MCF-7 cancers that are inoculated subcutaneously into the SCID mice and provokes resistance to doxorubicin in the implanted tumors. The effect can be blocked by injection of yohimbine, an alpha(2)-adrenergic inhibitor, but not by metyrapone, a corticosterone synthesis blocker. Therefore, we conclude that breast cancers may develop resistance against chemotherapeutic drugs under psychological distress by over-expressing mdr1 via adrenergic stimulation.     

Cell cycle arrest and induction of apoptosis by cajanin stilbene acid from Cajanus cajan in breast cancer cells

Background: The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown to kill estrogen receptor α positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and apoptosis-related mechanisms have not been analyzed yet.Material and methods: We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution, mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation assay (apoptosis), Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA microarray hybridization and Ingenuity pathway analysis.Results: CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 µM. The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray profiling revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal replication pathways.Conclusion: CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading to cell cycle arrest and apoptosis.     

Pharicin A,a novel natural ent-kaurene diterpenoid,induces mitotic arrest and mitotic catastrophe of cancer cells by interfering with BubR1 function

In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon xerophilus, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in leukemia and solid tumor-derived cells identified by their morphology, DNA content, and mitotic marker analyses. Pharicin A-induced mitotic arrest is associated with unaligned chromosomes, aberrant BubR1 localization, and deregulated spindle checkpoint activation. Pharicin A directly binds to BubR1 in vitro, which is correlated with premature sister chromatid separation in vivo. Pharicin A also induces mitotic arrest in paclitaxel-resistant Jurkat and U2OS cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing mitotic progression and initiating mitotic catastrophe, which merits further preclinical and clinical investigations for cancer drug development.     

Curcumin-treated cancer cells show mitotic disturbances leading to growth arrest and induction of senescence phenotype

Cellular senescence is recognized as a potent anticancer mechanism that inhibits carcinogenesis. Cancer cells can also undergo senescence upon chemo- or radiotherapy. Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, shows anticancer properties both in vitro and in vivo. Previously, we have shown that treatment with curcumin leads to senescence of human cancer cells. Now we identified the molecular mechanism underlying this phenomenon. We observed a time-dependent accumulation of mitotic cells upon curcumin treatment. The time-lapse analysis proved that those cells progressed through mitosis for a significantly longer period of time. A fraction of cells managed to divide or undergo mitotic slippage and then enter the next phase of the cell cycle. Cells arrested in mitosis had an improperly formed mitotic spindle and were positive for γH2AX, which shows that they acquired DNA damage during prolonged mitosis. Moreover, the DNA damage response pathway was activated upon curcumin treatment and the components of this pathway remained upregulated while cells were undergoing senescence. Inhibition of the DNA damage response decreased the number of senescent cells. Thus, our studies revealed that the induction of cell senescence upon curcumin treatment resulted from aberrant progression through the cell cycle. Moreover, the DNA damage acquired by cancer cells, due to mitotic disturbances, activates an important molecular mechanism that determines the potential anticancer activity of curcumin.     

Pharicin A,a novel natural ent-kaurene diterpenoid,induces mitotic arrest and mitotic catastrophe of cancer cells by interfering with BubR1 function

In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in leukemia and solid tumor-derived cells identified by their morphology, DNA content and mitotic marker analyses. Pharicin A-induced mitotic arrest is associated with unaligned chromosomes, aberrant BubR1 localization and deregulated spindle checkpoint activation. Pharicin A directly binds to BubR1 in vitro, which is correlated with premature sister chromatid separation in vivo. Pharicin A also induces mitotic arrest in paclitaxel-resistant Jurkat and U2OS cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing mitotic progression and initiating mitotic catastrophe, which merits further preclinical and clinical investigations for cancer drug development.Key words: pharicin A, mitotic arrest, leukemia, tumor cells, spindle checkpoint     

Salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells

Recently, salidroside (p-hydroxyphenethyl-β-d-glucoside) has been identified as one of the most potent compounds isolated from plants of the Rhodiola genus used widely in traditional Chinese medicine, but pharmacokinetic data on the compound are unavailable. We were the first to report the cytotoxic effects of salidroside on cancer cell lines derived from different tissues, and we found that human breast cancer MDA-MB-231 cells (estrogen receptor negative) were sensitive to the inhibitory action of low-concentration salidroside. To further investigate the cytotoxic effects of salidroside on breast cancer cells and reveal possible ER-related differences in response to salidroside, we used MDA-MB-231 cells and MCF-7 cells (estrogen receptor-positive) as models to study possible molecular mechanisms; we evaluated the effects of salidroside on cell growth characteristics, such as proliferation, cell cycle duration, and apoptosis, and on the expression of apoptosis-related molecules. Our results demonstrated for the first time that salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells and may be a promising candidate for breast cancer treatment.     

EGFR inhibition by pentacyclic triterpenes exhibit cell cycle and growth arrest in breast cancer cells

Aims

Pentacyclic triterpenes are a group of molecules with promising anticancer potential, although their precise molecular target remains elusive. The current work aims to investigate the antiproliferative and associated mechanisms of triterpenes in breast cancer cells in vitro.

Main methods

Effect of triterpenes on cell cycle distribution, ROS and key regulatory proteins were analyzed in three breast cancer cells in vitro. Growth inhibition, new DNA synthesis, colony formation assays and Western blot analysis were performed to assess the EGFR inhibitory effect of triterpenes. Molecular docking was performed to study the interaction between EGFR and triterpenes.

Key findings

We have demonstrated the ability of dimethyl melaleucate (DMM), a pentacyclic triterpene to exhibit cell cycle arrest at G0/G1 phase by down-regulation of cyclin D1 through PI3K/AKT inhibition. Further, to identify the upstream target of DMM, potential EGFR inhibitory activity of DMM and three structurally related pentacyclic triterpenes, ursolic acid, 18α-glycyrrhetinic acid and carbenoxolone was investigated. Interestingly, pentacyclic triterpenes limit EGF mediated breast cancer proliferation through sustained inhibition of EGFR and its downstream effectors STAT3 and cyclin D1 in breast cancer lines. We also show pentacyclic triterpenes to bind at the ATP binding pocket of tyrosine kinase domain of EGFR leading to the hypothesis that pentacyclic triterpenes could be a novel class of EGFR inhibitors. In conclusion, pentacyclic triterpenes inhibit EGFR activation through binding with tyrosine kinase domain thereby suppressing breast cancer proliferation.

Significance

Pentacyclic triterpenes may serve as a potential platform for development of novel drugs against breast cancer.     

BMP4 enhances anoikis resistance and chemoresistance of breast cancer cells through canonical BMP signaling

Bone morphogenetic proteins (BMPs) regulate cell fate during development and mediate cancer progression. In this study, we investigated the role of BMP4 in proliferation, anoikis resistance, metastatic migration, and drug resistance of breast cancer cells. We utilized breast cancer cell lines and clinical samples representing different subtypes to understand the functional effect of BMP4 on breast cancer. The BMP pathway was inhibited with the small molecule inhibitor LDN193189 hydrochloride (LDN). BMP4 signaling enhanced the expression of stem cell genes CD44, ALDH1A3, anti-apoptotic gene BCL2 and promoted anoikis resistance in MDA-MB-231 breast cancer cells. BMP4 enhanced self-renewal and chemoresistance in MDA-MB-231 by upregulating Notch signaling while LDN treatment abrogated anoikis resistance and proliferation of anoikis resistant breast cancer cells in the osteogenic microenvironment. Conversely, BMP4 downregulated proliferation, colony-forming ability, and suppressed anoikis resistance in MCF7 and SkBR3 cells, while LDN treatment promoted tumor spheroid formation and growth. These findings indicate that BMP4 has a context-dependent role in breast cancer. Further, our data with MDA-MB-231 cells representing triple-negative breast cancer suggest that BMP inhibition might impair its metastatic spread and colonization.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00649-9.     

miR-1275 targets MDK/AKT signaling to inhibit breast cancer chemoresistance by lessening the properties of cancer stem cells

Chemoresistance is a major obstacle in the neoadjuvant chemotherapy (NCT) of locally advanced breast cancer (LABC). Identification of miRNAs as prognostic biomarkers may help overcome chemoresistance of breast cancer (BC). This study aimed to evaluate the expression level of miR-1275 in plasma samples and its biological functions in the chemoresistance of BC. The expression levels of miR-1275 in plasma samples and cells were measured by RT-qPCR. CRISPR/Cas9-mediated gene editing was used to construct miR-1275 knock-out cells in MCF-7. We found that miR-1275 was significantly downregulated in plasma from patients resistant to chemotherapy and in chemoresistant BC cell lines, while patients with low levels of miR-1275 showed poor overall survival. miR-1275 knock-out promoted chemoresistance in BC cells by increasing the properties of cancer stem cells (CSCs). Mechanistically, we identified that MDK was determined to be direct downstream protein of miR-1275 which initiated PI3K/Akt signaling in breast cancer cells. We demonstrated that the high expression level of miR-1275 in plasma predicted better response to NCT. The reduction of miR-1275 promoted BC cells chemoresistance by increasing CSCs properties via targeting MDK/AKT axis. The potential of miR-1275 as a new prognostic biomarker and therapeutic target of BC patients was identified.