Parental MCF-7 Breast Cancer Cells Research Articles

Targeting hormone-resistant breast cancer cells with docetaxel: a look inside the resistance.

Aim: The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen (HT) on their sensitivity to tubulin polymerization inhibitor docetaxel. Methods: The analysis of cell viability was performed by the MTT method. The expression of signaling proteins was analyzed by immunoblotting and flow cytometry. ERα activity was evaluated by gene reporter assay. To establish hormone-resistant subline MCF7, breast cancer cells were treated with 4-hydroxytamoxifen for 12 months. Results: The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen, and the resistance index was 2. Increased Akt activity (2.2-fold) and decreased ERα expression (1.5-fold) were revealed in MCF7/HT cells. The activity of the estrogen receptor α was reduced (1.5-fold) in MCF7/HT. Evaluation of class III β-tubulin expression (TUBB3), a marker associated with metastasis, revealed the following trends: higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells (P < 0.05). The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells (MCF7/HT < MCF7 < MDA-MB-231, approximately 1:2:4). High TUBB3 expression strongly correlated with docetaxel resistance: IC50 value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells, whereas resistant MCF7/HT cells were the most sensitive to the drug. The accumulation of cleaved PARP (a 1.6-fold increase) and Bcl-2 downregulation (1.8-fold) were more pronounced in docetaxel-treated resistant cells (P < 0.05). The expression of cyclin D1 decreased (2.8-fold) only in resistant cells after 4 nM docetaxel treatment, while this marker was unchanged in parental MCF7 breast cancer cells. Conclusion: Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising, especially for cancers with low TUBB3 expression.

The SMAC mimetic LCL161 is a direct ABCB1/MDR1-ATPase activity modulator and BIRC5/Survivin expression down-regulator in cancer cells

Upregulation of ABCB1/MDR1 (P-gp) and BIRC5/Survivin promotes multidrug resistance in a variety of human cancers. LCL161 is an anti-cancer DIABLO/SMAC mimetic currently being tested in patients with solid tumors, but the molecular mechanism of action of LCL161 in cancer cells is still incompletely understood. It is still unclear whether LCL161 is therapeutically applicable for patients with ABCB1-overexpressing multidrug resistant tumors. In this study, we found that the potency of LCL161 is not affected by the expression of ABCB1 in KB-TAX50, KB-VIN10, and NTU0.017 cancer cells. Besides, LCL161 is equally potent towards the parental MCF7 breast cancer cells and its BIRC5 overexpressing, hormone therapy resistance subline MCF7-TamC3 in vitro. Mechanistically, we found that LCL161 directly modulates the ABCB1-ATPase activity and inhibits ABCB1 multi-drug efflux activity at low cytotoxic concentrations (i.e. 0.5xIC50 or less). Further analysis revealed that LCL161 also decreases intracellular ATP levels in part through BIRC5 downregulation. Therapeutically, co-treatment with LCL161 at low cytotoxic concentrations restored the sensitivity to the known ABCB1 substrate, paclitaxel, in ABCB1-expressing cancer cells and increased the sensitivity to tamoxifen in MCF7-TamC3 cells. In conclusion, LCL161 has the potential for use in the management of cancer patients with ABCB1 and BIRC5-related drug resistance. The findings of our study provide important information to physicians for designing a more “patient-specific” LCL161 clinical trial program in the future.

Design, synthesis and biological evaluation of a new thieno[2,3-d]pyrimidine-based urea derivative with potential antitumor activity against tamoxifen sensitive and resistant breast cancer cell lines

Breast cancer (BC) and endocrine resistance to chemotherapy are challenging problems where angiogenesis plays fundamental roles. Thus, targeting of VEGFR-2 signalling pathway has been an attractive approach. In this study, we synthesised a new sorafenib analogue, thieno[2,3-d]pyrimidine based urea derivative, KM6. It showed 65% inhibition of VEGF2 tyrosine kinase activity and demonstrated a potential antitumor activity in TAM-resistant, LCC2, and its parental MCF7 BC cells. KM6 retained the sensitivity of LCC2 through upregulation of key enzymes of apoptosis and proteins of cell death including caspases 3, 8, 9, P53, BAX/BCL-2 ratio and LDH in media. It downregulated mRNA expression of Ki-67, survivin, Akt, and reduced levels of ROS and glucose uptake. Moreover, KM6 reduced the levels of inflammation markers PGE2, COX2, IL-1β and IL6 and metastasis markers MMP-2 and MMP-9. In conclusion, KM6 is a promising compound for ER + and TAM-resistant BC with many potential antitumor and polypharmacological mechanisms.

Regulation of PERK expression by FOXO3: a vulnerability of drug-resistant cancer cells

The major impediment to effective cancer therapy has been the development of drug resistance. The tumour suppressive transcription factor FOXO3 promotes cell cycle arrest, senescence and cell death, and mediates the cytotoxic and cytostatic functions of cancer therapeutics. In consequence, FOXO3 is often downregulated as an adaptive response in cancer and particularly in chemotherapeutic drug-resistant cells. Consistently, we find that FOXO3 expression is attenuated in the drug-resistant MCF-7-EpiR and MCF-7-TaxR compared to the parental MCF-7 breast cancer cells. Using ChIP, short-interfering RNA (siRNA) knockdown, and overexpression assays as well as Foxo1/3/4−/− MEFs, we establish the endoplasmic reticulum (ER)-stress defence modulator PERK (eIF2AK3) as a direct downstream transcriptional target of FOXO3. In agreement, there is also a positive correlation between FOXO3 and PERK expression at the protein and RNA levels in breast cancer patient samples. We uncover that PERK expression is downregulated but its activity constitutively elevated in the drug-resistant cells. With this in mind, we exploit this adaptive response of low FOXO3 and PERK expression, and high PERK activity in drug-resistant breast cancer cells and show that these drug-resistant cells are specifically sensitive to PERK inhibition. In support of this finding, we show that ectopic overexpression of FOXO3 can reduce the sensitivity of the resistant cells to the PERK inhibitor GSK2606414, while the Foxo1/3/4−/− MEFs expressing lower levels of PERK are more sensitive to PERK inhibition compared to wild-type MEFs. PERK inhibitor-titration and -time course experiments showed that the drug-resistant cells, which express lower expression and higher activity levels of PERK, are more sensitive to the increasing concentrations of PERK inhibitor compared to parental MCF-7 cells. Our present work thus reveals a chemotherapeutic drug-resistant cancer cell vulnerability in PERK and suggests PERK as a potential target for cancer therapy, specifically in the context of drug-resistant cancers.

Everolimus downregulates estrogen receptor and induces autophagy in aromatase inhibitor-resistant breast cancer cells

BackgroundmTOR inhibition of aromatase inhibitor (AI)-resistant breast cancer is currently under evaluation in the clinic. Everolimus/RAD001 (Afinitor®) has had limited efficacy as a solo agent but is projected to become part of combination therapy for AI-resistant breast cancer. This study was conducted to investigate the anti-proliferative and resistance mechanisms of everolimus in AI-resistant breast cancer cells.MethodsIn this study we utilized two AI-resistant breast cancer cell lines, MCF-7:5C and MCF-7:2A, which were clonally derived from estrogen receptor positive (ER+) MCF-7 breast cancer cells following long-term estrogen deprivation. Cell viability assay, colony formation assay, cell cycle analysis and soft agar anchorage-independent growth assay were used to determine the efficacy of everolimus in inhibiting the proliferation and tumor forming potential of MCF-7, MCF-7:5C, MCF-7:2A and MCF10A cells. Confocal microscopy and transmission electron microscopy were used to evaluate LC3-II production and autophagosome formation, while ERE-luciferase reporter, Western blot, and RT-PCR analyses were used to assess ER expression and transcriptional activity.ResultsEverolimus inhibited the proliferation of MCF-7:5C and MCF-7:2A cells with relatively equal efficiency to parental MCF-7 breast cancer cells. The inhibitory effect of everolimus was due to G1 arrest as a result of downregulation of cyclin D1 and p21. Everolimus also dramatically reduced estrogen receptor (ER) expression (mRNA and protein) and transcriptional activity in addition to the ER chaperone, heat shock protein 90 protein (HSP90). Everolimus restored 4-hydroxy-tamoxifen (4OHT) sensitivity in MCF-7:5C cells and enhanced 4OHT sensitivity in MCF-7 and MCF-7:2A cells. Notably, we found that autophagy is one method of everolimus insensitivity in MCF-7 breast cancer cell lines.ConclusionThis study provides additional insight into the mechanism(s) of action of everolimus that can be used to enhance the utility of mTOR inhibitors as part of combination therapy for AI-resistant breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2490-z) contains supplementary material, which is available to authorized users.

In-depth characterization of breast cancer tumor-promoting cell transcriptome by RNA sequencing and microarrays.

Numerous studies have reported the existence of tumor-promoting cells (TPC) with self-renewal potential and a relevant role in drug resistance. However, pathways and modifications involved in the maintenance of such tumor subpopulations are still only partially understood. Sequencing-based approaches offer the opportunity for a detailed study of TPC including their transcriptome modulation. Using microarrays and RNA sequencing approaches, we compared the transcriptional profiles of parental MCF7 breast cancer cells with MCF7-derived TPC (i.e. MCFS). Data were explored using different bioinformatic approaches, and major findings were experimentally validated. The different analytical pipelines (Lifescope and Cufflinks based) yielded similar although not identical results. RNA sequencing data partially overlapped microarray results and displayed a higher dynamic range, although overall the two approaches concordantly predicted pathway modifications. Several biological functions were altered in TPC, ranging from production of inflammatory cytokines (i.e., IL-8 and MCP-1) to proliferation and response to steroid hormones. More than 300 non-coding RNAs were defined as differentially expressed, and 2,471 potential splicing events were identified. A consensus signature of genes up-regulated in TPC was derived and was found to be significantly associated with insensitivity to fulvestrant in a public breast cancer patient dataset. Overall, we obtained a detailed portrait of the transcriptome of a breast cancer TPC line, highlighted the role of non-coding RNAs and differential splicing, and identified a gene signature with a potential as a context-specific biomarker in patients receiving endocrine treatment.

Abstract 25: Loss of interferon-induced transmembrane protein 1 enhances estrogen-induced cell death in AI-resistant breast cancer cells

Abstract Estrogen deprivation using aromatase inhibitors (AIs) is the standard of care for postmenopausal women with hormone receptor-positive breast cancer. However, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. We have previously reported the development of an AI-resistant breast cancer cell line, MCF-7:5C, which was clonally derived from parental MCF-7 breast cancer cells following long term estrogen deprivation. A unique phenotype of AI-resistant MCF-7:5C cells is that they grow in the absence of estrogen; however, when they are treated with estradiol they undergo apoptosis/cell death through activation of the mitochondrial death pathway. More recently, we have also discovered that AI-resistant MCF-7:5C cells constitutively overexpress the interferon stimulated gene IFITM1. IFITM1 is a cell surface membrane protein that is most well-known for its ability to block viral replication, however, its role in breast cancer and AI-resistance is not known. We found that IFITM1 was constitutively overexpressed at the protein (&amp;gt; 20-fold) and mRNA (&amp;gt; 15-fold) level in AI-resistant MCF-7:5C breast cancer cells compared to parental MCF-7 cells and that its overexpression strongly correlated with enhanced growth and increased aggressiveness. Clinical data also indicated that IFITM1 was overexpressed in 36 out of 40 AI-resistant breast tumor samples compared to AI-sensitive tumors. In this study, we assessed the functional role of IFITM1 in AI-resistant MCF-7:5C cells. Notably, we found that knockdown of IFITM1 significantly induced cell death in MCF-7:5C cells and it reduced cell proliferation. The induction of cell death as a result of IFITM1 knockdown was associated with increases in p21, Bax, Noxa, and Puma expression; however, p53 was not altered in these cells. To validate the effect observed with IFITM1 knockdown in our resistant cells we used a second shRNA targeting IFITM1. We found that shRNA knockdown of IFITM1 induced PARP cleavage and p21, reduced cell proliferation, and induced cell death in AI-resistant MCF-7:5C cells. Moreover, we found that knockdown of IFITM1 dramatically enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells which was associated with induction of pro-apoptotic proteins and p21 via a p53-independent mechanism. Further analysis revealed that knockdown of STAT1 and STAT2, which are critical regulators of IFITM1, also significantly enhanced estradiol-induced apoptosis in MCF-7:5C cells. Taken together, these findings indicate that overexpression of IFITM1 plays a critical role in breast cancer and AI-resistance and that targeting this gene can sensitize AI-resistant cells to estrogen-induced death. Furthermore, these results suggest that IFITM1 can regulate the mitochondrial pathway through its interactions with anti-apoptotic proteins. Citation Format: Hye Joung Choi, Joan Lewis-Wambi. Loss of interferon-induced transmembrane protein 1 enhances estrogen-induced cell death in AI-resistant breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 25. doi:10.1158/1538-7445.AM2015-25

Aurora kinase A and B as new treatment targets in aromatase inhibitor-resistant breast cancer cells.

Aromatase inhibitors (AIs) are used for treatment of estrogen receptor α (ER)-positive breast cancer; however, resistance is a major obstacle for optimal outcome. This preclinical study aimed at identifying potential new treatment targets in AI-resistant breast cancer cells. Parental MCF-7 breast cancer cells and four newly established cell lines, resistant to the AIs exemestane or letrozole, were used for a functional kinase inhibitor screen. A library comprising 195 different compounds was tested for preferential growth inhibition of AI-resistant cell lines. Selected targets were validated by analysis of cell growth, cell cycle phase distribution, protein expression, and subcellular localization. We identified 24 compounds, including several inhibitors of Aurora kinases e.g., JNJ-7706621 and barasertib. Protein expression of Aurora kinase A and B was found upregulated in AI-resistant cells compared with MCF-7, and knockdown studies showed that Aurora kinase A was essential for AI-resistant cell growth. In AI-resistant cell lines, the clinically relevant Aurora kinase inhibitors alisertib and danusertib blocked cell cycle progression at the G2/M phase, interfered with chromosome alignment and spindle pole formation, and resulted in preferential growth inhibition compared with parental MCF-7 cells. Even further growth inhibition was obtained when combining the Aurora kinase inhibitors with the antiestrogen fulvestrant. Our study is the first to demonstrate that Aurora kinase A and B may be treatment targets in AI-resistant cells, and our data suggest that therapy targeting both ER and Aurora kinases may be a potent treatment strategy for overcoming AI resistance in breast cancer.

Abstract P2-09-08: Therapeutic potential of the dual HER1/2 inhibitor AZD8931 in circumventing endocrine resistance

Abstract Background: Crosstalk between ER and HER-family signaling pathways has been suggested to play a role in the development of endocrine resistance. Preclinical studies have shown that AZD8931, a dual tyrosine kinase inhibitor (TKI) of HER1 (EGFR) and HER2, elicits an equipotent inhibition of HER1, HER2, and HER3 signaling, and is consequently more effective in blocking ligand-dependent HER signaling than the dual HER 1/2 TKI lapatinib. Using in vitro and in vivo models we investigated AZD8931's therapeutic potential in enhancing endocrine therapy and in overcoming the growth of tumor cells resistant to tamoxifen (Tam). Materials and Methods: The effect of different TKIs (AZD8931 and lapatinib) on endocrine therapy [Tam and Fulvestrant (Ful)] was tested in ER+ MCF7 and T47D parental cells and their Tam-resistant derivatives (TamRes). In vitro growth, proliferation, and apoptosis were assessed using an in situ cell cytometer, EDU incorporation, and Annexin V-FITC/c-PARP, respectively. HER ligands in the parental and TamRes cells were profiled using RNASeq analysis of these lines. Western blot analysis was used to analyze the effect of AZD8931, lapatinib, and the HER1 TKI gefitinib on HER 1/2 pathway activation upon ligand stimulation. Nude mice with transplantable MCF7/TamRes xenografts at 200 mm3 were randomized to continued Tam, Tam+AZD8931, Fulvestrant (Ful), or Ful+AZD8931 treatments. Results: We found that neither lapatinib nor AZD8931 significantly enhanced endocrine sensitivity in parental MCF7 breast cancer cells, although AZD8931 did enhance endocrine sensitivity in parental T47D cells. Furthermore, AZD8931 combined with either Tam or Ful inhibited cell growth more profoundly than lapatinib in the T47D TamRes cell model, and was significantly, though modestly, more potent than lapatinib in the MCF7 TamRes model when combined with Tam. In both TamRes models, AZD8931 significantly inhibited cell proliferation and induced apoptosis, with the highest effects seen in combination with Ful. Interestingly, multiple HER ligands are upregulated in both MCF7 and T47D TamRes models, which could explain the superiority of AZD8931 over lapatinib in these models. Indeed, in EGF and heregulin (HRG) stimulated conditions, AZD8931 more potently inhibited HER signaling (i.e., phosphorylated (p) levels of HER1/2, MAPK, and AKT) than lapatinib or gefitinib. Finally, AZD8931 significantly delayed the growth of MCF7 TamRes xenografts in the presence of continued Tam or Ful. These tumors were also inhibited by Ful alone, but the strongest inhibition was achieved by Ful and AZD8931 in combination. However, despite the marked tumor growth delay, no tumor regression was found in any of these treatments. Conclusion: This study provides evidence that AZD8931 has greater inhibitory efficacy than lapatinib in endocrine resistant models that are dependent on ligand activation of the HER pathway. Although the AZD8931 combination with Ful robustly slowed growth of TamRes tumors in vivo, the absence of tumor regression suggests that additional escape pathways are also involved and should also be targeted to fully circumvent tamoxifen resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-08.

Abstract A008: HER2Δ16, the oncogenic isoform of HER2, suppresses microRNA-7 to promote breast tumor cell migration and proliferation

Abstract Of the five different molecular subtypes of breast cancer, HER2-positive patients have the worst overall prognosis and shortest relapse-free survival. We have shown that HER2-positive breast tumors co-express the oncogenic HER2 isoform, HER2Δ16, which is associated with metastasis and trastuzumab resistance. Here we hypothesize that HER2Δ16 represents the transforming form of HER2 and drives tumorigenesis through deregulation of microRNA (miR) expression. We compared global miR expression profiles between parental MCF-7 breast cancer cells (MCF-7/pcDNA) and a MCF-7 cell line with ectopic expression of HER2Δ16 (MCF-7/HER2Δ16) and found altered regulation of multiple miRs. Interestingly, we observed a 4.8 fold suppression of the tumor suppressor microRNA-7 (miR-7) in the MCF-7/HER2Δ16 cells. MiR-7 has been shown to regulate EGFR expression and clinically, the coexpression of HER2 and EGFR in primary breast tumors is associated with significantly worse prognosis than tumors with HER2 or EGFR expression alone. To determine the impact of miR-7 on EGFR expression and breast tumor cell proliferation and migration, we reestablished miR-7 expression in the highly invasive MCF-7/HER2Δ16 cell line (MCF-7/HER2Δ16/miR-7) and we suppressed miR-7 expression in the non-invasive MCF-7 cell line (MCF-7/miR-7KD). As predicted, reexpression of miR-7 in MCF-7/HER2Δ16 cells dramatically reduced EGFR protein levels, while suppression of miR-7 in MCF-7 cells increased EGFR protein. Furthermore, the reestablished miR-7 expression in MCF-7/HER2Δ16 cells significantly reduced the size and numbers of colonies in a colony formation assay, caused a G1 arrest of the cell cycle and abolished cell migration to the levels observed in noninvasive parental MCF-7 cells. Conversely, inhibition of miR-7 in MCF-7 cells resulted in an increase in colony formation and S-phase of the cell cycle. Taken together, these results indicate that suppression of miR-7 is necessary but not sufficient to promote breast tumor cell migration, while miR-7 directly regulates cellular proliferation. To investigate the contribution of EGFR to HER2Δ16 tumorigenesis, we silenced EGFR expression using shRNAs (MCF-7/HER2Δ16/EGFRKD). While the MCF-7/HER2Δ16/miR-7 cells inhibited colony formation, the MCF-7/HER2Δ16/EGFRKD cells showed enhanced colony formation. However, suppression of EGFR significantly reduced HER2Δ16-induced cell migration activity. These results indicate that EGFR is an essential component of the HER2Δ16 cell migration pathway, but is dispensable for HER2Δ16 colony formation activity. Interestingly, reexpression of miR-7 in the MCF-7/HER2Δ16 cell line resulted in a loss of Src kinase phosphorylated activation. Our preliminary results suggest that Src kinase activation in the absence of miR-7 drives HER2Δ16 tumor cell proliferation. We are currently investigating the contribution of miR-7 to HER2Δ16-induced resistance to both trastuzumab and lapatinib. In summary, HER2Δ16 represents a novel breast oncogene that promotes breast tumor cell proliferation and metastasis through suppression of miR-7 and cooperation with Src kinase. Our data suggests that modulation of miR-7 expression can be implemented as a therapeutic approach for the treatment of the most aggressive and therapeutically refractory HER2-positive metastatic breast tumors. Citation Format: Felicia C. Huynh, Frank E. Jones. HER2Δ16, the oncogenic isoform of HER2, suppresses microRNA-7 to promote breast tumor cell migration and proliferation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A008.

Role of Insulin-Like Growth Factor Binding Protein-3 in 1, 25-Dihydroxyvitamin-D3-Induced Breast Cancer Cell Apoptosis

Insulin-like growth factor I (IGF-I) is implicated in breast cancer development and 1, 25-dihydroxyvitamin D3 (1, 25-D3) has been shown to attenuate prosurvival effects of IGF-I on breast cancer cells. In this study the role of IGF binding protein-3 (IGFBP-3) in 1, 25-D3-induced apoptosis was investigated using parental MCF-7 breast cancer cells and MCF-7/VDR cells, which are resistant to the growth inhibitory effects of 1, 25-D3. Treatment with 1, 25-D3 increased IGFBP-3 mRNA expression in both cell lines but increases in intracellular IGFBP-3 protein and its secretion were observed only in MCF-7. 1, 25-D3-induced apoptosis was not associated with activation of any caspase but PARP-1 cleavage was detected in parental cells. IGFBP-3 treatment alone produced cleavage of caspases 7, 8, and 9 and PARP-1 in MCF-7 cells. IGFBP-3 failed to activate caspases in MCF-7/VDR cells; however PARP-1 cleavage was detected. 1, 25-D3 treatment inhibited IGF-I/Akt survival signalling in MCF-7 but not in MCF-7/VDR cells. In contrast, IGFBP-3 treatment was effective in inhibiting IGF-I/Akt pathways in both breast cancer lines. These results suggest a role for IGFBP-3 in 1, 25-D3 apoptotic signalling and that impaired secretion of IGFBP-3 may be involved in acquired resistance to vitamin D in breast cancer.

Different Modalities of Intercellular Membrane Exchanges Mediate Cell-to-cell P-glycoprotein Transfers in MCF-7 Breast Cancer Cells

Multi-drug resistance (MDR) is a phenomenon by which tumor cells exhibit resistance to a variety of chemically unrelated chemotherapeutic drugs. The classical form of multidrug resistance is connected to overexpression of membrane P-glycoprotein (P-gp), which acts as an energy dependent drug efflux pump. P-glycoprotein expression is known to be controlled by genetic and epigenetic mechanisms. Until now processes of P-gp gene up-regulation and resistant cell selection were considered sufficient to explain the emergence of MDR phenotype within a cell population. Recently, however, "non-genetic" acquisitions of MDR by cell-to-cell P-gp transfers have been pointed out. In the present study we show that intercellular transfers of functional P-gp occur by two different but complementary modalities through donor-recipient cells interactions in the absence of drug selection pressure. P-glycoprotein and drug efflux activity transfers were followed over 7 days by confocal microscopy and flow cytometry in drug-sensitive parental MCF-7 breast cancer cells co-cultured with P-gp overexpressing resistant variants. An early process of remote transfer was established based on the release and binding of P-gp-containing microparticles. Microparticle-mediated transfers were detected after only 4 h of incubation. We also identify an alternative mode of transfer by contact, consisting of cell-to-cell P-gp trafficking by tunneling nanotubes bridging neighboring cells. Our findings supply new mechanistic evidences for the extragenetic emergence of MDR in cancer cells and indicate that new treatment strategies designed to overcome MDR may include inhibition of both microparticles and Tunneling nanotube-mediated intercellular P-gp transfers.

Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells

BackgroundEnhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of the ErbB2 (ΔN-ErbB2).Methodology/Principal FindingsMass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic ΔN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however, normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm.Conclusions/SignificanceOur data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in autophagosome formation.