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Temporally sequenced anticancer drugs overcome adaptive resistance by targeting a vulnerable chemotherapy-induced phenotypic transition.

Goldman A, Majumder B, Dhawan A, Ravi S, Goldman D, Kohandel M, Majumder PK, Sengupta S - Nat Commun (2015)

Bottom Line: Understanding the emerging models of adaptive resistance is key to overcoming cancer chemotherapy failure.This state is associated with a clustering of CD44 and CD24 in membrane lipid rafts, leading to the activation of Src Family Kinase (SFK)/hemopoietic cell kinase (Hck) and suppression of apoptosis.The use of pharmacological inhibitors of SFK/Hck in combination with taxanes in a temporally constrained manner, where the kinase inhibitor is administered post taxane treatment, but not when co-administered, markedly sensitizes the chemotolerant cells to the chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA [2] Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts 02139, USA [3] Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

ABSTRACT
Understanding the emerging models of adaptive resistance is key to overcoming cancer chemotherapy failure. Using human breast cancer explants, in vitro cell lines, mouse in vivo studies and mathematical modelling, here we show that exposure to a taxane induces phenotypic cell state transition towards a favoured transient CD44(Hi)CD24(Hi) chemotherapy-tolerant state. This state is associated with a clustering of CD44 and CD24 in membrane lipid rafts, leading to the activation of Src Family Kinase (SFK)/hemopoietic cell kinase (Hck) and suppression of apoptosis. The use of pharmacological inhibitors of SFK/Hck in combination with taxanes in a temporally constrained manner, where the kinase inhibitor is administered post taxane treatment, but not when co-administered, markedly sensitizes the chemotolerant cells to the chemotherapy. This approach of harnessing chemotherapy-induced phenotypic cell state transition for improving antitumour outcome could emerge as a translational strategy for the management of cancer.

No MeSH data available.


Related in: MedlinePlus

Taxane chemotherapy induces phenotypic cell state transition and adaptive resistance.(a) Schematic of human explant model to evaluate response of refractory human tissue to anticancer agents. Tumour biopsies were cut into ~200 mM-thick sections and cultured in microwells coated with tumour matrix and media supplemented with autologous serum. (b) Representative immunohistochemistry (IHC) of primary human breast tumour explants shows induction of CD44 and CD24 cell surface expression following 72 h treatment with docetaxel versus vehicle. × 40 Scale bar, 50 μm inset show higher magnification, × 100 (c,d) Graph shows quantification of CD44 and CD24 levels in the primary tumour explant studies, (N=14 patients). Black and red points denote the protein levels measured by IHC score in a tumour explant in vehicle- and docetaxel-treated groups. Each number denotes a patient. The orange arrows denote patients who were taxane-treatment naive, whereas those denoted with black arrows received a taxane. (e) Representative IHC from explant culture shows effect of different drug treatments (3.4 μM docetaxel, 5.6 μM doxorubicin) on the expression of CD44 and cleaved (cl) caspase 3 in corresponding serial sections. Gem, gemcitabine. × 40 magnification Scale bar, 50 μm. Inset shows higher magnification × 100 (f) Graph shows the quantification of CD44 and cleaved caspase 3 expression in the explants treated with docetaxel (n=9) or a combination of gemcitabine+carboplatin (n=2). Data shows mean±s.e.m. (g) Schematic shows generation of drug-tolerant cells (DTCs) selected acutely using high-concentration docetaxel chemotherapy in vitro. Cells were cultured in 100 μM (~20X IC50) docetaxel. Cells surviving by day 4 were quiescent and considered as drug-tolerant cells (DTCs). Growing out the DTCs over 35 days resulted in restoring parental properties. (h) Graph shows MTS cell viability analysis of parental cells and DTCs generated from of MDA-MB-231 breast cancer cells following incubation (48 h) with different tubulin-binding chemotherapeutics at indicated concentration range. (i) Confocal images show expression levels of CD44 and CD24 in parental cells and DTCs generated from MDA-MB-468s. Scale bar, 18 μm (j) The population percentage of CD44HiCD24Hi cells in parental and DTCs generated from an array of luminal and basal breast cancer cell lines. Data shown are mean±s.e.m., n=3 (P<0.01 other than T47D cells). (k) Representative FACS plot of CD44 and CD24 in MDA-MB-231 parent cells and DTC. (l) Graph shows quantification of CD44Hi/CD24Lo and CD44Hi/CD24Hi as % of total population of MDA-MB-231 parent cells and DTCs (Data shown are mean±s.e.m., n=8, ANOVA analysis *P<0.01, ***P<0.001).
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f1: Taxane chemotherapy induces phenotypic cell state transition and adaptive resistance.(a) Schematic of human explant model to evaluate response of refractory human tissue to anticancer agents. Tumour biopsies were cut into ~200 mM-thick sections and cultured in microwells coated with tumour matrix and media supplemented with autologous serum. (b) Representative immunohistochemistry (IHC) of primary human breast tumour explants shows induction of CD44 and CD24 cell surface expression following 72 h treatment with docetaxel versus vehicle. × 40 Scale bar, 50 μm inset show higher magnification, × 100 (c,d) Graph shows quantification of CD44 and CD24 levels in the primary tumour explant studies, (N=14 patients). Black and red points denote the protein levels measured by IHC score in a tumour explant in vehicle- and docetaxel-treated groups. Each number denotes a patient. The orange arrows denote patients who were taxane-treatment naive, whereas those denoted with black arrows received a taxane. (e) Representative IHC from explant culture shows effect of different drug treatments (3.4 μM docetaxel, 5.6 μM doxorubicin) on the expression of CD44 and cleaved (cl) caspase 3 in corresponding serial sections. Gem, gemcitabine. × 40 magnification Scale bar, 50 μm. Inset shows higher magnification × 100 (f) Graph shows the quantification of CD44 and cleaved caspase 3 expression in the explants treated with docetaxel (n=9) or a combination of gemcitabine+carboplatin (n=2). Data shows mean±s.e.m. (g) Schematic shows generation of drug-tolerant cells (DTCs) selected acutely using high-concentration docetaxel chemotherapy in vitro. Cells were cultured in 100 μM (~20X IC50) docetaxel. Cells surviving by day 4 were quiescent and considered as drug-tolerant cells (DTCs). Growing out the DTCs over 35 days resulted in restoring parental properties. (h) Graph shows MTS cell viability analysis of parental cells and DTCs generated from of MDA-MB-231 breast cancer cells following incubation (48 h) with different tubulin-binding chemotherapeutics at indicated concentration range. (i) Confocal images show expression levels of CD44 and CD24 in parental cells and DTCs generated from MDA-MB-468s. Scale bar, 18 μm (j) The population percentage of CD44HiCD24Hi cells in parental and DTCs generated from an array of luminal and basal breast cancer cell lines. Data shown are mean±s.e.m., n=3 (P<0.01 other than T47D cells). (k) Representative FACS plot of CD44 and CD24 in MDA-MB-231 parent cells and DTC. (l) Graph shows quantification of CD44Hi/CD24Lo and CD44Hi/CD24Hi as % of total population of MDA-MB-231 parent cells and DTCs (Data shown are mean±s.e.m., n=8, ANOVA analysis *P<0.01, ***P<0.001).

Mentions: To elucidate the mechanisms underlying adaptive resistance to anticancer therapy, we used three-dimensional explants derived from fresh tumour biopsies from patients. Three-dimensional tumour explants are emerging as powerful models to study tumour biology, as they preserve the tumour heterogeneity and microenvironment15. In a recent study, we have observed that culturing the explants in autologous serum and in grade-matched tumour matrix conserves the parental tumour genotypic and phenotypic characteristics16. We included breast cancers of different stages and receptor status, including those that were taxanes-treatment naive (Supplementary Table 1). We used 200 μm tumour explants in this study as drugs can diffuse through such thickness17 (Fig. 1a). CD44, a membrane glycoprotein, has been associated with chemorefractory, more mesenchymal stem-like characteristics818. In contrast, CD24-positive breast cancer cells have been reported to be more of the differentiated luminal and a Her2+ subtype, whereas basal-like tumours were classified as CD24−/Lo (ref. 19). We observed a significant inter-tumoral heterogeneity in the baseline expression of CD44 and CD24, and the distribution was normal between tumours from taxane-treated and taxane-naive patients (Fig. 1b–d). Interestingly, incubating the explants with high-concentration DTX (3.4 μM)20 for 72 h resulted in an increase in the median expression of both CD44 and CD24 as compared with vehicle-treated explants (P<0.01) (Fig. 1b–d), irrespective of the tumour type. In addition, the DTX-induced increase in expression of CD24 and CD44 was similar in explants generated from tumours that had progressed clinically on taxanes and those generated from taxanes-treatment naive patients, indicating that the phenotypic plasticity did not rely on the acquisition of resistance. The upregulation of CD44 following DTX treatment was correlated with reduced apoptosis as seen in decreased cleaved caspase-3 levels compared with baseline (Fig. 1e,f). Treatment with doxorubicin, which is widely used in the adjuvant or metastatic settings in breast cancer, similarly induced CD44 expression with reduced cleaved caspase-3 levels. Interestingly, in contrast, treatment with carboplatin (100 μM) and gemcitabine (100 μM) induced apoptosis without any upregulation of CD44 expression (Fig. 1e,f). Indeed, in a recent study, a combination of gemcitabine and carboplatin was found to be effective for pretreated patients with metastatic breast cancer21.


Temporally sequenced anticancer drugs overcome adaptive resistance by targeting a vulnerable chemotherapy-induced phenotypic transition.

Goldman A, Majumder B, Dhawan A, Ravi S, Goldman D, Kohandel M, Majumder PK, Sengupta S - Nat Commun (2015)

Taxane chemotherapy induces phenotypic cell state transition and adaptive resistance.(a) Schematic of human explant model to evaluate response of refractory human tissue to anticancer agents. Tumour biopsies were cut into ~200 mM-thick sections and cultured in microwells coated with tumour matrix and media supplemented with autologous serum. (b) Representative immunohistochemistry (IHC) of primary human breast tumour explants shows induction of CD44 and CD24 cell surface expression following 72 h treatment with docetaxel versus vehicle. × 40 Scale bar, 50 μm inset show higher magnification, × 100 (c,d) Graph shows quantification of CD44 and CD24 levels in the primary tumour explant studies, (N=14 patients). Black and red points denote the protein levels measured by IHC score in a tumour explant in vehicle- and docetaxel-treated groups. Each number denotes a patient. The orange arrows denote patients who were taxane-treatment naive, whereas those denoted with black arrows received a taxane. (e) Representative IHC from explant culture shows effect of different drug treatments (3.4 μM docetaxel, 5.6 μM doxorubicin) on the expression of CD44 and cleaved (cl) caspase 3 in corresponding serial sections. Gem, gemcitabine. × 40 magnification Scale bar, 50 μm. Inset shows higher magnification × 100 (f) Graph shows the quantification of CD44 and cleaved caspase 3 expression in the explants treated with docetaxel (n=9) or a combination of gemcitabine+carboplatin (n=2). Data shows mean±s.e.m. (g) Schematic shows generation of drug-tolerant cells (DTCs) selected acutely using high-concentration docetaxel chemotherapy in vitro. Cells were cultured in 100 μM (~20X IC50) docetaxel. Cells surviving by day 4 were quiescent and considered as drug-tolerant cells (DTCs). Growing out the DTCs over 35 days resulted in restoring parental properties. (h) Graph shows MTS cell viability analysis of parental cells and DTCs generated from of MDA-MB-231 breast cancer cells following incubation (48 h) with different tubulin-binding chemotherapeutics at indicated concentration range. (i) Confocal images show expression levels of CD44 and CD24 in parental cells and DTCs generated from MDA-MB-468s. Scale bar, 18 μm (j) The population percentage of CD44HiCD24Hi cells in parental and DTCs generated from an array of luminal and basal breast cancer cell lines. Data shown are mean±s.e.m., n=3 (P<0.01 other than T47D cells). (k) Representative FACS plot of CD44 and CD24 in MDA-MB-231 parent cells and DTC. (l) Graph shows quantification of CD44Hi/CD24Lo and CD44Hi/CD24Hi as % of total population of MDA-MB-231 parent cells and DTCs (Data shown are mean±s.e.m., n=8, ANOVA analysis *P<0.01, ***P<0.001).
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f1: Taxane chemotherapy induces phenotypic cell state transition and adaptive resistance.(a) Schematic of human explant model to evaluate response of refractory human tissue to anticancer agents. Tumour biopsies were cut into ~200 mM-thick sections and cultured in microwells coated with tumour matrix and media supplemented with autologous serum. (b) Representative immunohistochemistry (IHC) of primary human breast tumour explants shows induction of CD44 and CD24 cell surface expression following 72 h treatment with docetaxel versus vehicle. × 40 Scale bar, 50 μm inset show higher magnification, × 100 (c,d) Graph shows quantification of CD44 and CD24 levels in the primary tumour explant studies, (N=14 patients). Black and red points denote the protein levels measured by IHC score in a tumour explant in vehicle- and docetaxel-treated groups. Each number denotes a patient. The orange arrows denote patients who were taxane-treatment naive, whereas those denoted with black arrows received a taxane. (e) Representative IHC from explant culture shows effect of different drug treatments (3.4 μM docetaxel, 5.6 μM doxorubicin) on the expression of CD44 and cleaved (cl) caspase 3 in corresponding serial sections. Gem, gemcitabine. × 40 magnification Scale bar, 50 μm. Inset shows higher magnification × 100 (f) Graph shows the quantification of CD44 and cleaved caspase 3 expression in the explants treated with docetaxel (n=9) or a combination of gemcitabine+carboplatin (n=2). Data shows mean±s.e.m. (g) Schematic shows generation of drug-tolerant cells (DTCs) selected acutely using high-concentration docetaxel chemotherapy in vitro. Cells were cultured in 100 μM (~20X IC50) docetaxel. Cells surviving by day 4 were quiescent and considered as drug-tolerant cells (DTCs). Growing out the DTCs over 35 days resulted in restoring parental properties. (h) Graph shows MTS cell viability analysis of parental cells and DTCs generated from of MDA-MB-231 breast cancer cells following incubation (48 h) with different tubulin-binding chemotherapeutics at indicated concentration range. (i) Confocal images show expression levels of CD44 and CD24 in parental cells and DTCs generated from MDA-MB-468s. Scale bar, 18 μm (j) The population percentage of CD44HiCD24Hi cells in parental and DTCs generated from an array of luminal and basal breast cancer cell lines. Data shown are mean±s.e.m., n=3 (P<0.01 other than T47D cells). (k) Representative FACS plot of CD44 and CD24 in MDA-MB-231 parent cells and DTC. (l) Graph shows quantification of CD44Hi/CD24Lo and CD44Hi/CD24Hi as % of total population of MDA-MB-231 parent cells and DTCs (Data shown are mean±s.e.m., n=8, ANOVA analysis *P<0.01, ***P<0.001).
Mentions: To elucidate the mechanisms underlying adaptive resistance to anticancer therapy, we used three-dimensional explants derived from fresh tumour biopsies from patients. Three-dimensional tumour explants are emerging as powerful models to study tumour biology, as they preserve the tumour heterogeneity and microenvironment15. In a recent study, we have observed that culturing the explants in autologous serum and in grade-matched tumour matrix conserves the parental tumour genotypic and phenotypic characteristics16. We included breast cancers of different stages and receptor status, including those that were taxanes-treatment naive (Supplementary Table 1). We used 200 μm tumour explants in this study as drugs can diffuse through such thickness17 (Fig. 1a). CD44, a membrane glycoprotein, has been associated with chemorefractory, more mesenchymal stem-like characteristics818. In contrast, CD24-positive breast cancer cells have been reported to be more of the differentiated luminal and a Her2+ subtype, whereas basal-like tumours were classified as CD24−/Lo (ref. 19). We observed a significant inter-tumoral heterogeneity in the baseline expression of CD44 and CD24, and the distribution was normal between tumours from taxane-treated and taxane-naive patients (Fig. 1b–d). Interestingly, incubating the explants with high-concentration DTX (3.4 μM)20 for 72 h resulted in an increase in the median expression of both CD44 and CD24 as compared with vehicle-treated explants (P<0.01) (Fig. 1b–d), irrespective of the tumour type. In addition, the DTX-induced increase in expression of CD24 and CD44 was similar in explants generated from tumours that had progressed clinically on taxanes and those generated from taxanes-treatment naive patients, indicating that the phenotypic plasticity did not rely on the acquisition of resistance. The upregulation of CD44 following DTX treatment was correlated with reduced apoptosis as seen in decreased cleaved caspase-3 levels compared with baseline (Fig. 1e,f). Treatment with doxorubicin, which is widely used in the adjuvant or metastatic settings in breast cancer, similarly induced CD44 expression with reduced cleaved caspase-3 levels. Interestingly, in contrast, treatment with carboplatin (100 μM) and gemcitabine (100 μM) induced apoptosis without any upregulation of CD44 expression (Fig. 1e,f). Indeed, in a recent study, a combination of gemcitabine and carboplatin was found to be effective for pretreated patients with metastatic breast cancer21.

Bottom Line: Understanding the emerging models of adaptive resistance is key to overcoming cancer chemotherapy failure.This state is associated with a clustering of CD44 and CD24 in membrane lipid rafts, leading to the activation of Src Family Kinase (SFK)/hemopoietic cell kinase (Hck) and suppression of apoptosis.The use of pharmacological inhibitors of SFK/Hck in combination with taxanes in a temporally constrained manner, where the kinase inhibitor is administered post taxane treatment, but not when co-administered, markedly sensitizes the chemotolerant cells to the chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA [2] Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts 02139, USA [3] Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

ABSTRACT
Understanding the emerging models of adaptive resistance is key to overcoming cancer chemotherapy failure. Using human breast cancer explants, in vitro cell lines, mouse in vivo studies and mathematical modelling, here we show that exposure to a taxane induces phenotypic cell state transition towards a favoured transient CD44(Hi)CD24(Hi) chemotherapy-tolerant state. This state is associated with a clustering of CD44 and CD24 in membrane lipid rafts, leading to the activation of Src Family Kinase (SFK)/hemopoietic cell kinase (Hck) and suppression of apoptosis. The use of pharmacological inhibitors of SFK/Hck in combination with taxanes in a temporally constrained manner, where the kinase inhibitor is administered post taxane treatment, but not when co-administered, markedly sensitizes the chemotolerant cells to the chemotherapy. This approach of harnessing chemotherapy-induced phenotypic cell state transition for improving antitumour outcome could emerge as a translational strategy for the management of cancer.

No MeSH data available.


Related in: MedlinePlus