Overcoming the chemo-resistance in ovarian cancer

Transcription

1 Overcoming the chemo-resistance in ovarian cancer Department Obstetrics & Gynecology Keimyung University, School of Medicine Daegu, Korea Chi-Heum Cho M.D., Ph.D

2 Agenda Ovarian cancer overview Multidrug resistance Mechanisms of resistance of Pt-based antitumor agents Cancer stem cell and chemotherapeutic drug resistance Regulation of cancer cell metabolism Metabolic regulation by p53 Preliminary data for overcoming the chemo-resistance

3 Ovarian Cancer: Overview Ovarian cancers are the second most frequent malignancy in the United States 2008 estimates: 21,650 new cases, 15,520 deaths [1] Account for 54% of all deaths from gynecologic cancers Ovarian cancers present as stage III/IV disease in ~ 75% of cases [2] No effective early diagnostic tests Requires systemic therapy as the mainstay of treatment Although the success rate with initial therapy has improved, most patients will exhibit recurrent or persistent disease and will require management beyond initial therapy 1. American Cancer Society Jelic S, et al. ESMO

4 Recurrent Ovarian Cancer: Magnitude of the Clinical Problem Stage I/II Essentially all patients will achieve a clinical CR after surgery and chemotherapy 20% to 25% will relapse Optimal stage III > 90% will achieve a clinical CR 75% will recur Suboptimal stage III/IV 50% will achieve a clinical CR > 90% will recur

5 Debulking surgery remains key in ovarian cancer treatment Residual tumor size of greater than 2 cm is associated with a reduced survival of months, compared with months if the tumor is less than 2 cm *Sandercock et al, First-line treatment for advanced ovarian cancer: paclitaxel, platinum and the evidence. Br. J. Cancer 87, (2002) * Mutch, D. G. Surgical management of ovarian cancer. Semin. Oncol. 29, 3 8 (2002).

6 platinum paclitaxel combination regimen as firstline treatment for advanced ovarian cancer, yielding response rates of over 80% and 40 60% complete responses However, most of these patients will eventually relapse with a median progression-free survival of 18 months Greenlee et al, Cancer statistics, CA Cancer J. Clin. 51, (2001

7 Efforts have been made to cure ovarian cancer over the past decade using different classes of chemotherapy agents in various combinations, dosages and schedules to overcome chemoresistance following front-line paclitaxel platinum treatment Despite these efforts, clinical responses remain short lived and have merely led to marginal improvements in the survival of patients with platinum-resistant disease 1. De Placido, S. et al. Topotecan compared with no therapy after response to surgery and carboplatin/paclitaxel in patients with ovarian cancer: Multicenter Italian Trials in Ovarian Cancer (MITO-1) randomized study. J. Clin. Oncol. 22, (2004)

8 MDR (Multidrug Resistance) ATP-binding cassette (ABC) transport superfamily Energy requiring efflux pump decreased cellular accumulation of hydrophobic anticancer chemotherapeutic drugs Solute carrier transport superfamily Mediate the cellular uptake of anticancer drug drug resistance may result from decreased activity of these transporters

9 MDR (Multidrug Resistance) ATP-binding cassette (ABC) transport superfamily nucleotide-binding domain (NBD): efflux pump Transmembrane domain (TMD) : translocation from the cytoplasm to the cell membrane ABC family : divide 7 subfamily (A-G) P-glycoprotein (P-gp; MDR1/ABCB1) MDR-associated protein (MRP1; ABCC1) breast cancer resistance protein (BCRP; ABCG2)

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12 P-gylcoprotein (P-gp, MDR1, ABCB1) 1 st identified ABC transporter, Chromosome 7q21 Mutation of the p53 gene and over expression of the p63 gene and/or the p73 gene in certain tumors may facilitate P-gp expression ABCB1 promoter activation by a nuclear protein, MDR1 promoter- enhancing factor, or a component of the multiprotein complex, RNA helicase A, regulate P-gp expression No clear therapeutic strategy to overcome the actions of this classical drug efflux pump in tumors Anti-CD44 antibody not only blocks HA-CD44 binding but also inhibits ABCB1-mediated efflux activity. Thus, anti-cd44 antibody may be used in combination with chemotherapy to enhance chemosensitivity Bourguignon et al. J Biol Chem 2008;283:

13 P-gylcoprotein

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15 MDR-associated protein (MRP1, ABCC1) 1531 AA, Chromosome 16p13.1 located in the apical membranes of epithelial cells overexpressing MRP1 protein are resistant to a wide variety of anticancer drugs, e.g. doxorubicin, epirubicin, vinblastine, vincristine, and etoposide MRP1 cannot transport the unmodified anticancer drugs without the presence of glutathione (GSH). This implies that MRP1 may cotransport the anticancer drugs with GSH Until now few MRP1-specific inhibitors have been developed Chang XB. Cancer Metastasis Rev 2007;26:15 37

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18 Breast cancer resistance protein (BCRP, ABCG2) 655 AA, Chromosome 4q21-22 first cloned from a heavily drug-selected breast cancer cell line (MCF- 7/AdrVp) abundantly in the liver and intestinal epithelium, the placenta, the blood brain barrier, and various stem cells hypoxia can regulate ABCG2 expression stem cells or tumor cells in hypoxic environments may be protected from chemotherapeutic agents because of increased levels of ABCG2 induced by hypoxia Robey et al: determining its relevance in clinical drug resistance. Cancer Metastasis Rev 2007;26:39 57.

19 Mechanisms of resistance of Pt-based antitumor agents Although P-gp, MRP1, and ABCG2 are responsible for tumor resistance to many chemotherapeutic drugs currently used in cancer therapy, no roles in resistance to Pt-based antitumor agents such as cisplatin, carboplatin, and oxaliplatin taken up into the cells through a member of the solute carrier transporter superfamily, hctr1, copper transporter at the cell membrane. Three pathway : copper efflux transporters, ATP7A and ATP7B, interact with GSH in the cytoplasm to form Pt(GS)2 complex, enter the nucleus and form Pt-DNA adducts Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov 2005;4:307 20

20 Mechanisms of resistance of Pt-based antitumor agents Expression of hctr1, ATP7A and ATP7B: regulated by intracellular copper homeostasis. increased expression of ATP7A mediates resistance to cisplatin, carboplatin, and oxaliplatin in ovarian cancer cells copper-binding proteins : cellular thiol-containing proteins (e.g. metallothioneins and GSH) bind not only copper but also Pt; increased intracellular concentration of GSH may result in inactivation of cisplatin Increased levels of MRP2 are associated with elevated cisplatin resistance, decreased intracellular accumulation of cisplatin, and decreased DNA adduct formation

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22 Tumor resistance to cisplatin and carboplatin mediated by inadequate levels of platinum reaching target DNA

23 FDA-approved platinum drugs and the main platinum drugs in development

24 Tumor resistance to cisplatin and carboplatin mediated after DNA binding

25 Major ongoing strategies to circumvent cisplatin and carboplatin resistance.

26 The current platinum drug family tree and generalized rationale underlying their development

27 Future directions and prospects: Pt-based cancer chemotherapy three approved platinum drugs, cisplatin, carboplatin and oxaliplatin, continue to have a major role in contemporary medical oncology satraplatin and picoplatin might further broaden their applicability to tumour types such as prostate cancer and small-cell lung cancer bevacizumab in combination with carboplatin and paclitaxel in patients with lung cancer; breast cancer provides additional possibilities

28 Cancer stem cell and chemotherapeutic drug resistance Cancer stem cells also retain the essential property of self-protection through the activity of MDR transporters These cancer stem cells make up as few as 1% of the total tumor cells, making them difficult to detect and study the existence of cancer stem cells provides a tumor reservoir that is the source of disease recurrence and metastasis

29 Cancer stem cell and chemotherapeutic drug resistance Type I, chemo-resistant and Type II, chemosensitive EOC cells Type I cells are characterized by slower growth, which is inhibited upon cell-to cell contact, constitutive NFκB activity and constitutively secrete IL6, IL8, MCP-1 and GROα Type II EOC cells represent the classical ovarian cancer cells characterized by fast growth and cell division, and lack of cell-to-cell contact inhibition

30 Cellular morphology of ovarian cancer stem cells

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32 bulk of ovarian cancer tumors arise from the CD44+ progenitor cancer cells, which have the capacity to self-renew (red arrow) and differentiate (yellow arrow)

33 chemotherapy targets only the CD44- cancer cells while CD44+ Type I EOC stem cells persist

34 recurrence maybe associated with a larger pool of CD44+ Type I EOC stem cells

35 Regulation of cancer cell metabolism Metabolic changes are a common feature of cancerous tissues, although it is unclear to what extent these metabolic changes are important in low-grade slow growing tumors Warburg effect, which is a shift from ATP generation through oxidative phosphorylation to ATP generation through glycolysis, even under normal oxygen concentrations regulated by the PI3K, hypoxia-indicible factor (HIF), p53, MYC and AMP-activated protein kinase (AMPK) liver kinase B1 (LKB1) pathways

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37 Molecular mechanisms driving the Warburg effect

38 Determinants of the tumor metabolic phenotype

39 Regulation of cancer cell metabolism Metabolic adaptation in tumors extends beyond the Warburg effect key molecule produced as a result of altered cancer metabolism is reduced nicotinamide adenine dinucleotide phosphate (NADPH), which functions as a cofactor and provides reducing power in many enzymatic reactions that are crucial for macromolecular biosynthesis NADPH is also an antioxidant and forms part of the defence against reactive oxygen species (ROS) that are produced during rapid proliferation

40 PKM2 and its effect on glycolysis and the pentose phosphate pathway

41 Mechanisms of redox control and their alterations in cancer

42 Regulation of cancer cell metabolism High levels of ROS can cause damage to macromolecules, which can induce senescence and apoptosis Cells counteract the detrimental effects of ROS by producing antioxidant molecules, such as reduced glutathione (GSH) and thioredoxin (TRX) GSH and TRX, rely on the reducing power of NADPH to maintain their activities

43 Relationship between the levels of ROS and cancer

44 Regulation of cancer cell metabolism genetic changes that alter tumor cell metabolism, the abnormal tumor microenvironment such as hypoxia, ph and low glucose concentrations have a major role in determining the metabolic phenotype of tumor cells Mutations in oncogenes and tumor suppressor genes cause alterations to multiple intracellular signalling pathways that affect tumor cell metabolism

45 Metabolic regulation by p53 p53 is an important regulator of metabolic pathways p53 is able to contribute to the regulation of glycolysis, oxidative phosphorylation, glutaminolysis, insulin sensitivity, nucleotide biosynthesis, mitochondrial integrity, fatty acid oxidation, antioxidant response, autophagy and mtor signalling p53 is emerging as an important regulator of metabolic homeostasis

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49 Signalling between p53 and the IGF-AKT-mTORC1 pathway

50 Key signalling pathways involved in the progression of ovarian cancer and potential targets for anticancer therapy

51 The Seven Hallmarks of Cancer and Their Links to Tumor Metabolism

52 A2780 & A2780 cp cell : MTS 120 A2780 % of cellv viability * ** ** A2780 cp 0 control 100uM 200uM 400uM cisplatin * p<0.05, ** p < 0.01

53 ** ** Con Cg 10 Cg 20 Cg 30 + CDDP 100 µm/l Anti-proliferative effect of Ciglitizone (Cg) on Cisplatin resistant cell A2780. Cells were treated with Ciglitizone (10,20 and30µm/l) & CDDP 100 µm/l for 24 hours. The cell viability was measured using the Titer Cell Proliferation Assay (Promega Madison, WI). ** p < 0.01

54 Glucose Uptake (A2780 vs. A2780 CP) CP PM A2780 A2780 CP

55 Glucose Uptake (A2780cp) CP PM CTL Ciglitazone 1 μm Ciglitazone 10 μm

56 Figure 3 A2780cis A2780cp + CDDP 100 µm/l Ciglitizone µm SP1 Akt Cdk2 β -actin

57 Ciglitizone µm+ CDDP 100 µm/l SP1 P-AMPK β Catenin Akt Cdk2 β -Actin The relative expression of different protein changes after the treatment of Ciglitizone (5,10, and 20uM) in A2780 cisplatin resistant ovarian cancer cells. After 24 hour s treatment cell extracts were prepared and subjected to immunoblotting for various proteins as mentioned in fig. β-actin was used as an internal loading control.

58 Ciglitizone µm Glut 1 + CDDP 100 µm/l GAPDH Reverse transcription-polymerase chain reaction analysis of dose dependent effect of ciglitizone (5, 10 and 20 um) on RNA level of Glut1 in A2780 cisplatin resistant ovarian cancer cell line. Cells were treated with ciglitizone for 24 hours as an indicated doses. Expression levels presented were normalized to GAPDH.

59 A2780 & A2780 cp cell Cisplatin 48hrs. A2780 A2780 cp C c (μm ) Nrf2 130kd

60 Nrf2 IF Control ISL 10 µm ISL 20 µm ISL 50 µm

61 Conclusions Successful cancer therapy will depend on the ability to discern the subtle differences between the tumor and normal stem cells so tha t approaches can be developed to eliminate the tumor stem cells wi thout excessive toxicity to normal stem cells alterations in cancer cell metabolism are intricately linked to the principal hallmarks of cancer dual hits are exemplified by the inhibition of HIF (which inhibits angiogenesis), reestablishment of p53 function (which restores apoptosis and senescence), or suppression of the PI3K/Akt/mTOR pathway (which inhibits growth), three interventions that should also normalize metabolic functions

62 Thank you for your attention