Final Report Summary - CISTAX (The inverse-resistance relationship between platinum and taxane chemotherapy)
Lay Description of Completed Project – CISTAX – Dr. Britta Stordal.
People who have cancer are given medicine by their doctor to try and kill the cancer. I grow cancer cells in the laboratory and give them the same medicines given to people to understand how they work to kill the cancer. In the future if research like my project goes well, there will be many tests doctors can use to pick the best medicine for each person. We will be able to predict if a particular medicine will cure your cancer. This will be different for each person and it will mean people won’t have to take medicine that won’t work for them. This will mean a lot more people will live longer after being treated for cancer.
Many people who have been treated for cancer experience a relapse, the cancer has returned. I have been examining cancer cells in the laboratory that gave been given chemotherapy treatment similar to what a cancer patient receives. The surviving resistant cells in the laboratory are similar to the cancer cells in a cancer patient who has relapsed. I have identified several genes which may be related to resistance to a medicine called cisplatin which is used in the treatment of ovarian cancer. I have examined these genes in publically available data from lots of ovarian cancer patients as well as in a small study from patients treated at our hospital. The results suggest that these genes may be a promising way to predict if cisplatin will work to treat ovarian cancer in an individual patient. We are now applying for more funding to continue this research project.
Understanding the mechanism of Collateral Sensitivity to Cisplatin in KB-8-5-11
The KB-8-5-11 taxane resistant cell line was developed in the laboratory of Prof. Michael Gottesman at the National Cancer Institute (1, 2). These cells are taxane resistant as they overexpress P-glycoprotein (Figure 1A). However, these cells exhibit collateral sensitivity to cisplatin and the mechanism for this sensitivity is unknown. Understanding the mechanism of collateral sensitivity to cisplatin may lead to biomarkers for platinum sensitivity in patients with cancer. We have published in Anticancer Research a separate study on platinum sensitivity in KB-8-5-11 cells.
A Taqman low-density array was used to characterize the expression of 380 genes previously associated with chemoresistance. Identified pathways were further analysed using cytotoxicity assays, metabolomics and western blots. KB-8-5-11 cells were sensitive to CuSO4 and the glutathione inhibitor buthionine sulphoximine. Expression of ATPase, Cu2+ transporting alpha (ATP7A) (Figure 1B) and ATP7B were decreased at the protein and gene level respectively in KB-8-5-11. KB-8-5-11 had decreased gene expression of glutathione S-transferase pi 1 (GSTP1), GSTA4 and GSTK1. Cisplatin treatment significantly lowered total cellular glutathione in parental KB-3-1 cells. Glutathione also tended to be lower in KB-8-5-11 cells compared to KB-3-1 cells (Figure 1C). KB-8-5-11 cells have alterations in their copper transporters and glutathione metabolism, which contribute to their cisplatin-sensitive phenotype.
Examining the P-glycoprotein substrate status of Parp Inhibitors
The BRCA1 and BRCA2 proteins play a critical role in DNA damage repair and deleterious mutations in these genes increase the risk of developing breast and ovarian cancer. Synthetic lethality is the phenomenon whereby cell death results from the loss of function of two different gene products, when loss of either gene product in isolation does not. This concept has led to the idea that a new class of agents, PARP inhibitors, could be effective at treating BRCA1/2 dysfunctional cancers, such as breast and ovarian cancer. For any new chemotherapy agents it is important to establish if they are substrates of the classical ABC transporters, such as P-glycoprotein (P-gp, ABCB1, MDR1). Agents that are not P-gp substrates may be more useful clinically, as if drug resistance develops the cells are unlikely to be resistant to the wide range of chemotherapy drugs that are also P-gp substrates. We examined the PARP inhibitors olaparib, veliparib and CEP-8983 in two cell models of acquired drug resistance where the major mechanism of drug resistance is overexpression of P-glycoprotein, IGROVCDDP and KB-8-5-11. This work has been published in the Journal of Pharmaceutical Sciences.
IGROVCDDP and KB-8-5-11 were both resistant to olaparib and resistance was reversible with the P-glycoprotein inhibitors elacridar, zosuquidar and valspodar (Figures 2A and B). In contrast, the P-glycoprotein overexpressing models were not resistant to veliparib or CEP-8983. Olaparib and veliparib did not induce protein expression of P-glycoprotein in IGROVCDDP or KB-8-5-11 at doses which successfully inhibit PARP (Figures 2C and D). Olaparib therefore appears to be a P-glycoprotein substrate. Veliparib and CEP-8983 do not appear to be substrates. Veliparib and CEP-8983 may therefore be more useful in combined chemotherapy regimens with P-glycoprotein substrates and may be active in platinum and taxane-resistant ovarian cancer.
Validation of identified molecular markers in cell line panel and clinical ovarian cancer samples.
Both the IGROVCDDP and KB-8-5-11 cells are taxane resistant and have overexpression of the drug pump P-glycoprotein as their dominant mechanism of taxane resistance. The IGROVCDDP and KB-8-5-11 cells differ in their platinum resistance status. By looking for genes that change in opposite directions in the two cell line pairs we can identify potential biomarkers of cisplatin sensitivity in taxane resistant cells. The Affymetrix whole genome array revealed 11 gene changes that go in opposite directions and are therefore potential biomarkers of platinum resistance.
These 11 potential biomarkers were then screened using publically available Ovarian Cancer Data in collaboration with Dr. Stephen Madden at Dublin City University. 6 of the 11 identified biomarkers in above were positively validated in the OvMark dataset. An increase in expression associated with platinum resistance in the cell lines. We also performed a pilot study on samples from our ovarian cancer biobank. 11 formalin fixed paraffin embedded (FFPE) blocks from patients with serous papillary adenocarcinoma of the ovary were selected from the archives of the histology department of St James Hospital. While significant results were not obtained in this small pilot study, 4 out of the 6 biomarkers showed a fold change consistent, with the cell lines and Ovmark data. These results will form pilot data for future grant applications.
Development of new models of drug resistance in ovarian carcinoma, investigating the role of BRCA1 mutation in the inverse-resistance relationship between cisplatin and paclitaxel.
The development of resistant models has been completed and my PhD student is currently investigating mechanisms of drug resistance to platinum and taxane chemotherapy to complete his PhD as well as publish a characterisation study of the drug-resistant models.
Figure 3 shows the development of drug resistance to carboplatin and taxol in the UPN251 and OVCAR8 cell lines. OVCAR8 did not develop stable drug resistance and will not be used for further studies (Figure 3B and D). This lack of development of resistance may be due to the BRCA1 methylation in the cell line or lower intrinsic resistance in the cell lines. Higher levels of resistance was developed to taxol compared to carboplatin, this may be due to the cells recovering more easily from taxol treatment or that the taxol resistance developed was due to overexpression of the drug pump P-glycoprotein.
We hypothesised that alternating treatments of chemotherapy would slow down the development of drug resistance. Figure 4A and B shows the fold resistance achieved to carboplatin and taxol respectively after 6 treatment cycles. The R6-U-T and R6-U-C cells treated with the single agent show a higher level of resistance than the cell lines which received alternating therapy. However, it appears that we have delayed rather than prevented resistance occurring. If the sublines treated with a single-agent are compared at treatment 3 with the alternating sublines which have received 3 treatments of the given agent the levels of fold resistance are similar (Figure 4C and D).
People who have cancer are given medicine by their doctor to try and kill the cancer. I grow cancer cells in the laboratory and give them the same medicines given to people to understand how they work to kill the cancer. In the future if research like my project goes well, there will be many tests doctors can use to pick the best medicine for each person. We will be able to predict if a particular medicine will cure your cancer. This will be different for each person and it will mean people won’t have to take medicine that won’t work for them. This will mean a lot more people will live longer after being treated for cancer.
Many people who have been treated for cancer experience a relapse, the cancer has returned. I have been examining cancer cells in the laboratory that gave been given chemotherapy treatment similar to what a cancer patient receives. The surviving resistant cells in the laboratory are similar to the cancer cells in a cancer patient who has relapsed. I have identified several genes which may be related to resistance to a medicine called cisplatin which is used in the treatment of ovarian cancer. I have examined these genes in publically available data from lots of ovarian cancer patients as well as in a small study from patients treated at our hospital. The results suggest that these genes may be a promising way to predict if cisplatin will work to treat ovarian cancer in an individual patient. We are now applying for more funding to continue this research project.
Understanding the mechanism of Collateral Sensitivity to Cisplatin in KB-8-5-11
The KB-8-5-11 taxane resistant cell line was developed in the laboratory of Prof. Michael Gottesman at the National Cancer Institute (1, 2). These cells are taxane resistant as they overexpress P-glycoprotein (Figure 1A). However, these cells exhibit collateral sensitivity to cisplatin and the mechanism for this sensitivity is unknown. Understanding the mechanism of collateral sensitivity to cisplatin may lead to biomarkers for platinum sensitivity in patients with cancer. We have published in Anticancer Research a separate study on platinum sensitivity in KB-8-5-11 cells.
A Taqman low-density array was used to characterize the expression of 380 genes previously associated with chemoresistance. Identified pathways were further analysed using cytotoxicity assays, metabolomics and western blots. KB-8-5-11 cells were sensitive to CuSO4 and the glutathione inhibitor buthionine sulphoximine. Expression of ATPase, Cu2+ transporting alpha (ATP7A) (Figure 1B) and ATP7B were decreased at the protein and gene level respectively in KB-8-5-11. KB-8-5-11 had decreased gene expression of glutathione S-transferase pi 1 (GSTP1), GSTA4 and GSTK1. Cisplatin treatment significantly lowered total cellular glutathione in parental KB-3-1 cells. Glutathione also tended to be lower in KB-8-5-11 cells compared to KB-3-1 cells (Figure 1C). KB-8-5-11 cells have alterations in their copper transporters and glutathione metabolism, which contribute to their cisplatin-sensitive phenotype.
Examining the P-glycoprotein substrate status of Parp Inhibitors
The BRCA1 and BRCA2 proteins play a critical role in DNA damage repair and deleterious mutations in these genes increase the risk of developing breast and ovarian cancer. Synthetic lethality is the phenomenon whereby cell death results from the loss of function of two different gene products, when loss of either gene product in isolation does not. This concept has led to the idea that a new class of agents, PARP inhibitors, could be effective at treating BRCA1/2 dysfunctional cancers, such as breast and ovarian cancer. For any new chemotherapy agents it is important to establish if they are substrates of the classical ABC transporters, such as P-glycoprotein (P-gp, ABCB1, MDR1). Agents that are not P-gp substrates may be more useful clinically, as if drug resistance develops the cells are unlikely to be resistant to the wide range of chemotherapy drugs that are also P-gp substrates. We examined the PARP inhibitors olaparib, veliparib and CEP-8983 in two cell models of acquired drug resistance where the major mechanism of drug resistance is overexpression of P-glycoprotein, IGROVCDDP and KB-8-5-11. This work has been published in the Journal of Pharmaceutical Sciences.
IGROVCDDP and KB-8-5-11 were both resistant to olaparib and resistance was reversible with the P-glycoprotein inhibitors elacridar, zosuquidar and valspodar (Figures 2A and B). In contrast, the P-glycoprotein overexpressing models were not resistant to veliparib or CEP-8983. Olaparib and veliparib did not induce protein expression of P-glycoprotein in IGROVCDDP or KB-8-5-11 at doses which successfully inhibit PARP (Figures 2C and D). Olaparib therefore appears to be a P-glycoprotein substrate. Veliparib and CEP-8983 do not appear to be substrates. Veliparib and CEP-8983 may therefore be more useful in combined chemotherapy regimens with P-glycoprotein substrates and may be active in platinum and taxane-resistant ovarian cancer.
Validation of identified molecular markers in cell line panel and clinical ovarian cancer samples.
Both the IGROVCDDP and KB-8-5-11 cells are taxane resistant and have overexpression of the drug pump P-glycoprotein as their dominant mechanism of taxane resistance. The IGROVCDDP and KB-8-5-11 cells differ in their platinum resistance status. By looking for genes that change in opposite directions in the two cell line pairs we can identify potential biomarkers of cisplatin sensitivity in taxane resistant cells. The Affymetrix whole genome array revealed 11 gene changes that go in opposite directions and are therefore potential biomarkers of platinum resistance.
These 11 potential biomarkers were then screened using publically available Ovarian Cancer Data in collaboration with Dr. Stephen Madden at Dublin City University. 6 of the 11 identified biomarkers in above were positively validated in the OvMark dataset. An increase in expression associated with platinum resistance in the cell lines. We also performed a pilot study on samples from our ovarian cancer biobank. 11 formalin fixed paraffin embedded (FFPE) blocks from patients with serous papillary adenocarcinoma of the ovary were selected from the archives of the histology department of St James Hospital. While significant results were not obtained in this small pilot study, 4 out of the 6 biomarkers showed a fold change consistent, with the cell lines and Ovmark data. These results will form pilot data for future grant applications.
Development of new models of drug resistance in ovarian carcinoma, investigating the role of BRCA1 mutation in the inverse-resistance relationship between cisplatin and paclitaxel.
The development of resistant models has been completed and my PhD student is currently investigating mechanisms of drug resistance to platinum and taxane chemotherapy to complete his PhD as well as publish a characterisation study of the drug-resistant models.
Figure 3 shows the development of drug resistance to carboplatin and taxol in the UPN251 and OVCAR8 cell lines. OVCAR8 did not develop stable drug resistance and will not be used for further studies (Figure 3B and D). This lack of development of resistance may be due to the BRCA1 methylation in the cell line or lower intrinsic resistance in the cell lines. Higher levels of resistance was developed to taxol compared to carboplatin, this may be due to the cells recovering more easily from taxol treatment or that the taxol resistance developed was due to overexpression of the drug pump P-glycoprotein.
We hypothesised that alternating treatments of chemotherapy would slow down the development of drug resistance. Figure 4A and B shows the fold resistance achieved to carboplatin and taxol respectively after 6 treatment cycles. The R6-U-T and R6-U-C cells treated with the single agent show a higher level of resistance than the cell lines which received alternating therapy. However, it appears that we have delayed rather than prevented resistance occurring. If the sublines treated with a single-agent are compared at treatment 3 with the alternating sublines which have received 3 treatments of the given agent the levels of fold resistance are similar (Figure 4C and D).