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Djerf, Emelie
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Publications (7 of 7) Show all publications
Severinsson, E. (2012). Experimental studies on ErbB targeted therapy in malignant melanoma. (Doctoral dissertation). Linköping: Linköping University Electronic Press
Open this publication in new window or tab >>Experimental studies on ErbB targeted therapy in malignant melanoma
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Malignant melanoma has one of the fastest increasing incidences among the different types of cancer in the Western world. This raise can partly be ascribed to the change in sun habits that has taken place during the last decades, since the major external risk factor for melanoma is exposure to ultraviolet radiation. In the case of patients with early stages of melanoma, the prognosis is usually good and the disease may be cured by surgery alone. However, with conventional anti-cancer treatments, patients diagnosed with unresectable or metastatic melanoma have a very low 5-year survival rate ranging from less than 10 percent to about 20 percent, depending on the location and extent of metastatic spread. Despite the development of novel promising targeted drugs, such as the immunomodulating antibody ipilimumab and the B-raf inhibitor vemurafenib, that have been shown to significantly extend patient survival, there is still an urgent need for new and improved treatment strategies which can further increase the survival of patients with advanced malignant melanoma.

The aim of this thesis was to investigate the anti-tumor effect of two different tyrosine kinase inhibitors (TKIs), gefitinib and canertinib, on human malignant melanoma cell lines with wild-type BRAF and NRAS. We investigated the effect of these two drugs on cell proliferation, survival and on the ErbB1-4 receptor phosphorylation, as well as the downstream signaling molecules Akt, Erk1/2 and Stat3. We also established a melanoma cell line resistant to gefitinib treatment and studied the resistance mechanisms developed by the cells.

The aim of this thesis was to investigate the anti-tumor effect of two different tyrosine kinase inhibitors (TKIs), gefitinib and canertinib, on human malignant melanoma cell lines with wild-type BRAF and NRAS. We investigated the effect of these two drugs on cell proliferation, survival and on the ErbB1-4 receptor phosphorylation, as well as the downstream signaling molecules Akt, Erk1/2 and Stat3. We also established a melanoma cell line resistant to gefitinib treatment and studied the resistance mechanisms developed by the cells.

In conclusion, gefitinib and canertinib display promising anti-tumor effects on ErbB-expressing malignant melanoma and might be used in future studies in combination with conventional chemotherapy or other targeted therapies in the treatment of malignant melanoma patients not harboring BRAF or NRAS mutations.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. p. 104
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1336
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-85665 (URN)978-91-7519-775-3 (ISBN)
Public defence
2012-12-13, Eken, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2012-11-27 Created: 2012-11-27 Last updated: 2013-03-11Bibliographically approved
Djerf Svenningsson, E., Olausson, P., Ghafouri, B., Stål, O., Hallbeck, A.-L. & Walz, T. (2012). Resistance to gefitinib in malignant melanoma cells is related to increased expression of Met and the insulin receptor and sustained Akt signaling.
Open this publication in new window or tab >>Resistance to gefitinib in malignant melanoma cells is related to increased expression of Met and the insulin receptor and sustained Akt signaling
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2012 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Acquired resistance to cancer therapy, including targeted therapies such as epidermal growth factor receptor (ErbB) tyrosine kinase inhibitors (TKIs), constitutes a major clinical problem in treating patients with malignant disease. Several drug resistance mechanisms for ErbB1 TKIs involving abnormal activation of growth factor receptors or activation of intracellular signaling pathways have been discovered. ErbB TKIs have recently been shown to inhibit growth in melanoma cells. This study was undertaken to develop a gefitinib-resistant melanoma cell line in order to find any resistance mechanism to gefitinib in melanoma cells lacking activating mutation in BRAF or NRAS.

Material and methods: A malignant melanoma cell line (RaH5) was made resistant to the ErbB1 TKI gefitinib by continuous culture with stepwise increasing concentrations of the drug up to 10 μM. The phosphorylation status of 42 different human receptor tyrosine kinases was screened in a protein array in resistant (RaH5ZDR) and wild-type RaH5 cells treated with or without gefitinib. The PI3K, MAPK and Stat3 signaling pathways were studied in an analogous way by Western blot analysis; 2-D gel electrophoresis was performed to determine other potential proteins involved in gefitinib resistance in RaH5 cells. In addition, the effect of the pan-ErbB TKI canertinib on gefitinib-resistant cells was investigated.

Results: Protein array experiments showed that only Met and the insulin receptor (IR) exhibited substantially increased activation in RaH5ZDR cells as compared to their nonresistant counterparts. Interestingly, following gefitinib treatment ErbB2 and ErbB3 receptor signaling in resistant cells were equally well suppressed as in non-resistant cells. However, downstream Akt and Erk1/2 phosphorylation was inhibited to a greater extent in non-resistant RaH5 cells.

Conclusion: Resistance to gefitinib in RaH5 cells appears to be related to an increased expression of Met and IR and linked to a more persistent signaling through Akt and Erk1/2. However, additional studies are required to further elucidate the resistance to gefitinib in our experimental system.

Keywords
Malignant melanoma, gefitinib, canertinib, gefitinib resistance, ErbB, Erk1/2, Akt, Stat3, Met, IR
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-85664 (URN)
Available from: 2012-11-27 Created: 2012-11-27 Last updated: 2012-11-27Bibliographically approved
Djerf, E., Trinks, C., Green, H., Abdiu, A., Hallbeck, A.-L., Stål, O. & Walz, T. (2011). The pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo. Biochemical and Biophysical Research Communications - BBRC, 414(3), 563-568
Open this publication in new window or tab >>The pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo
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2011 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 414, no 3, p. 563-568Article in journal (Refereed) Published
Abstract [en]

The ErbB receptor family has been suggested to constitute a therapeutic target for tumor-specific treatment of malignant melanoma. Here we investigate the effect of the pan-ErbB tyrosine kinase inhibitor canertinib on cell growth and survival in human melanoma cells in vitro and in vivo. Canertinib significantly inhibited growth of cultured melanoma cells, RaH3 and RaH5, in a dose-dependent manner as determined by cell counting. Half-maximum growth inhibitory dose (IC(50)) was approximately 0.8 mu M and by 5 mu M both cell lines were completely growth-arrested within 72 h of treatment. Incubation of exponentially growing RaH3 and RaH5 with 1 mu M canertinib accumulated the cells in the G(1)-phase of the cell cycle within 24 h of treatment without induction of apoptosis as determined by flow cytometry. Immunoblot analysis showed that 1 mu M canertinib inhibited ErbB1-3 receptor phosphorylation with a concomitant decrease of Akt-, Erk1/2- and Stat3 activity in both cell lines. In contrast to the cytostatic effect observed at doses less than= 5 mu M canertinib, higher concentrations induced apoptosis as demonstrated by the Annexin V method and Western blot analysis of PARP cleavage. Furthermore, canertinib significantly inhibited growth of RaH3 and RaH5 melanoma xenografts in nude mice. Pharmacological targeting of the ErbB receptors may prove successful in the treatment of patients with metastatic melanoma.

Place, publisher, year, edition, pages
Elsevier, 2011
Keywords
Malignant melanoma; Tyrosine kinase inhibitor; Canertinib; ErbB-receptor; Apoptosis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-73740 (URN)10.1016/j.bbrc.2011.09.118 (DOI)000298519500021 ()
Note

On the day of the defence date the status of this article was Manuscript and the title "The pan-ErbB receptor tyrosine kinase inhibitor Canertinib (CI-1033)promotes cell cycle arrest and apoptosis of human malignantmelanoma in vitro".

Available from: 2012-01-12 Created: 2012-01-12 Last updated: 2017-12-08
Trinks, C., Severinsson, E. A., Holmlund, B., Gréen, A., Green, H., Jönsson, J.-I., . . . Walz, T. (2011). The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells. Biochemical and Biophysical Research Communications - BBRC, 410(3), 422-427
Open this publication in new window or tab >>The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells
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2011 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 410, no 3, p. 422-427Article in journal (Refereed) Published
Abstract [en]

Canertinib is a novel ErbB-receptor inhibitor currently in clinical development for the treatment of solid tumors overexpressing ErbB-receptors. We have recently demonstrated that canertinib displays anti-proliferative and pro-apoptotic effects in human myeloid leukemia cells devoid of ErbB-receptors. The mechanism mediating these effects are however unknown. In this study, we show that canertinib is able to act as a multi-kinase inhibitor by inhibition of several intracellular kinases involved in T-cell signaling such as Akt, Erk1/2 and Zap-70, and reduced Lck protein expression in the human T-cell leukemia cell line Jurkat. Treatment with canertinib at a concentration of 2 mu M caused accumulation of Jurkat cells in the G(1) cell cycle phase and increased doses induced apoptosis in a time-dependent manner. Apoptotic signs of treated cells were detected by Annexin V staining and cleavage of PARP, caspase-3, -8, -9, -10 and Bid. A subset of the pro-apoptotic signals mediated by canertinib could be significantly reduced by specific caspase inhibitors. Taken together, these results demonstrate the dual ability of canertinib to downregulate important signaling pathways and to activate caspase-mediated intrinsic apoptosis pathway in human T-cell leukemia cells.

Place, publisher, year, edition, pages
Elsevier Science B.V., Amsterdam, 2011
Keywords
T-cell leukemia; Canertinib; ErbB-receptor; Apoptosis; Caspase; Intracellular signaling
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-69797 (URN)10.1016/j.bbrc.2011.05.148 (DOI)000292797700009 ()
Available from: 2011-08-10 Created: 2011-08-08 Last updated: 2017-12-08
Trinks, C., Djerf, E., Hallbeck, A.-L., Jönsson, J.-I. & Walz, T. (2010). The pan-ErbB receptor tyrosine kinase inhibitor canertinib induces ErbB-independent apoptosis in human leukemia (HL-60 and U-937) cells. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 393(1), 6-10
Open this publication in new window or tab >>The pan-ErbB receptor tyrosine kinase inhibitor canertinib induces ErbB-independent apoptosis in human leukemia (HL-60 and U-937) cells
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2010 (English)In: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ISSN 0006-291X, Vol. 393, no 1, p. 6-10Article in journal (Refereed) Published
Abstract [en]

Epidermal growth factor (EGF) receptor tyrosine kinase inhibitors have recently been shown to display anti-neoplastic effects in human malignant myeloid cells. Our study was initiated in order to determine the effect of the pan-ErbB receptor tyrosine kinase inhibitor, canertinib (CI-1033), on growth and survival of human leukemia (HL-60 and U-937) cells. We show that treatment of HL-60 and U-937 cells with canertinib significantly inhibits growth of both cell lines in a dose-dependent manner; half maximal effective dose (IC50) in HL-60 and U-937 cells was approximately 2.5 mu M and 1.0 mu M, respectively. Treatment with 2 mu M canertinib promoted a G(1) cell cycle arrest, whereas doses of 5 mu M or more induced apoptosis as determined by the Annexin V method and cleavage of poly-(ADP-ribose) polymerase (PARP). HL-60 and U-937 cells lacked EGF-receptor transcript but expressed ErbB2-4 mRNA as determined by RTPCR. However, none of the corresponding ErbB-receptor proteins could be detected by Western blot analysis. We conclude that canertinib induces apoptosis in HL-60 and U-937 cells devoid of functional ErbB1-4 receptors. Our results suggest that canertinib could be of potential clinical interest in the treatment of acute myeloid leukemia.

Keywords
Leukemia, Tyrosine kinase inhibitor, CI-1033, ErbB-receptor, Growth inhibition, Apoptosis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-54619 (URN)10.1016/j.bbrc.2010.01.055 (DOI)000275371300002 ()
Available from: 2010-03-26 Created: 2010-03-26 Last updated: 2012-10-30
Djerf, E., Trinks, C., Abdiu, A., Thunell, L., Hallbeck, A.-L. & Walz, T. M. (2009). ErbB receptor tyrosine kinases contribute to proliferation of malignant melanoma cells: inhibition by gefitinib (ZD1839). Melanoma research, 19(3), 156-166
Open this publication in new window or tab >>ErbB receptor tyrosine kinases contribute to proliferation of malignant melanoma cells: inhibition by gefitinib (ZD1839)
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2009 (English)In: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, ISSN 0960-8931, Vol. 19, no 3, p. 156-166Article in journal (Refereed) Published
Abstract [en]

Members of the epidermal growth factor (EGF) family of structurally related tyrosine kinase receptors, known as the ErbB receptors (EGFR/ErbB1/HER1, ErbB2/HER2/neu, ErbB3/HER3 and ErbB4/HER4) and their respective ligands, have been suggested to be involved in the development and progression of malignant melanoma. Here we investigate the effects of the ErbB1 tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) on human malignant melanoma cells (RaH3 and RaH5) in vitro. ZD1839 inhibited proliferation of exponentially growing RaH3 and RaH5 cells in a dose-dependent manner with a half-maximally effective dose of 3.5 and 2.0 mu mol/l, respectively. Cell growth was inhibited at 0.1 mu mol/l ZD1839 in both cell lines. Maximal inhibition was accomplished at 10 mu mol/l ZD1839; however, the effect was not complete as both cell lines showed a continuous slow growth during the treatment period. Flow cytometry analysis of cell-cycle distribution showed that ZD1839 treatment caused accumulation of RaH3 and RaH5 cells in the G, phase. The growth arrest induced by ZD1839 coincided with upregulation of the cyclin-dependent kinase inhibitor p27(KIP1). There was no increase in apoptosis as determined by analysis of plasma phosphatidyl serine redistribution. Western blot analysis revealed that ZD1839 substantially reduced tyrosine phosphorylation of ErbB1 as well as ErbB2 and ErbB3. This was accompanied by a concomitant decrease in Akt-phosphorylation, Erk1/2-phosphorylation, and Stat3-phosphorylation. Our results show that ZD1839 interferes with the growth of human malignant melanoma cells by cytostatic effects. These findings indicate the possible use of ErbB receptor kinase inhibitors as a novel treatment strategy in malignant melanoma.

Keywords
antiproliferative, ErbB receptors, gefitinib, melanoma, tyrosine kinase inhibitor
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-19129 (URN)10.1097/CMR.0b013e32832c6339 (DOI)
Available from: 2009-06-12 Created: 2009-06-12 Last updated: 2017-12-13Bibliographically approved
Djerf, E. (2009). Studies on the effect of ErbB tyrosine kinase inhibitors on malignant melanoma growth and survival in vitro. (Licentiate dissertation). Linköping: Linköping University Electronic Press
Open this publication in new window or tab >>Studies on the effect of ErbB tyrosine kinase inhibitors on malignant melanoma growth and survival in vitro
2009 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Malignant melanoma has one of the fastest increasing incidences among the different types of cancerin the Western world. This raise can partly be ascribed to the change in sun habits that has takenplace during the last decades, since the major external risk factor for melanoma is exposure toultraviolet radiation. Patients with early stages of melanoma can often be cured by surgery, howeverfor patients suffering from metastatic melanoma there are only a few treatment options available.Unfortunately malignant melanoma is often resistant to radio-, bio- and chemotherapy and treatmentwith the currently most frequently used agent, dacarbazine, is characterized by a very low clinicalresponse rate. Therefore, there is an urgent need for new treatment strategies which can increase theoverall survival and cause less severe side effects.

The aim of this thesis was to investigate the anti-tumor effect of two different tyrosine kinaseinhibitors (TKIs), gefitinib and canertinib, on two different human malignant melanoma (RaH3 andRaH5) cell lines. We investigate the effect of these two drugs on cell proliferation and survival andstudied the effect of gefitinib and canertinib on ErbB1-4 receptor phosphorylation, as well as Akt,Erk1/2 and Stat3 activity.

Our results showed that phosphorylation of ErbB1, ErbB2 and ErbB3 decreased followingtreatment with both gefitinib and canertinib and that the subsequent downstream signaling via Akt,Erk1/2 and Stat3 was inhibited after TKI treatment. However, it was noted that the gefitinibinducedinhibition of Akt, and particularly Erk1/2, was transient and only a weak inhibition of Stat3phosphorylation was seen. Gefitinib treatment of the RaH3 and RaH5 cells resulted in anaccumulation of the cells in the G1 phase of the cell cycle without any induction of apoptosis.Canertinib caused a more pronounced inhibition of Akt, Erk1/2, and Stat3 phosphorylation thangefitinib. This might be one explanation to why canertinib induced apoptosis in RaH3 and RaH5cells whereas gefitinib only caused cell cycle arrest. In conclusion, gefitinib and canertinib displaypromising anti-tumor effects on ErbB expressing malignant melanoma and might be used in futurestudies in combination with conventional chemotherapy or other targeted therapies in the treatmentof malignant melanoma.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. p. 56
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 100
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-19949 (URN)978-91-7393-610-1 (ISBN)
Presentation
2009-08-28, Karl-Johan salen, Onkologen, Campus US, Linköpings universitet, Linköping, 10:00 (Swedish)
Opponent
Supervisors
Available from: 2009-09-21 Created: 2009-08-19 Last updated: 2012-10-30Bibliographically approved
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