Abstract
Small cell lung cancer (SCLC) is a highly malignant disease with no current satisfactory treatments.
Identification of new therapeutic targets and treatment strategies are therefore in great demand for the improvement or replacement of current available treatment regimes. Molecular mechanisms altered in SCLC include loss of tumor suppressor genes (TSGs), making restoration of normal TSG function a potential therapeutic strategy. One of the TSGs most frequently inactivated in SCLC is the transcription factor p53 (>90%). The majority of the mutations found in the p53 gene are in the DNA binding core domain, disrupting specific DNA binding and transactivation of target genes involved in cell cycle arrest and/or apoptosis. In addition, loss-off-heterozygosity (LOH) on chromosome 3p, in particular in the region of 3p[14-25], is also commonly seen in SCLC, suggesting that the 3p arm carries several TSGs. Indeed, a number of candidate TSGs has been identified in this region, including FHIT and FUS1.
Loss or reduced protein expression of both FUS1 and FHIT is seen in the majority of SCLC, a feature compatible with TSG function. Therefore, restoration of FUS1, FHIT and p53 function might serve as potential therapeutic strategies for SCLC. However, as mutant p53 proteins tend to
accumulate in SCLC cells, reintroduction of wild-type p53 may not be effective due to dominant-negative effects of the mutant protein. Therefore, a more effective approach would be to reactivate the endogenous mutant p53 proteins in the cells using small molecules such as PRIMA-1 Met, which has been shown to reactivate mutant p53 in various cancer cells.
The aim of this PhD project was to investigate the effect of FUS1, FHIT and PRIMA-1 Met alone or in combination in SCLC cells to see if this could influence their cancer-associated phenotypes.
PRIMA-1 Met was found to efficiently inhibit growth of SCLC cell lines expressing mutant p53 and to induce apoptosis in the cells. The cytotoxic effect of PRIMA-1 Met in the SCLC cells was dependent on mutant p53, as efficient knock-down of mutant p53 by siRNA reduced the effect of PRIMA-1 Met
markedly. PRIMA-1 Met also induced significant tumor growth delay in human SCLC mouse models without any signs of toxicity. These results suggest that PRIMA-1 Met can reactivate mutant p53 in SCLC cells, leading to the induction of apoptosis and tumor growth delay.
To study the growth inhibitory effect of FUS1 in SCLC cell lines, cells lacking FUS1 protein expression were transiently transfected with a FUS1 expression vector and the effect of exogenous FUS1 expression on SCLC cell growth and apoptosis was examined. Ectopic expression of FUS1
inhibited growth of the SCLC cell lines by inducing apoptosis as compared to vector or mutant-inactive FUS1 controls. These results demonstrate that FUS1 has a tumor suppressive function in SCLC and suggest that FUS1-mediated gene therapy may be explored as a therapeutic strategy for the treatment of SCLC.
The effect of FHIT gene overexpression was studied by adenoviral-mediated gene transfer in SCLC cells expressing various levels of endogenous FHIT protein. FHIT overexpression led to growth inhibition in all of the SCLC cell lines studied; although more effectively in cell lines with high levels of endogenous FHIT protein and transduction efficiency. FHIT-induced inhibition of cell growth seemed to be associated with activation of the apoptotic pathway. Furthermore, combination of FHIT with PRIMA-1 Met resulted in a synergistic cell growth inhibition at certain dose ranges in SCLC cell lines. These results suggest that overexpression of FHIT by adenoviral vector-mediated gene transfer can effectively suppress SCLC cell growth and may therefore be used as a potential
therapeutic gene either alone or in combination with p53-reactivating molecules for the treatment of SCLC.
Identification of new therapeutic targets and treatment strategies are therefore in great demand for the improvement or replacement of current available treatment regimes. Molecular mechanisms altered in SCLC include loss of tumor suppressor genes (TSGs), making restoration of normal TSG function a potential therapeutic strategy. One of the TSGs most frequently inactivated in SCLC is the transcription factor p53 (>90%). The majority of the mutations found in the p53 gene are in the DNA binding core domain, disrupting specific DNA binding and transactivation of target genes involved in cell cycle arrest and/or apoptosis. In addition, loss-off-heterozygosity (LOH) on chromosome 3p, in particular in the region of 3p[14-25], is also commonly seen in SCLC, suggesting that the 3p arm carries several TSGs. Indeed, a number of candidate TSGs has been identified in this region, including FHIT and FUS1.
Loss or reduced protein expression of both FUS1 and FHIT is seen in the majority of SCLC, a feature compatible with TSG function. Therefore, restoration of FUS1, FHIT and p53 function might serve as potential therapeutic strategies for SCLC. However, as mutant p53 proteins tend to
accumulate in SCLC cells, reintroduction of wild-type p53 may not be effective due to dominant-negative effects of the mutant protein. Therefore, a more effective approach would be to reactivate the endogenous mutant p53 proteins in the cells using small molecules such as PRIMA-1 Met, which has been shown to reactivate mutant p53 in various cancer cells.
The aim of this PhD project was to investigate the effect of FUS1, FHIT and PRIMA-1 Met alone or in combination in SCLC cells to see if this could influence their cancer-associated phenotypes.
PRIMA-1 Met was found to efficiently inhibit growth of SCLC cell lines expressing mutant p53 and to induce apoptosis in the cells. The cytotoxic effect of PRIMA-1 Met in the SCLC cells was dependent on mutant p53, as efficient knock-down of mutant p53 by siRNA reduced the effect of PRIMA-1 Met
markedly. PRIMA-1 Met also induced significant tumor growth delay in human SCLC mouse models without any signs of toxicity. These results suggest that PRIMA-1 Met can reactivate mutant p53 in SCLC cells, leading to the induction of apoptosis and tumor growth delay.
To study the growth inhibitory effect of FUS1 in SCLC cell lines, cells lacking FUS1 protein expression were transiently transfected with a FUS1 expression vector and the effect of exogenous FUS1 expression on SCLC cell growth and apoptosis was examined. Ectopic expression of FUS1
inhibited growth of the SCLC cell lines by inducing apoptosis as compared to vector or mutant-inactive FUS1 controls. These results demonstrate that FUS1 has a tumor suppressive function in SCLC and suggest that FUS1-mediated gene therapy may be explored as a therapeutic strategy for the treatment of SCLC.
The effect of FHIT gene overexpression was studied by adenoviral-mediated gene transfer in SCLC cells expressing various levels of endogenous FHIT protein. FHIT overexpression led to growth inhibition in all of the SCLC cell lines studied; although more effectively in cell lines with high levels of endogenous FHIT protein and transduction efficiency. FHIT-induced inhibition of cell growth seemed to be associated with activation of the apoptotic pathway. Furthermore, combination of FHIT with PRIMA-1 Met resulted in a synergistic cell growth inhibition at certain dose ranges in SCLC cell lines. These results suggest that overexpression of FHIT by adenoviral vector-mediated gene transfer can effectively suppress SCLC cell growth and may therefore be used as a potential
therapeutic gene either alone or in combination with p53-reactivating molecules for the treatment of SCLC.
| Originalsprog | Engelsk |
|---|
| Udgivelsessted | København |
|---|---|
| Antal sider | 136 |
| Status | Udgivet - jun. 2011 |