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The role of the TGFβ signaling pathway in clinically non-functioning pituitary adenoma and the potential role of inhibitors of this pathway as a new target in pituitary tumor therapy
The role of the TGFβ signaling pathway in clinically non-functioning pituitary adenoma and the potential role of inhibitors of this pathway as a new target in pituitary tumor therapy
The parenchyme of the anterior pituitary gland consists of various distinct endocrine cells which second to the hypothalamic -releasing hormones regulates all other endocrine organs with few exceptions. About 10-15% of all intracranial tumors derive from one of those endocrine cells, leading to symptoms mainly due to the disruption of the hypothalamic-hypophysial-endocrine axis. Nonfunctioning pituitary adenomas (NFPAs) account for about 30 to 40 % of all pituitary tumors and the majority of them originate from gonadotroph pituitary cells. NFPAs typically become symptomatic due to mass effects, the endocrine function of the adenohypophysis remains intact as long as the functioning hormone-secreting cells are still not compromised due to tumor volume. Surgical removal is the first line of therapy, if no contraindications against surgery exist. However, as they start to invade neighboring extrasellar structures, complete resection is often challenging if not impossible for the surgeon. Repeated surgeries neoadjuvant and adjuvant radiotherapy and sometimes chemotherapy are often necessary to treat recurrent NFPA. Consequently, although they are rarely aggressive, they lead to a significant morbidity for the patient. Therefore, alternate pharmacological treatment concepts for NFPA, are needed, also to prevent the transformation of these tumors to more aggressive ones. Members of the transforming growth factor β (TGFβ) protein family, among them TGFβ-1 and TGFβ-3 isoforms, play a contrary role in the process of tumor formation. They are well-known as potential promoters of the growth of solid epithelial tumors, though can also exhibit tumor suppressive effects. Initially they block the development of tumors, whereas in advanced stage, in particular after epithelial tumors had undergone epithelial mesenchymal transition (EMT), they stimulate tumor progression. Only recently, inhibitors of the action of TGFβ isoforms have been developed, among them the TGFβ receptor suppressor SB431542, and SIS3, an inhibitor of the TGFβ induced intracellular signaling protein Smad3. In the present thesis I want to study the influence of the TGFβ inhibitors on the growth of hormonally inactive pituitary tumors, using two hormonally inactive murine pituitary tumor cell lines: (1) TtT/GF cells as a model of folliculo-stellate cells, which show supportive regulatory within the pituitary gland and which play a key role in the formation of tumor microenvironment. (2) Gonadotroph-like αT3-1 cells, as the majority of NFPA seem to derive from gonadotropin-producing cells. Stimulating these cells with TGFβ-1/-3, alone or in combination with the inhibitors SIS3 and SB431542 I measured the difference in cell viability using the 3H-thymidine incorporation assay. I compared these results with those obtained with corresponding experiments on TGFβ-signaling in a small series of 6 human NFPA-samples in primary cell culture. I further studied the effect of the respective chosen TGFβ inhibitors on the production of vascular endothelial growth factor (VEGF), a key factor of intratumoral vessel formation and thus tumor expansion, in the two murine cell lines. Due to the limited tissue material available, I did omit from VEGF-experiments in human NFPA cell cultures. Both SB431542 and SIS3 significantly inhibited the proliferation of TtT/GF and αT3-1 cells in a dose dependent manner both in the absence and presence of TGFβ-1/-3. However, even at high concentrations the external stimulation with the TGFβ isoforms, the stimulatory influence on the proliferation of the murine cell lines and primary cell cultures was not as high as expected beforehand. The small growth stimulatory effect of TGFβ- 1 and TGFβ- 3 alone and the clearly visible suppressive action of the inhibitors on the basal growth of the cell lines without external application of TGFβ suggests, that for reasons that are so far not known, the TGFβ signaling system is intrinsically and constitutively activated in rapidly growing TtT/GF and αT3-1 cells. Using northern blot analysis I could show the mRNA-expression of not only the TGFb receptors, but also the subsequent signaling proteins (Smad 2,3 and 4) and the tested TGFb isoforms (TGFb-1,TGFb-3) in both TtT/GF and αT3-1 cells. This suggests that TGFb may be produced by the cell lines and that the activation of this pathway may thus affect growth and function of aT3-1 and TtT/GF cells in an autocrine manner as well. This stresses the potential importance of this signaling pathway in these distinct pituitary cell-types. Either way, the two inhibitors SIS3 and SB431542 seem like potent suppressors of the proliferation of the cell lines. The situation is different in primary cell cultures derived from 6 human NFPAs. Here TGFβ-1 significantly inhibited the proliferation in one tumor cell culture, slightly but not significantly stimulated cell growth in 2 cases, and significantly stimulated cell proliferation in 3 tumor cell cultures. The role of TGFb-3 could be tested only in 2 adenoma cell cultures and in none of them a stimulatory or inhibitory effect on cell proliferation could be detected. In most cases the inhibitors suppressed the proliferation of the adenoma cells already in the absence of TGFb isoforms. In the cell culture of NFPA1, in which TGFb-1 inhibited the cell proliferation, the combined application of TGFb-1 and the inhibitors led to a growth stimulatory effect. SB431542 alone led to a small spurt in tumor growth in the NFPA1-sample, that was potentiated in combination with TGFb-1, whereas SIS3 suppressed cell growth – this effect decreased though at the higher dosage, which corresponded with the results of the combined treatment of SIS3 with TGFb-1. In the remaining tumor cell cultures, in which TGFb-1 had growth-stimulatory effects, the treatment with the inhibitors combined or alone led to a significant reduction of proliferation rate. Although TGFb-3 alone had no effect on cell proliferation, the combined application of TGFb-3 and the inhibitors abolished the growth suppressive effects of the inhibitors. As in the murine tumor cell lines, a suppressive effect of the two inhibitors was also seen in the absence of external TGFβ-1. The results obtained from the limited number of tumors suggest that TGFβ inhibitors may suppress the proliferation and thus the expansion of NFPAs only under certain conditions, probably in advanced stages of tumor development. It would be particularly interesting to compare the patient outcome and clinical behavior of the tumors from which our or new samples derive with these experimental results and to find molecular or clinical markers predicting the response towards TGFβ- isoforms and the individual inhibitors of the corresponding pathway. Regarding VEGF secretion, both TGFβ-1 and -3 stimulated the production of this angiogenic factor in TtT/GF and αT3-1 cells. The tested inhibitors significantly inhibited both TGFβ- promoted and basal VEGF secretion in the cell lines. Although no corresponding studies could be done in human NFPAs due to lack of tissue, SB431542 and SIS3 may probably also suppress the VEGF production in NFPAs and may therefore suppress the development of these tumors by inhibiting tumor vascularization. Taken together, the data suggest that TGFβ inhibitors may slow down or block the progression of NFPAs, in particular those that have passed epithelial mesenchymal - transition (EMT) and have developed a more aggressive phenotype. In any case, this research suggests that with the recently developed specific inhibitors, SIS3 and SB431542 it is possible to reach a further in-depth understanding of the various respective elements involved in the TGFβ signaling cascade and their alterations during tumor progression, leading to possible new targeted therapeutic options. This, however, implies further studies and experimental designs than this small study encompasses.
Not available
Saan, Marie Céline van
2022
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Saan, Marie Céline van (2022): The role of the TGFβ signaling pathway in clinically non-functioning pituitary adenoma and the potential role of inhibitors of this pathway as a new target in pituitary tumor therapy. Dissertation, LMU München: Faculty of Medicine
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Abstract

The parenchyme of the anterior pituitary gland consists of various distinct endocrine cells which second to the hypothalamic -releasing hormones regulates all other endocrine organs with few exceptions. About 10-15% of all intracranial tumors derive from one of those endocrine cells, leading to symptoms mainly due to the disruption of the hypothalamic-hypophysial-endocrine axis. Nonfunctioning pituitary adenomas (NFPAs) account for about 30 to 40 % of all pituitary tumors and the majority of them originate from gonadotroph pituitary cells. NFPAs typically become symptomatic due to mass effects, the endocrine function of the adenohypophysis remains intact as long as the functioning hormone-secreting cells are still not compromised due to tumor volume. Surgical removal is the first line of therapy, if no contraindications against surgery exist. However, as they start to invade neighboring extrasellar structures, complete resection is often challenging if not impossible for the surgeon. Repeated surgeries neoadjuvant and adjuvant radiotherapy and sometimes chemotherapy are often necessary to treat recurrent NFPA. Consequently, although they are rarely aggressive, they lead to a significant morbidity for the patient. Therefore, alternate pharmacological treatment concepts for NFPA, are needed, also to prevent the transformation of these tumors to more aggressive ones. Members of the transforming growth factor β (TGFβ) protein family, among them TGFβ-1 and TGFβ-3 isoforms, play a contrary role in the process of tumor formation. They are well-known as potential promoters of the growth of solid epithelial tumors, though can also exhibit tumor suppressive effects. Initially they block the development of tumors, whereas in advanced stage, in particular after epithelial tumors had undergone epithelial mesenchymal transition (EMT), they stimulate tumor progression. Only recently, inhibitors of the action of TGFβ isoforms have been developed, among them the TGFβ receptor suppressor SB431542, and SIS3, an inhibitor of the TGFβ induced intracellular signaling protein Smad3. In the present thesis I want to study the influence of the TGFβ inhibitors on the growth of hormonally inactive pituitary tumors, using two hormonally inactive murine pituitary tumor cell lines: (1) TtT/GF cells as a model of folliculo-stellate cells, which show supportive regulatory within the pituitary gland and which play a key role in the formation of tumor microenvironment. (2) Gonadotroph-like αT3-1 cells, as the majority of NFPA seem to derive from gonadotropin-producing cells. Stimulating these cells with TGFβ-1/-3, alone or in combination with the inhibitors SIS3 and SB431542 I measured the difference in cell viability using the 3H-thymidine incorporation assay. I compared these results with those obtained with corresponding experiments on TGFβ-signaling in a small series of 6 human NFPA-samples in primary cell culture. I further studied the effect of the respective chosen TGFβ inhibitors on the production of vascular endothelial growth factor (VEGF), a key factor of intratumoral vessel formation and thus tumor expansion, in the two murine cell lines. Due to the limited tissue material available, I did omit from VEGF-experiments in human NFPA cell cultures. Both SB431542 and SIS3 significantly inhibited the proliferation of TtT/GF and αT3-1 cells in a dose dependent manner both in the absence and presence of TGFβ-1/-3. However, even at high concentrations the external stimulation with the TGFβ isoforms, the stimulatory influence on the proliferation of the murine cell lines and primary cell cultures was not as high as expected beforehand. The small growth stimulatory effect of TGFβ- 1 and TGFβ- 3 alone and the clearly visible suppressive action of the inhibitors on the basal growth of the cell lines without external application of TGFβ suggests, that for reasons that are so far not known, the TGFβ signaling system is intrinsically and constitutively activated in rapidly growing TtT/GF and αT3-1 cells. Using northern blot analysis I could show the mRNA-expression of not only the TGFb receptors, but also the subsequent signaling proteins (Smad 2,3 and 4) and the tested TGFb isoforms (TGFb-1,TGFb-3) in both TtT/GF and αT3-1 cells. This suggests that TGFb may be produced by the cell lines and that the activation of this pathway may thus affect growth and function of aT3-1 and TtT/GF cells in an autocrine manner as well. This stresses the potential importance of this signaling pathway in these distinct pituitary cell-types. Either way, the two inhibitors SIS3 and SB431542 seem like potent suppressors of the proliferation of the cell lines. The situation is different in primary cell cultures derived from 6 human NFPAs. Here TGFβ-1 significantly inhibited the proliferation in one tumor cell culture, slightly but not significantly stimulated cell growth in 2 cases, and significantly stimulated cell proliferation in 3 tumor cell cultures. The role of TGFb-3 could be tested only in 2 adenoma cell cultures and in none of them a stimulatory or inhibitory effect on cell proliferation could be detected. In most cases the inhibitors suppressed the proliferation of the adenoma cells already in the absence of TGFb isoforms. In the cell culture of NFPA1, in which TGFb-1 inhibited the cell proliferation, the combined application of TGFb-1 and the inhibitors led to a growth stimulatory effect. SB431542 alone led to a small spurt in tumor growth in the NFPA1-sample, that was potentiated in combination with TGFb-1, whereas SIS3 suppressed cell growth – this effect decreased though at the higher dosage, which corresponded with the results of the combined treatment of SIS3 with TGFb-1. In the remaining tumor cell cultures, in which TGFb-1 had growth-stimulatory effects, the treatment with the inhibitors combined or alone led to a significant reduction of proliferation rate. Although TGFb-3 alone had no effect on cell proliferation, the combined application of TGFb-3 and the inhibitors abolished the growth suppressive effects of the inhibitors. As in the murine tumor cell lines, a suppressive effect of the two inhibitors was also seen in the absence of external TGFβ-1. The results obtained from the limited number of tumors suggest that TGFβ inhibitors may suppress the proliferation and thus the expansion of NFPAs only under certain conditions, probably in advanced stages of tumor development. It would be particularly interesting to compare the patient outcome and clinical behavior of the tumors from which our or new samples derive with these experimental results and to find molecular or clinical markers predicting the response towards TGFβ- isoforms and the individual inhibitors of the corresponding pathway. Regarding VEGF secretion, both TGFβ-1 and -3 stimulated the production of this angiogenic factor in TtT/GF and αT3-1 cells. The tested inhibitors significantly inhibited both TGFβ- promoted and basal VEGF secretion in the cell lines. Although no corresponding studies could be done in human NFPAs due to lack of tissue, SB431542 and SIS3 may probably also suppress the VEGF production in NFPAs and may therefore suppress the development of these tumors by inhibiting tumor vascularization. Taken together, the data suggest that TGFβ inhibitors may slow down or block the progression of NFPAs, in particular those that have passed epithelial mesenchymal - transition (EMT) and have developed a more aggressive phenotype. In any case, this research suggests that with the recently developed specific inhibitors, SIS3 and SB431542 it is possible to reach a further in-depth understanding of the various respective elements involved in the TGFβ signaling cascade and their alterations during tumor progression, leading to possible new targeted therapeutic options. This, however, implies further studies and experimental designs than this small study encompasses.