Reciprocal Activation Of Tumor-associated Fibroblasts And Pituitary Adenomas Through The TGFβ1/Smad-SDF-1α/CXCR4Pathway

For most of patients with pituitary adenomas, tumor mass effects and endocrinological symptoms could be relieved or even be cured completely following the therapies of surgery, medicine, radiation or comprehensive treatments. However, some tumors resisted these standard treatments, some of which manifested malignant phenotype with rapid cell proliferation, high Ki-67labeling index, positive p53staining and more ratios of fibrous tissues following surgery or gammar-knife therapy. Meta analysis indicated that nonfunctional pituitary adenomas (NFPAs) are the most common type of recurrent PAs,12%-58%of which would relapse followed by GH-secreting PAs,2%-3%. Since the recurrent PA tissues are characterized with stiffness, severe adhesion with the adjacent important blood vessels or nerves, it is much more difficult to remove the tumor lesions, no matter what operative approaches the neurosurgeons choose. Therefore, recurrent fibrous pituitary adenomas (RFPAs) are placing a huge burden on patients’families and society, and it is important and imperative to investigate the mechanism of RFPA’s tumorigenesis and progression for further therapy studies.Recent studies have revealed that beside the alteration of genome, the tumour stroma, that is, microenvironment surrounding tumour cells, can contribute to the tumorigenesis and the increasing invasiveness of tumour cells during tumor progression. Fibroblasts in tumor stroma that are considered to play an essential role in regulating tumour cells’ biological behavior and are subsequently named as "cancer-associated fibroblasts"(CAFs), were similar to those present in sites of wound healing and chronic fibrosis. Transforming growth factor β1(TGFβ1), a inflammation-relative factor, has been one and only known cytokine that can attract the fibroblasts in epidermis to migrate into tumor lesion through chemotaxis and act on silent fibroblasts to translate them into activated state in wound or in tumor lesions through canonical TGFβ1/Smad pathway. On the other hand, the activated fibroblasts could secrete other cytokines, such as IL-6and SDF-la, to contribute to tumor angiogenesis, cell proliferation, invasiveness/migration, or drug-resistence. Hence, it is very important and necessary to investigate the role of fibroblasts in RFPA stroma.Based on the phenomenon above and the previous studies results, we designed this research:5paired primary and recurrent fibrous PAs (PPAs and RFPAs) paraffin-embedding samples from the same patientsand another22primary PAs, as well as12RFPAs were included. With the help of laser capture microdissection, we collected pituitary adenoma cells (PACs) and pituitary adenoma-associated fibroblasts (PAFs) from unpaired PPA and RFPA tissues, respectively. First of all, we evaluated the differential expression of TGFβ1, collagen Ⅰ, Ⅲ, a-SMA, FAP, pSmad2and3, and other proteins related to cell proliferation, invasiveness/migration, between PACs and PAFs from PPA and RFPA tissues, to verify that there were more activated fibroblasts that have function of secretion of collagen I and III through pituitary adenoma cell-secreted TGFβ1and TGFβ1/Smad signaling pathway. And these were confirmed by in vitro murine PA and fibroblast cell line test. Furthermore, the TGFβ1-activated fibroblasts could secrete higher levels of SDF-la and there were also an autocrine/paracrine signaling loop of TGFβ1/Smad and SDF-la/CXCR4pathway in activated fibroblasts which could contribute to the maintenance of fibroblast activation and its function of fibrosis. On the other hand, in vitro and in vivo investigation also indicated that the activated fibroblast-derived SDF-1α could promote PAC’s cell proliferation, invasiveness/migration, hormone secretion, tumor formation and tumor cell sternness. Lastly, we discovered that two miR-134and miR-370were decreased and their target gene-encoded proteins VEGFA and HMGA2were increased by the treatment of SDF-la in14NFPA primary cultured cells.In conclusion, there are reciprocal activation between fibroblasts and pituitary adenoma cells through TGFβ1/Smad-SDF-la/CXCR4signaling pathway, which can contribute to each cell’s proliferation and functions. Therefore, this signaling loop may play an important role in the tumorigenesis and progression of RFPA, and it may be a novel therapy for RFPA to block this positive feedback loop.Section One Differential analysis of the phenotype of pituitary adenoma-associated fibroblasts (PAFs) and their expression of TGFβ1/Smad signaling pathway between PPA and RFPA tissuesObjective:To verify the existence of activated pituitary adenoma-associated fibroblasts and its fibrosis functions in RFPA tissues; to analyze the differential expression of TGFβ1of pituitary adenoma cells between PPA and RFPA tissues; to identify the effects of TGFβ1/Smad signaling pathway on the transformation of activated pituitary adenoma-associated fibroblasts and its collagen-producing abilities.Methods:5paired primary and recurrent fibrous PAs (PPAs and RFPAs) paraffin-embedding samples from the same patientswere included for IHC staining of collagen I, III in tumor stroma and a-SMA, FAP, TGFβRI, TGFβRIII, pSmad2and pSmad3in fibroblasts. Another22primary PAs, as well as12RFPAs were also included for further examinations of qRT-PCR and IHC of a-SMA, FAP, collagen I and III in fibroblasts and TGFβ1in pituitary adenoma cells, with the help of laser capture microdissection (LCM).Results:Compared with the paired PPA tissues, the RFPA tissues contained more proportion of activated fibroblasts with elevated function of collagen secretion (p<0.001). TGFβRI. TGFβRIII. pSmad2and pSmad3were all positive stained in the activated fibroblasts. Meanwhile, the mRNA levels of TGFβ1in tumor cells, α-SMA、 FAP、 COL1A1、 COL1A2and COL3A1in fibroblasts from RFPAs were higher than those in PPAs (p<0.05).Conclusions:Activated pituitary adenoma-associated fibroblasts with the ability of collagen-producing abound in the tissues of RFPAs. The increased TGFβ1-secreting pituitary adenoma cells could mediate the stimulation of their adjacent fibroblasts through TGFβ1/Smad signaling pathway. Section Two The effects of TGFβ1/SDF-la autocrine/paracrine signaling loop on the maintenance of myofibroblast differentiation and fibrous functions of fibroblasts in RFPAsObjective:To identify the autocrine of TGFβ1in the TGFβ1/Smad-activated RPAFs in RFPAs; to analyze the effects of TGFβ1/Smad signaling pathway on the expression of SDF-la and its receptor CXCR4in RPAFs; to search the effects of SDF-la/CXCR4on the autocrine/paracrine of SDF-la and TGFβ1by RPAFs.Methods:the two types of fibroblasts from22primary PAs, as well as12RFPAs were obtained by LCM for the examination of SDF-la and CXCR4through qRT-PCR. Meanwhile, the fibroblasts from the above patients were cultured and treated by vehicle control, recombinant SDF-la or TGFβ1, the matched inhibitors to analyze the differential expression of autocrine SDF-1α and TGFβ1by themselves using qRT-PCR and ELISA. Furthermore, murine cell lines were treated as mentioned above to confirm the results of the former examinations by qRT-PCR, western blot and ELISA.Results:The expression level of SDF-la and CXCR4in the RPAFs were elevated significantly, compared with that in PPAs (p<0.05). TGFβ1or SDF-la could enhance the expression of TGFβ1、 SDF-la and CXCR4in PPAFs, which could be inhibited by TGFβ1receptor or CXCR4inhibitors (p<0.05). In vitro murine cell line examination showed the same results as above.Conclusions:Exogenous TGFβ1and SDF-1α could enhance the expression of endogenous TGFβ1and SDF-la in activated RPAFs in RFPA tissues, thus form the TGFβ1/SDF-1α autocrine/paracrine signaling loop for the maintenance of activation of fibroblasts in RFPAs. This positive-feedback loop may be one reason why numbers of activated fibroblasts arise in the tissues of RFPAs and a large quantity of fibroblasts appear in the culture medium at the later period of primary human pituitary adenoma culture. Section Three The effects of the activated fibroblasts-secreted SDF-la on pituitary adenoma cells’proliferation, invasiveness/migration, hormone secretion, tumor cell stemness and tumor formation in vivoObjective:to study the effects of the activated fibroblasts-secreted SDF-la on pituitary adenoma cells’ proliferation, invasiveness/migration, hormone secretion, tumor cell stemness and tumor xenografts in nude mice for further investigation of molecular mechanism of RFPAs’ tumorigenesis and therapy targets.Methods:The differential expression of SDF-la, CXCR4and the biomarkers related to cell proliferation, invasiveness/migration, tumor stem cell properties between PPAs and RFPAs were examined by IHC, qRT-PCR and LCM. Treated by vehicle control, recombinant SDF-la, activated fibroblast-CM, or inhibitors, αT3-1or GH3were examined for their cell proliferation rate, invasiveness/migration abilities, hormone secretion levels, the proportion of tumor stem cells through the studies of CCK8, ELISA, Live Cell Imaging System, Flow Cytometry. The protein levels of CXCR4and some biomarkers were also tested by western blot. Moreover, GH3treated by various reagents mentioned above with Factor Reduced Matrigel were implanted into30nude mice, to observe tumor growth, peripheral serum GH levels and protein expressions related to cell proliferation, invasiveness/migration by IHC.Results:The IHC results displayed that the expression levels of SDF-la and proteins related to tumor cell proliferation, invasiveness/migration, tumor stem cell properties and CXCR4in RFPAs were all much higher than those in PPAs (p<0.05). Compared with other treatment groups, tumor cells exposed to SDF-la and activated fibroblast-CM manifested higher cell proliferation rates, more powerful invasiveness/migration abilities, stronger hormone secretion, larger population of tumor stem cells, with the increased expressions of the relative proteins (p<0.05). Meanwhile, the tumor volumes of SDF-1α and activated fibroblast-CM-treated groups were much larger than other groups (p<0.05), and the peripheral GH levels from both the treatment groups were also increased, with the differential expression of the relative proteins by IHC.Conclusion:The activated fibroblast-over-secreted SDF-la could promote pituitary adenoma cells’proliferation, invasiveness/migration, hormone secretion, tumor stem cell generation and tumor formation in vivo. The elevated expression of SDF-la may be one pivotal mechanism of RFPA tumorigenesis and progression clinically, and to inhibit SDF-la/CXCR4signaling in pituitary adenomas may be a promising approach for the treatments of RFPAs. Section Four The effects of SDF-la on the expression of microRNAs located in the imprinted DLK1-MEG3locus, as well as their target gene-encoded proteins in human clinically nonfunctioning pituitary adenomasObjective:To investigate the effects of exogenous SDF-la on the expressions of the microRNAs located in the imprinted DLK1-MEG3locus in human clinically nonfunctioning pituitary adenomas and find out the significant changed miRNAs and their target gene-encoded proteins, for providing molecular mechanisms of SDF-1α’s effects on tumor cell proliferation and invasiveness/migration.Methods:14clinically nonfunctioning pituitary adenomas were included. The tumor cells, following the removal of fibroblasts and erythrocytes, were exposed to vehicle control or exogenous SDF-la for96h. Six miRNAs (miR-134, miR-299-5p, miR-329, miR-370, miR-377-5p�'�miR-432), reported as being related to the tumorigenesis of NFPA within in the imprinted DLK1-MEG3locus, were examined by qRT-PCR to evaluate the change of their expression. The significantly changed miRNAs were confirmed by statistical methods and their valid target mRNA would be identified through web of miRBase. Western blots examined whether there would be significant changes of these proteins between pre-and post-treatment of SDF-la. At last, a correlation analysis between the changes of the proteins and that of miRNAs were carried out.Results:Both the expression levels of miR-134and miR-370were attenuated following SDF-la treatment (p<0.05). The search results on miRBase said that VEGFA (vascular endothelial growth factor A) and ABCC1(ATP-binding cassette, sub-family C, member1) are the miR-134’s valid target mRNAs, while MAP3K8(mitogen-activated protein kinase kinase kinase8) and HMGA2(high mobility group AT-hook2) are miR-370’s. The expression levels of VEGFA and HMGA2were both significant increased after SDF-la treatment (p<0.05), and there was negative correlation between the changes of VEGFA and those of miR-134(R=-0.758, p=0.002). The IHC analysis also showed that there was elevated expression of VEGFA and HMGA2in recurrent pituitary adenoma cells.Conclusions:SDF-la may promote tumor cell’s proliferation, invasiveness/migration and angiogenesis through decreasing the expression levels of miR-370and miR-134, which may impact the expression of VEGFA and HMGA2. Hence, to enhance the expression levels of miR-134and miR-370in PA cells may be novel therapy for RFPAs.