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Role of TGFβ family members on physiological angiogenesis and ovarian cancer

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Universitat de Barcelona. Departament de Ciències Fisiològiques II

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[eng] Role of TGFβ in physiological angiogenesis and ovarian cancer Transforming Growth Factor Beta (TGFβ) is a signalling pathway involved in a wide range of cellular processes such as proliferation, differentiation, angiogenesis, migration and homeostasis. In addition, it has been broadly related with pathological situations like cancer or other diseases. In this thesis we have been focused on two of its receptors; the TβRI called ALK5, ubiquitously expressed, and ALK1, an endothelial cell-restricted receptor. In the first part of this work we have studied the role of ALK1 in angiogenesis. Loss of function mutations in ALK1 cause a subtype of hereditary haemorrhagic telangiectasias (HHTs) characterized by vasculature malformations. Currently, there is no suitable treatment to cure these patients. To study the ALK1 role in vascular development, we have used the retina mouse model and seeking to find a treatment for these patients, we have used a mouse model for HHTs. This mouse model has a heterozygous alteration in ALK1, as in homozygosis die at +/- mice resulted in abnormal endothelial cell proliferation mid-gestion. The alteration on ALK1 and increased retinal vessel width without affecting pericyte coverage, migration or elongation of the ECs. We have shown that genetic and pharmacological inhibition of PI3K signalling +/- abolished the increase in vessel width in ALK1 retinas and normalized vasculature. Overall, our results suggest the potential for using PI3K inhibitors as new therapeutic agents for treating HHTs. In the second part of this work we have searched for new therapeutic targets for treating epithelial ovarian cancer. Firstly, we analysed the TGFβ signalling pathway in these tumours. Using a Tissue Micro Array (TMA) with patient samples we found high Smad2 phosphorylation, a read-out of ALK5 activation, in epithelial ovarian cancer tumoral cells, independently of tumour subtype (high-grade serous or endometrioid). To evaluate the impact of TGFβ receptor inhibition on tumoral growth, we used different models of human epithelial ovarian cancer orthotopically grown in nude mice (OVAs); two high-grade serous carcinoma and one endometrioid carcinoma. We have confirmed that the histological properties and the levels of phosphoSmad2 expression pattern were maintained on the ovarian tumour mouse models compared to its human primary tumours samples. Treatment with a TGFβRI&II dual inhibitor, LY2109761, caused a significant reduction in tumour size in all these models, affecting cell proliferation rate. On the contrary, TGFβ inhibition did not affect ovarian tumour cell capacity to disseminate in our models. We identified Insulin Growth Factor (IGF)1 receptor as the signal positively regulated by TGFβ implicated in ovarian tumour cell proliferation. We have demonstrated that our TMA samples had high IGF1R protein levels and that phosphoSmad2 and IGF1R protein levels significantly positively correlated. Inhibition of IGF1R activity by treatment with a blocker antibody (IMC- A12) or with a tyrosine kinase inhibitor (linsitinib) inhibited ovarian tumoral growth in vivo. At the molecular level TGFβ signalling positively controls IGF1R levels by two mechanisms. Induction of mRNA IGF1R levels in some cell lines or tumour samples, whereas a change on IGF1R protein localization was observed in others. In fact, TGFβ inhibition decreased IGF1R protein levels by controlling its stability; increasing IGF1R internalization and degradation through the lysosome. When IGF1R levels were decreased by shRNA treatment, ovarian cancer cell lines grew less and LY2109761 lost its capacity to block tumoral ovarian cell proliferation. Overall, our results suggest an important role for the TGFβ signalling pathway in ovarian tumour cell growth through the control of IGF1R signalling pathway. Moreover, it identifies anti-TGFβ inhibitors as being of potential use in new therapies for ovarian cancer patients as an alternative to IGF1R inhibition, being used in clinical trials.

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[cat] Aquest treball estudia el paper de la família del Factor de Creixement Transformant (TGFβ) en el càncer d’ovari i en una altra patologia, la telangectàsia hemorràgica hereditària (HHT). Mutacions de pèrdua de funció del receptor ALK1 causen un subtipus de HHT caracteritzat per malformacions vasculars. Per analitzar el rol d'ALK1 en el desenvolupament vascular, hem utilitzat un model animal d'HHT. Les alteracions obtingudes han estat una proliferació anormal de les cèl.lules endotelials i un augment de l'amplitud dels vasos de la retina dels ratolins ALK1+/-. Hem demostrat que una inhibició genètica o farmacològica de la via de senyalització de PI3K aboleix l'augment de l'amplitud de vasos i la vasculatura queda normalitzada, suggerint el potencial ús dels inhibidors de PI3K com a nous agents terapèutics pel tractament d'aquests pacients. En la segona part d'aquest treball, hem buscat nous agents terapèutics pel tractament del càncer d'ovari. Utilitzant mostres de pacients, hem observat alts nivells de Smad2 fosforilat en les cèl.lules tumorals de càncer d'ovari, independentment del tipus de tumor. El tractament amb l'inhibidor de TGFβRI&II, LY2109761, en els models ortotòpics de càncer d'ovari causa una reducció significativa de la mida tumoral, afectant la proliferació cellular. Hem identificat el receptor del Factor de Creixement de la Insulina (IGF)1 com la via implicada en la proliferació de les cèl.lules tumorals d'ovari, la qual és regulada positivament per TGFβ. La inhibició de l'activitat d'IGF1R inhibeix el creixement del tumor d'ovari in vivo. A nivell molecular, TGFβ controla els nivells de mRNA d'IGF1R i també la seva internalització i degradació proteica a través del lisosoma. Quan els nivells d'IGF1R són disminuïts per la utilització d'un shRNA, LY2109761 perd la capacitat de bloquejar la proliferació de la cèl.lula tumoral d'ovari. Aquests resultats postulen que TGFβ. té un paper important en el creixement cellular del tumor d'ovari a través del control de la senyalització d'IGF1R. A més, identifica els inhibidors contra TGFβ com a noves teràpies pels pacients de càncer d'ovari com una alternativa als inhibidors d'IGF1R.

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Tesis Doctorals - Departament - Ciències Fisiològiques II

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Role of TGFβ family members on physiological angiogenesis and ovarian cancer