Periodic Reporting for period 1 - MAtChinG (Mechanically Activated Channels in Glioma: the role of mechanoreceptor Piezo1 and hnRNP K in cancer as novel oncoregulators)
Reporting period: 2021-09-01 to 2023-08-31
Glioma is a broad category of brain tumours from glial cell. Amongst them, glioblastoma multiforme (GBM) is the most malignant and aggressive subtype in adults, having an incidence of 2-3 per 100,000 adults per year and affecting more males than females up to a ratio 3:1. This subtype is a grade 4 tumour, with fast growth rate, and sometimes spread to other parts of the brain. Current treatments include surgery, radiotherapy and chemotherapy; however, prognosis of most glioma is not optimistic, with survival rate normally ranging 14-18 months, with only around 10% of the patients living up to 5 years after diagnosis. This is partly due to the existence of glioma ‘cancer stem cells’ (CSCs), a subtype of tumour-initiating cells found in glioma with stem cell-like properties and resistant to conventional therapeutic treatments, being the cause of most relapses after treatment. Its aggressiveness is due to the complexity and the lack of full understanding of the biochemical nature and mechanisms of these type of tumours.
Recently, the importance of the mechanical properties of tissues in health and disease has become evident and has opened a new research field in biology. It is known that the mechanical environment of tumours is very hard, and that tumour cells overexpress proteins that respond to these changes in the mechanical properties, named mechanoreceptors. Stretch-activated cation channels (SACs) are a broad family of mechanosensitive channels that when activated by stretch, allows the influx of cations. Piezo1 –also known as FAM38A-, belongs to this subtype of mechanoreceptors.
This project is focused on understanding the importance of the mechanical properties of brain tumour environment and investigating new therapies tackling these changes. It has been observed that tumour cells contribute to the stiffening of their surroundings, and we have studied that when tumour cells sense these mechanical changes, it contributes to the progression of the cancer. Thus, our main objective is to fully understand why it happens and to develop new therapeutic strategies that could help combat gliomas, so that later on they could be translated into the clinic for the patients to benefit from them.
Recently, the importance of the mechanical properties of tissues in health and disease has become evident and has opened a new research field in biology. It is known that the mechanical environment of tumours is very hard, and that tumour cells overexpress proteins that respond to these changes in the mechanical properties, named mechanoreceptors. Stretch-activated cation channels (SACs) are a broad family of mechanosensitive channels that when activated by stretch, allows the influx of cations. Piezo1 –also known as FAM38A-, belongs to this subtype of mechanoreceptors.
This project is focused on understanding the importance of the mechanical properties of brain tumour environment and investigating new therapies tackling these changes. It has been observed that tumour cells contribute to the stiffening of their surroundings, and we have studied that when tumour cells sense these mechanical changes, it contributes to the progression of the cancer. Thus, our main objective is to fully understand why it happens and to develop new therapeutic strategies that could help combat gliomas, so that later on they could be translated into the clinic for the patients to benefit from them.
During these two years of project we have achieved most of our objectives with positive results, shedding light into the role of Piezo1 in glioma. We have successfully developed a transgenic mouse model overexpressing Piezo1 in GFAP-expressing cells with and without deletion of Tp53, and we have analysed and described the phenotype of this model, observing that the overexpression of Piezo1 leads to the onset of glioma both by itself and with loss of Tp53 (Milestone 1). This new in vivo tool is unique on itself and moreover, there are very few transgenic rodent models of primary glioma, so our mouse model could be a very useful tool for the scientific community. More importantly, we are the first ones demonstrating that Piezo1 is a novel and uncharacterized bona fide oncogene, as it can lead to glioma on its own.
We have also investigated the molecular role of Piezo1 and hnRNP K in glioma cells in vitro, using different human glioma cell lines (i.e. U87 and U251) and genetically knocking or overexpressing both hnRNP K and Piezo1 through CRISPR-Cas9 or CRISPR-Cas9/SAM technology, respectively. With these genetically engineered tools we have analysed the proliferation, migration and clonality capacity of the glioma cells, understanding the role of Piezo1 in these key aspects of tumour cells (Milestone 2.1) and partially deciphered the molecular mechanisms underlying. Additionally, not only did we study the effects of the overexpression or knock-out (KO) of Piezo1, but we also studied the effects of the pharmacological modulation of this mechanoreceptor through its activator Yoda-1 and the inhibitor GsMTx4. Finally, we have done an RNAseq of the Piezo1-KO and overexpressing cells to identify the molecular pathways involved, and we have validated them via western-blot and qPCR to understand the molecular processes underlying the phenotype observed in our cell lines (Milestone 2.2).
Lastly, during this grant secondment in the Universita’ di Parma, under the supervision of Prof. Fabio Sonvico, I have been trained in the development and production of nanoparticles with drug encapsulation for nasal delivery. During this time, we designed and successfully produced nanoparticles encapsulating Yoda-1 for the targeting of Piezo1 in glioma cells. We performed the physico-chemical analysis of the properties of these nanoparticles and we have already performed the assessment of their efficacy in vitro using our Piezo1-KO and overexpressing U251 cell lines (Milestone 3).
We have also investigated the molecular role of Piezo1 and hnRNP K in glioma cells in vitro, using different human glioma cell lines (i.e. U87 and U251) and genetically knocking or overexpressing both hnRNP K and Piezo1 through CRISPR-Cas9 or CRISPR-Cas9/SAM technology, respectively. With these genetically engineered tools we have analysed the proliferation, migration and clonality capacity of the glioma cells, understanding the role of Piezo1 in these key aspects of tumour cells (Milestone 2.1) and partially deciphered the molecular mechanisms underlying. Additionally, not only did we study the effects of the overexpression or knock-out (KO) of Piezo1, but we also studied the effects of the pharmacological modulation of this mechanoreceptor through its activator Yoda-1 and the inhibitor GsMTx4. Finally, we have done an RNAseq of the Piezo1-KO and overexpressing cells to identify the molecular pathways involved, and we have validated them via western-blot and qPCR to understand the molecular processes underlying the phenotype observed in our cell lines (Milestone 2.2).
Lastly, during this grant secondment in the Universita’ di Parma, under the supervision of Prof. Fabio Sonvico, I have been trained in the development and production of nanoparticles with drug encapsulation for nasal delivery. During this time, we designed and successfully produced nanoparticles encapsulating Yoda-1 for the targeting of Piezo1 in glioma cells. We performed the physico-chemical analysis of the properties of these nanoparticles and we have already performed the assessment of their efficacy in vitro using our Piezo1-KO and overexpressing U251 cell lines (Milestone 3).
At the end of this project, we expect to have identified and corroborated the role of Piezo1 and hnRNPK in the regulation of glioma and cancer stem cells. To do so, we are working at a cellular level and in vivo level.
At cellular level, we are so far developing the CRISPR/Cas9 modified Piezo1 knocked-out and overexpressing U87 and U251 cell lines. We have validated the modified protein and mRNA expression, as well as the functionality with calcium imaging. This cellular tools will allow us to investigate whether Piezo1 modulates cellular proliferation, migration, epithelial-mesenquimal transition, as well as whether there is a link between hnRNP K regulation and Piezo1.
At in vivo level, we have so far started the transgenic mouse colony Tg.Tomato-Piezo1/GFAP-creERT2 and Tg.Tomato-Piezo1/GFAP-cre/p53lox which will allow us to investigate whether overexpression of Piezo1 in glial cells leads to glioma, and if so, if it contributes to an increased number in cancer stem cells and worse prognosis.
At the end of thi project, we expect to have shed some light into these research questions as well as developed nasal delivered nanoparticles that could encapsulate drugs to modulate Piezo1 or hnRNP K and thus become a new therapeutic possibility for the clinic.
The innovative aspect of this research resides in the novel insight in new mutations responsible for glioma formation, and the mechanotransduction aspects of glioma progression and migration, two of the main big issues that make gliomas like GBM so mortal, with usual 14-18 months of life expectancy after diagnosis, with an only 30% survival rate after 2 years of diagnosis. Furthermore, one of the main issues to treat pathologies in the central nervous system is the difficulty in drug delivery. However, this project will address this issue by developing and testing a novel non-invasive way of delivering drugs into the brain via nasal spray. The impact of this technique could be revolutionary in the field of neuroscience once stablished, having a great impact in the clinic.
At cellular level, we are so far developing the CRISPR/Cas9 modified Piezo1 knocked-out and overexpressing U87 and U251 cell lines. We have validated the modified protein and mRNA expression, as well as the functionality with calcium imaging. This cellular tools will allow us to investigate whether Piezo1 modulates cellular proliferation, migration, epithelial-mesenquimal transition, as well as whether there is a link between hnRNP K regulation and Piezo1.
At in vivo level, we have so far started the transgenic mouse colony Tg.Tomato-Piezo1/GFAP-creERT2 and Tg.Tomato-Piezo1/GFAP-cre/p53lox which will allow us to investigate whether overexpression of Piezo1 in glial cells leads to glioma, and if so, if it contributes to an increased number in cancer stem cells and worse prognosis.
At the end of thi project, we expect to have shed some light into these research questions as well as developed nasal delivered nanoparticles that could encapsulate drugs to modulate Piezo1 or hnRNP K and thus become a new therapeutic possibility for the clinic.
The innovative aspect of this research resides in the novel insight in new mutations responsible for glioma formation, and the mechanotransduction aspects of glioma progression and migration, two of the main big issues that make gliomas like GBM so mortal, with usual 14-18 months of life expectancy after diagnosis, with an only 30% survival rate after 2 years of diagnosis. Furthermore, one of the main issues to treat pathologies in the central nervous system is the difficulty in drug delivery. However, this project will address this issue by developing and testing a novel non-invasive way of delivering drugs into the brain via nasal spray. The impact of this technique could be revolutionary in the field of neuroscience once stablished, having a great impact in the clinic.