Ignite Creation Date:
2024-05-06 @ 12:34 PM
Last Modification Date:
2024-10-26 @ 1:01 PM
Study NCT ID:
NCT03798587
Status:
UNKNOWN
Last Update Posted:
2019-11-22
First Post:
2019-01-07
Brief Title:
Inhibition of SENP1 for the Suppression of OS Growth and Metastasis
Sponsor:
IRCCS Ospedale Galeazzi-SantAmbrogio
Organization:
IRCCS Ospedale Galeazzi-SantAmbrogio
Study Overview
Official Title:
PNA-mediated Inhibition of the SENP1 Molecular Hub as a Potential Therapeutic Approach for the Suppression of Osteosarcoma Growth and Metastasis PNA-OS
Status:
UNKNOWN
Status Verified Date:
2019-01
Last Known Status:
NOT_YET_RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The aim of this project is to test a new powerful PNA-based SENP1 inhibitor previously characterized in an in vitro model of OS cell lines
The most effective PNA conjugated with a cell-permeable CPP which is able to inhibit OS cells viability and invasiveness in both normoxia and hypoxia through SENP1-mediated inhibition of HIF1α ZEB1 and Akt will be investigated for its ability to penetrate and silence SENP1 expression in ex vivo human OS tissues
Primary aim
To determine the ability of PNA-CPP to penetrate into an ex vivo tridimensional tissue of OS derived from wasted biological material obtained during OS eradication surgery and to exert its biological function of inhibiting SENP1 within the tissue
The most effective PNA conjugated with a cell-permeable CPP which is able to inhibit OS cells viability and invasiveness in both normoxia and hypoxia through SENP1-mediated inhibition of HIF1α ZEB1 and Akt will be investigated for its ability to penetrate and silence SENP1 expression in ex vivo human OS tissues
Primary aim
To determine the ability of PNA-CPP to penetrate into an ex vivo tridimensional tissue of OS derived from wasted biological material obtained during OS eradication surgery and to exert its biological function of inhibiting SENP1 within the tissue
Detailed Description:
Background
Osteosarcoma OS is the most common type of primary malignant bone tumor in children and adolescents The overall survival rate is dramatically reduced by the development of metastases often pulmonary Solid malignant tumors such as OS often develop a hypoxic microenvironment which contributes to tumor growth metastasis treatment failure and patient mortality Adaptation to hypoxia as well as to other environmental conditions is often associated with modifications in the post-transcriptional regulation of key effectors Among these SUMOylation is carried out by small ubiquitin-like modifier SUMO proteins and is dynamically reversed deSUMOylation by SentrinSUMO-specific proteases SENPs SENP1 the best characterized SENP is upregulated in multiple tumors being involved in tumorigenesis and tumor progression Through deSUMOylation SENP1 acts as a molecular hub that stabilizes and activates key regulator factors such as hypoxia-inducible factor 1α HIF1α zinc finger E-box binding homeobox 1 ZEB1 and Akt responsible for tumor cells adaptation to hypoxic microenvironment induction of cell proliferation invasion and migration and inhibition of apoptosis thus contributing to tumor progression and metastasis
HIF1α is the master transcriptional regulator for cellular adaptation and survival under hypoxic conditions and contributes to enhance the cell metastatic potential SENP1-mediated HIF1α deSUMOylation prevents HIF1α degradation by proteasome thus activating the HIF1α signaling pathway SENP1 is overexpressed in OS cells under hypoxic condition and siRNA-mediated silencing of SENP1 decreases tumor cell viability promotes cell apoptosis reduces invasiveness and inhibits the epithelial-mesenchymal transition EMT
ZEB1 is involved in tumorigenesis progression invasion and metastases in several tumors eg glioblastoma prostate lung liver and colorectal ZEB1 silencing in OS cells leads to a reduced caspase-3 activity NF-κB and iNOS inhibition overall reduced cell proliferation and increased apoptosis SENP1 knockdown in hepatocellular carcinoma HCC cells decreases ZEB1 and inhibits EMT
Akt hyper-activation is essential for the onset and progression of tumors including OS In astroglioma cells siRNA-mediated inhibition of SENP1 is associated to Akt hypophosphorylation accompanied by the inhibition of its downstream targets Bcl-xL and cyclinD1 and p21 upregulation leading to cell-cycle arrest and increased apoptosis
Altogether these studies suggest that SENP1 acts as a hub whose inhibition reflects on multiple targets some of which ie HIF1α ZEB1 Akt are key factors in tumor progression and metastasis in both normoxia and hypoxia While it is known the effect of SENP1 on HIF1α in OS it is reasonable to assume that SENP1 might mediate ZEB1 downregulation and Akt inactivation also in OS Thus novel SENP1 inhibition strategies are potentially effective therapeutic approaches to block OS growth and metastasis
SENP1 inhibition can be achieved by gene silencing mediated by siRNAs However naked siRNAs are highly unstable and liposome-based delivery systems are poorly efficient and cytotoxic both in vitro and in vivo A promising approach for inhibition of SENP1 expression is gene silencing mediated by peptide nucleic acids PNA nucleobase oligomers with the phosphate backbone replaced by a pseudopeptide backbone of repeated units of N-2-aminoethyl glycine Because of their unnatural backbone PNAs are definitively resistant to both nuclease and protease activities form a more specific and stable binding with the complementary DNA or RNA allowing an efficient and persistent silencing effect Although PNA cell permeability is very poor it can be effectively enhanced by their conjugation with cell-penetrating peptides CPP In the last years PNA have emerged as really promising tools for cancer diagnosis and therapy and above all as effective candidates for stable gene silencing in gene therapy
Rational and preliminary study
3 different PNA sequences targeting different SENP1 mRNA regions will be designed and tested PNAs will be conjugated to an octa-arginine R8 CPP that efficiently mediate the intracellular delivery of PNAs The uptake will be studied with a scrambled-sequence R8- and fluorescein Fl-conjugated PNA scrPNA-R8-Fl
An in vitro characterization of the ability of the designed PNA-CPP to penetrate intracellular and to silence the target SENP1 will be performed in cell lines of OS
To study the PNA-R8 uptake in OS cells different OS cell lines SaOS-2 MG-63 U2OS with different invasive potential and all expressing SENP1 and primary human osteoblasts hOb as negative control for SENP1 expression will be used Following incubation with scrPNA-R8-Fl at different concentrations the uptake will be determined at consecutive time-points by flow cytometry while the cytoplasmic localization will be confirmed by fluorescence microscopy A scrPNA-Fl not conjugated to R8 will function as a negative control as it is not expected to enter the cell Cytotoxicity of scrPNA-R8 will be assayed by Alamar Blue Cell Viability assay The silencing effectiveness of the different anti-SENP1 PNA-R8 conjugates senpPNA-R8 will be assayed in all cell lines in both normoxia and hypoxia 1 O2 5 CO2 and 94 N2 The senpPNA-R8-mediated SENP1 silencing efficiency will be assessed by RT-qPCR and western-blot WB scrPNA-R8 will serve as negative control while cells transfected with siRNA targeting SENP1 will serve as positive control In this part the most efficient silencing senpPNA-R8 compound will be selected
The senpPNA-R8-mediated downregulation of HIF1α and potentially of ZEB1 expression and Akt phosphorylation inhibitions as consequence of SENP1 inhibition in OS cells will be assayed by WB Thus reduced cell viability migration and invasion induction of apoptosis and EMT inhibition will be assayed and compared to the effects in hOb Cell viability will be determined by Alamar Blue assay whereas apoptosis will be assayed by flow cytometry by staining of Annexin V and with propidium iodide The residual migration and invasion ability will be assessed by wound-healing assay and transwell invasion assay respectively Downregulation of vimentin and N-cadherin and upregulation of E-cadherin EMT markers and of the downstream targets of ZEB1 caspase-3 NF-κB and Akt cyclinD1 and Bcl-xL will be determined by WB
The in vitro characterization of the penetration and silencing ability of the designed PNA-CPP in OS cell lines is the preliminary step of the study An ex vivo analysis of the ability of the PNA-CPP to penetrate into a 3D tissue and silence the target SENP in an OS tissue explant from patients will follow
Aims of the study
The aim of this project is to test a new powerful PNA-based SENP1 inhibitor previously characterized in an in vitro model of OS cell lines
The most effective PNA conjugated with a cell-permeable CPP which is able to inhibit OS cells viability and invasiveness in both normoxia and hypoxia through SENP1-mediated inhibition of HIF1α ZEB1 and Akt will be investigated for its ability to penetrate and silence SENP1 expression in ex vivo human OS tissues
Primary aim
To determine the ability of PNA-CPP to penetrate into an ex vivo tridimensional tissue of OS derived from wasted biological material obtained during OS eradication surgery and to exert its biological function of inhibiting SENP1 within the tissue
Study design
For this study wasted biological material derived from osteosarcoma eradication surgery will be collected which comprise only a small proportion of the removed tumor mass other than that used for histological and molecular diagnosis
15 patients with primary OS will be recruited The target group will comprise patients hospitalized at IRCCS Istituto Ortopedico Galeazzi who will be subjected to surgical eradication of primary OS
The study will be presented to patients with age 18 years that can be recruited also in the IRCCS Istituto Ortopedico Galeazzi BioBanca protocol ethical committee approval n 29INT2017 by the surgeon These patients will sign two Informed Consents one for the BioBanca and one for the PNA-OS study
Patients with age 18 years will be additionally recruited besides the IRCCS Istituto Ortopedico Galeazzi BioBanca These patients will be considered eligible for the study if the legal tutor will sign the Informed Consents relative to the PNA-OS study
Also samples of OS already existing in the BioBanca as frozen samples preserved in liquid nitrogen at BioRep Service-Provider BioRep Srl Via Olgettina 60 20132 Milano will be used Every reasonable effort will be done to call these patients to sign a specific informed consent relative to this study
Since several practical issues eg unsuitableness or low amount of biological material can occur we envision the possibility to recruit additional patients until the achievement of 15 complete samples
The Study will start after approval of Ethical Committee and the estimated duration is 36 months divided as following
Timing for enrolment 24 month
Data analysis 12 month
Experimental design
Ex vivo analysis of the PNA-R8 silencing ability in osteosarcoma samples We will investigate whether senpPNA-R8 is able to penetrate into a tridimensional OS tissue and to exert its silencing effect
15 OS samples will be collected either in the context of the IRCCS Galeazzi BioBanca ethical committee approval n 29INT2017 or from newly recruited patients in collaboration with the CCOORR equip
Only wasted biological material derived from surgery will be used without any additional harm to the patients other than the surgery itself and the study does not involve any diagnostic aim or genetic profiling of the samples collected
OS samples already existing in the BioBanca as frozen samples preserved in liquid nitrogen at BioRep Service-Provider BioRep Srl Via Olgettina 60 20132 Milano will be used to determine the initial expression levels of SENP1 in OS by RT-qPCR For this samples will be homogenized total RNA will be extracted and RT-qPCR will be performed assaying for SENP1 expression levels
The remaining samples out of 15 freshly collected will be preserved in physiological solution until usage The OS samples will be cut in 3 mm3 pieces placed in a 24-multiwell culture plate and cultivated ex vivo as organotypic OS cultures in both normoxia and hypoxia-induced microenvironment under orbital rotation 21-23 Organotypic tumor tissue maintain the complexity of the original tissue with tumor cells being surrounded by their original microenvironment rather than artificial matrices and this system is particularly advantageous for ex vivo drug screening for studying drug uptake and molecular processes OS cultures will be treated with senpPNA-R8 and SENP1 expression in naïve and PNA-treated samples will be determined by RT-qPCR and immunohistochemistry in paraffin-embedded sections The ability of the PNA-R8 to penetrate within the hypoxic core of the OS samples will be assessed after incubation with scrPNA-R8-Fl sections will be immediately frozen -80C processed and analyzed by immunofluorescence
Samples will be analyzed and stored at Laboratorio di Biochimica Sperimentale e Biologia Molecolare at the Istituto Ortopedico Galeazzi for the whole duration of the study At the end of the study every residual material will be destroyed
Osteosarcoma OS is the most common type of primary malignant bone tumor in children and adolescents The overall survival rate is dramatically reduced by the development of metastases often pulmonary Solid malignant tumors such as OS often develop a hypoxic microenvironment which contributes to tumor growth metastasis treatment failure and patient mortality Adaptation to hypoxia as well as to other environmental conditions is often associated with modifications in the post-transcriptional regulation of key effectors Among these SUMOylation is carried out by small ubiquitin-like modifier SUMO proteins and is dynamically reversed deSUMOylation by SentrinSUMO-specific proteases SENPs SENP1 the best characterized SENP is upregulated in multiple tumors being involved in tumorigenesis and tumor progression Through deSUMOylation SENP1 acts as a molecular hub that stabilizes and activates key regulator factors such as hypoxia-inducible factor 1α HIF1α zinc finger E-box binding homeobox 1 ZEB1 and Akt responsible for tumor cells adaptation to hypoxic microenvironment induction of cell proliferation invasion and migration and inhibition of apoptosis thus contributing to tumor progression and metastasis
HIF1α is the master transcriptional regulator for cellular adaptation and survival under hypoxic conditions and contributes to enhance the cell metastatic potential SENP1-mediated HIF1α deSUMOylation prevents HIF1α degradation by proteasome thus activating the HIF1α signaling pathway SENP1 is overexpressed in OS cells under hypoxic condition and siRNA-mediated silencing of SENP1 decreases tumor cell viability promotes cell apoptosis reduces invasiveness and inhibits the epithelial-mesenchymal transition EMT
ZEB1 is involved in tumorigenesis progression invasion and metastases in several tumors eg glioblastoma prostate lung liver and colorectal ZEB1 silencing in OS cells leads to a reduced caspase-3 activity NF-κB and iNOS inhibition overall reduced cell proliferation and increased apoptosis SENP1 knockdown in hepatocellular carcinoma HCC cells decreases ZEB1 and inhibits EMT
Akt hyper-activation is essential for the onset and progression of tumors including OS In astroglioma cells siRNA-mediated inhibition of SENP1 is associated to Akt hypophosphorylation accompanied by the inhibition of its downstream targets Bcl-xL and cyclinD1 and p21 upregulation leading to cell-cycle arrest and increased apoptosis
Altogether these studies suggest that SENP1 acts as a hub whose inhibition reflects on multiple targets some of which ie HIF1α ZEB1 Akt are key factors in tumor progression and metastasis in both normoxia and hypoxia While it is known the effect of SENP1 on HIF1α in OS it is reasonable to assume that SENP1 might mediate ZEB1 downregulation and Akt inactivation also in OS Thus novel SENP1 inhibition strategies are potentially effective therapeutic approaches to block OS growth and metastasis
SENP1 inhibition can be achieved by gene silencing mediated by siRNAs However naked siRNAs are highly unstable and liposome-based delivery systems are poorly efficient and cytotoxic both in vitro and in vivo A promising approach for inhibition of SENP1 expression is gene silencing mediated by peptide nucleic acids PNA nucleobase oligomers with the phosphate backbone replaced by a pseudopeptide backbone of repeated units of N-2-aminoethyl glycine Because of their unnatural backbone PNAs are definitively resistant to both nuclease and protease activities form a more specific and stable binding with the complementary DNA or RNA allowing an efficient and persistent silencing effect Although PNA cell permeability is very poor it can be effectively enhanced by their conjugation with cell-penetrating peptides CPP In the last years PNA have emerged as really promising tools for cancer diagnosis and therapy and above all as effective candidates for stable gene silencing in gene therapy
Rational and preliminary study
3 different PNA sequences targeting different SENP1 mRNA regions will be designed and tested PNAs will be conjugated to an octa-arginine R8 CPP that efficiently mediate the intracellular delivery of PNAs The uptake will be studied with a scrambled-sequence R8- and fluorescein Fl-conjugated PNA scrPNA-R8-Fl
An in vitro characterization of the ability of the designed PNA-CPP to penetrate intracellular and to silence the target SENP1 will be performed in cell lines of OS
To study the PNA-R8 uptake in OS cells different OS cell lines SaOS-2 MG-63 U2OS with different invasive potential and all expressing SENP1 and primary human osteoblasts hOb as negative control for SENP1 expression will be used Following incubation with scrPNA-R8-Fl at different concentrations the uptake will be determined at consecutive time-points by flow cytometry while the cytoplasmic localization will be confirmed by fluorescence microscopy A scrPNA-Fl not conjugated to R8 will function as a negative control as it is not expected to enter the cell Cytotoxicity of scrPNA-R8 will be assayed by Alamar Blue Cell Viability assay The silencing effectiveness of the different anti-SENP1 PNA-R8 conjugates senpPNA-R8 will be assayed in all cell lines in both normoxia and hypoxia 1 O2 5 CO2 and 94 N2 The senpPNA-R8-mediated SENP1 silencing efficiency will be assessed by RT-qPCR and western-blot WB scrPNA-R8 will serve as negative control while cells transfected with siRNA targeting SENP1 will serve as positive control In this part the most efficient silencing senpPNA-R8 compound will be selected
The senpPNA-R8-mediated downregulation of HIF1α and potentially of ZEB1 expression and Akt phosphorylation inhibitions as consequence of SENP1 inhibition in OS cells will be assayed by WB Thus reduced cell viability migration and invasion induction of apoptosis and EMT inhibition will be assayed and compared to the effects in hOb Cell viability will be determined by Alamar Blue assay whereas apoptosis will be assayed by flow cytometry by staining of Annexin V and with propidium iodide The residual migration and invasion ability will be assessed by wound-healing assay and transwell invasion assay respectively Downregulation of vimentin and N-cadherin and upregulation of E-cadherin EMT markers and of the downstream targets of ZEB1 caspase-3 NF-κB and Akt cyclinD1 and Bcl-xL will be determined by WB
The in vitro characterization of the penetration and silencing ability of the designed PNA-CPP in OS cell lines is the preliminary step of the study An ex vivo analysis of the ability of the PNA-CPP to penetrate into a 3D tissue and silence the target SENP in an OS tissue explant from patients will follow
Aims of the study
The aim of this project is to test a new powerful PNA-based SENP1 inhibitor previously characterized in an in vitro model of OS cell lines
The most effective PNA conjugated with a cell-permeable CPP which is able to inhibit OS cells viability and invasiveness in both normoxia and hypoxia through SENP1-mediated inhibition of HIF1α ZEB1 and Akt will be investigated for its ability to penetrate and silence SENP1 expression in ex vivo human OS tissues
Primary aim
To determine the ability of PNA-CPP to penetrate into an ex vivo tridimensional tissue of OS derived from wasted biological material obtained during OS eradication surgery and to exert its biological function of inhibiting SENP1 within the tissue
Study design
For this study wasted biological material derived from osteosarcoma eradication surgery will be collected which comprise only a small proportion of the removed tumor mass other than that used for histological and molecular diagnosis
15 patients with primary OS will be recruited The target group will comprise patients hospitalized at IRCCS Istituto Ortopedico Galeazzi who will be subjected to surgical eradication of primary OS
The study will be presented to patients with age 18 years that can be recruited also in the IRCCS Istituto Ortopedico Galeazzi BioBanca protocol ethical committee approval n 29INT2017 by the surgeon These patients will sign two Informed Consents one for the BioBanca and one for the PNA-OS study
Patients with age 18 years will be additionally recruited besides the IRCCS Istituto Ortopedico Galeazzi BioBanca These patients will be considered eligible for the study if the legal tutor will sign the Informed Consents relative to the PNA-OS study
Also samples of OS already existing in the BioBanca as frozen samples preserved in liquid nitrogen at BioRep Service-Provider BioRep Srl Via Olgettina 60 20132 Milano will be used Every reasonable effort will be done to call these patients to sign a specific informed consent relative to this study
Since several practical issues eg unsuitableness or low amount of biological material can occur we envision the possibility to recruit additional patients until the achievement of 15 complete samples
The Study will start after approval of Ethical Committee and the estimated duration is 36 months divided as following
Timing for enrolment 24 month
Data analysis 12 month
Experimental design
Ex vivo analysis of the PNA-R8 silencing ability in osteosarcoma samples We will investigate whether senpPNA-R8 is able to penetrate into a tridimensional OS tissue and to exert its silencing effect
15 OS samples will be collected either in the context of the IRCCS Galeazzi BioBanca ethical committee approval n 29INT2017 or from newly recruited patients in collaboration with the CCOORR equip
Only wasted biological material derived from surgery will be used without any additional harm to the patients other than the surgery itself and the study does not involve any diagnostic aim or genetic profiling of the samples collected
OS samples already existing in the BioBanca as frozen samples preserved in liquid nitrogen at BioRep Service-Provider BioRep Srl Via Olgettina 60 20132 Milano will be used to determine the initial expression levels of SENP1 in OS by RT-qPCR For this samples will be homogenized total RNA will be extracted and RT-qPCR will be performed assaying for SENP1 expression levels
The remaining samples out of 15 freshly collected will be preserved in physiological solution until usage The OS samples will be cut in 3 mm3 pieces placed in a 24-multiwell culture plate and cultivated ex vivo as organotypic OS cultures in both normoxia and hypoxia-induced microenvironment under orbital rotation 21-23 Organotypic tumor tissue maintain the complexity of the original tissue with tumor cells being surrounded by their original microenvironment rather than artificial matrices and this system is particularly advantageous for ex vivo drug screening for studying drug uptake and molecular processes OS cultures will be treated with senpPNA-R8 and SENP1 expression in naïve and PNA-treated samples will be determined by RT-qPCR and immunohistochemistry in paraffin-embedded sections The ability of the PNA-R8 to penetrate within the hypoxic core of the OS samples will be assessed after incubation with scrPNA-R8-Fl sections will be immediately frozen -80C processed and analyzed by immunofluorescence
Samples will be analyzed and stored at Laboratorio di Biochimica Sperimentale e Biologia Molecolare at the Istituto Ortopedico Galeazzi for the whole duration of the study At the end of the study every residual material will be destroyed
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None