Primary Osteosarcoma of Bone Clinical Trial
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)
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.
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 Sentrin/SUMO-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
(e.g. 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, i.e. 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.
29/INT/2017) 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 S.r.l. 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 (e.g. 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. 29/INT/2017) or from newly recruited patients, in collaboration with
the C.C.O.O.R.R. 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 S.r.l. 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 (-80°C),
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.
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