The insulin-like growth factor (IGF) system is a dynamic network of proteins, which includes cognate ligands, membrane receptors, ligand binding proteins and functional downstream effectors. It plays ...a critical role in regulating several important physiological processes including cell growth, metabolism and differentiation. Importantly, alterations in expression levels or activation of components of the IGF network are implicated in many pathological conditions including diabetes, obesity and cancer initiation and progression. In this review we will initially cover some general aspects of IGF action and regulation in cancer and then focus in particular on the role of transcriptional regulators and novel interacting proteins, which functionally contribute in fine tuning IGF1R signaling in several cancer models. A deeper understanding of the biological relevance of this network of IGF1R modulators might provide novel therapeutic opportunities to block this system in neoplasia.
Osteosarcoma, the most common primary bone tumor, is characterized by an aggressive behavior with high tendency to develop lung metastases as well as by multiple genetic aberrations that have ...hindered the development of targeted therapies. New therapeutic approaches are urgently needed; however, novel combinations with immunotherapies and checkpoint inhibitors require suitable preclinical models with intact immune systems to be properly tested.
We have developed immunocompetent osteosarcoma models that grow orthotopically in the bone and spontaneously metastasize to the lungs, mimicking human osteosarcoma. These models have been used to test the efficacy of trabectedin, a chemotherapeutic drug utilized clinically for sarcomas and ovarian cancer.
Trabectedin, as monotherapy, significantly inhibited osteosarcoma primary tumor growth and lung metastases by both targeting neoplastic cells and reprogramming the tumor immune microenvironment. Specifically, trabectedin induced a striking differentiation of tumor cells by favoring the recruitment of Runx2, the master genetic regulator of osteoblastogenesis, on the promoter of genes involved in the physiologic process of terminal osteoblast differentiation. Differentiated neoplastic cells, as expected, showed reduced proliferation rate. Concomitantly, trabectedin enhanced the number of tumor-infiltrating T lymphocytes, with local CD8 T cells, however, likely post-activated or exhausted, as suggested by their high expression of the inhibitory checkpoint molecule PD-1. Accordingly, the combination with a PD-1-blocking antibody significantly increased trabectedin efficacy in controlling osteosarcoma progression.
These results demonstrate the therapeutic efficacy of trabectedin in osteosarcoma treatment, unveiling its multiple activities and providing a solid rationale for its combination with immune checkpoint inhibitors.
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Ewing’s sarcoma family of tumors (ESFT) are aggressive neoplasms with scant tumor-infiltrating lymphocytes. We analyzed the immunohistochemical (IHC) expression of PD-L1 and PD-1 and their prognostic ...significance in clinically localized neoplasms in a cohort of 370 ESFT. Slides prepared from tissue microarrays were stained for PD-L1, PD-1, and CD8. Membranous/cytoplasmic staining over 5% of tumor cells was regarded as positive for PD-L1 and PD-1. Prognostic analysis was done considering only clinically localized tumors (
n
= 217). PD-L1 expression was present in 19% of ESFT, while PD-1 was expressed in 26%. Forty-eight percent of tumors were negative and 12% were positive for both PD-L1 and PD-1. Metastatic tumors displayed higher expression of PD-L1 (
p
< 0.0001). Histological subtypes were not correlated with PD-L1 or PD-1 positivity. ESFT with elevated proliferation index (Ki-67) were associated with higher PD-L1 expression (
p
= 0.049). Regarding prognosis, no significant association was found between PD-L1 expression and progression-free survival (PFS) or overall survival (OS), whereas lack of PD-1 expression in tumor cells was correlated with both poor PFS (
p
= 0.02) and poor OS (
p
= 0.004). Tumor-infiltrating CD8(+) T lymphocytes were observed in 15.4% of ESFT with informative results (347 tumors). No correlation was found between tumor-infiltrating CD8(+) T lymphocytes and ESFT histological subtypes, tumor location, or PD-1 and PD-L1 expression, nor with PFS (
p
= 0.473) or OS (
p
= 0.087). PD-L1 expression was not significantly related to prognosis. PD-1 was expressed in 26% of ESFT tumor cells and may have prognostic and therapeutic implications. CD8 expression in tumor-infiltrating lymphocytes was not related to prognosis.
Lamin A, a main constituent of the nuclear lamina, is involved in mechanosignaling and cell migration through dynamic interactions with the LINC complex, formed by the nuclear envelope proteins SUN1, ...SUN2 and the nesprins. Here, we investigated lamin A role in Ewing Sarcoma (EWS), an aggressive bone tumor affecting children and young adults. In patients affected by EWS, we found a significant inverse correlation between LMNA gene expression and tumor aggressiveness. Accordingly, in experimental in vitro models, low lamin A expression correlated with enhanced cell migration and invasiveness and, in vivo, with an increased metastatic load. At the molecular level, this condition was linked to altered expression and anchorage of nuclear envelope proteins and increased nuclear retention of YAP/TAZ, a mechanosignaling effector. Conversely, overexpression of lamin A rescued LINC complex organization, thus reducing YAP/TAZ nuclear recruitment and preventing cell invasiveness. These effects were also obtained through modulation of lamin A maturation by a statin-based pharmacological treatment that further elicited a more differentiated phenotype in EWS cells. These results demonstrate that drugs inducing nuclear envelope remodeling could be exploited to improve therapeutic strategies for EWS.
Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A ...panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient's tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches.
Synovial sarcoma (SyS) is a rare aggressive soft tissue sarcoma carrying the chromosomal translocation t(X;18), encoding the fusion transcript SS18::SSX. The fusion oncoprotein interacts with both ...BAF enhancer complexes and polycomb repressor complexes, resulting in genome-wide epigenetic perturbations and a unique altered genetic signature. Over 80% of the patients are initially diagnosed with localized disease and have a 5-year survival rate of 70-80%, but metastatic relapse occurs in 50% of the cases. Advanced, unresectable, or metastatic disease has a 5-year survival rate below 10%, representing a critical issue. This review summarizes the molecular mechanisms behind SyS and illustrates current treatments in front line, second line, and beyond settings. We analyze the use of immune check point inhibitors (ICI) in SyS that do not behave as an ICI-sensitive tumor, claiming the need for predictive genetic signatures and tumor immune microenvironment biomarkers. We highlight the clinical translation of innovative technologies, such as proteolysis targeting chimera (PROTAC) protein degraders or adoptive transfer of engineered immune cells. Adoptive cell transfer of engineered T-cell receptor cells targeting selected cancer/testis antigens has shown promising results against metastatic SyS in early clinical trials and further improvements are awaited from refinements involving immune cell engineering and tumor immune microenvironment enhancement.
Ewing sarcoma (ES) is an aggressive neoplasm with variable morphology. It has no specific immunoprofile or molecular signature. Neither CD99, NKX2.2 nor PAX7 immunoreactivity alone is completely ...specific, although diagnostic specificity improves when combined. The purpose of the present study was to investigate the immunohistochemical (IHC) expression of PAX7 in a large series of genetically confirmed ES. Existing results for CD99 and NKX2.2 immunoexpression, morphological findings and molecular studies (fusion gene subtypes) were retrieved from a previous study. Survival analyses were performed in cases with available clinical follow-up. PAX7 was positive in 95.5% of ES with diffuse staining (> 50%) in all positive cases and moderate or strong intensity for most cases. Nineteen ES displayed both PAX7 and CD99 immunoreactivity but lacked NKX2.2 immunoexpression. No relationships could be found between PAX7 expression and the histological types or ES gene fusion subtypes. Univariant/multivariate analysis showed that lack of PAX7 and/or NKX2.2 immunoexpression constitute independent poor prognostic factors for progression free survival (PFS) and overall survival (OS). In conclusion, IHC for CD99, NKX2.2, and PAX7 may be useful in daily practice for ES diagnosis, particularly in hospitals lacking facilities for molecular studies. In addition, the combination of strong CD99 membranous positivity and nuclear PAX7 and NKX2.2 immunoreactivity seems to be very reliable for ES diagnosis when supported by a corroborating histomorphologic and clinical picture. Although PAX7 is not entirely specific for ES, it seems to have a more extensive and strong nuclear immunoreactivity than NKX2.2 expression, even in tumors with decalcification artifact. Considering the prognostically significant data herein reported, we strongly recommend validation in prospective ES series that include localized and disseminated tumors.
Cisplatin (CDDP) is a drug for high-grade osteosarcoma (HGOS) treatment. Several germline pharmacogenetic studies have revealed associations between single nucleotide polymorphisms (SNPs) and ...CDDP-based therapy response or CDDP-related toxicity in patients with HGOS. Whether these variants could play a biological role in HGOS cells has not been studied so far. The aim of this study was to explore 28 SNPs of 14 genes in 6 CDDP-resistant and 12 drug-sensitive human HGOS cell lines. An innovative multimodal targeted next generation sequencing (mmNGS) approach with custom primers designed for the most commonly reported SNPs of genes belonging to DNA repair, CDDP transport or detoxification, or associated with CDPP-related toxicity was applied. The mmNGS approach was validated by TaqMan genotyping assays and emerged to be an innovative, reliable tool to detect genetic polymorphisms at both the DNA and RNA level. Allele changes in three SNPs (ERCC2 rs13181 and rs1799793, ERCC1 rs11615) were identified on both DNA and RNA derived libraries in association with CDDP resistance. A change of the GSTP1 rs1695 polymorphism from AA to AG genotype was observed in the RNA of all six CDDP-resistant variants. These SNPs emerged to be causally associated with CDDP resistance in HGOS cells.
Synovial sarcomas (SyS) are rare malignant tumors predominantly affecting children, adolescents, and young adults. The genetic hallmark of SyS is the t(X;18) translocation encoding the SS18-SSX ...fusion gene. The fusion protein interacts with both the BAF enhancer and polycomb repressor complexes, and either activates or represses target gene transcription, resulting in genome-wide epigenetic perturbations and altered gene expression. Several experimental in in vivo models, including conditional transgenic mouse models expressing the SS18-SSX fusion protein and spontaneously developing SyS, are available. In addition, patient-derived xenografts have been estab-lished in immunodeficient mice, faithfully reproducing the complex clinical heterogeneity. This review focuses on the main molecular features of SyS and the related preclinical in vivo and in vitro models. We will analyze the different conditional SyS mouse models that, after combination with some of the few other recurrent alterations, such as gains in BCL2, Wnt-β-catenin signaling, FGFR family, or loss of PTEN and SMARCB1, have provided additional insight into the mechanisms of synovial sarcomagenesis. The recent advancements in the understanding of SyS biology and improvements in preclinical modeling pave the way to the development of new epigenetic drugs and immunotherapeutic approaches conducive to new treatment options.
High-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase ...inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in
cocultures of sarcoma.
The human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.
Along with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.
Taken together, our
data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.
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