Lucia D'Amico

The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL

Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)-ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

Microenvironment modulated metastatic CD133+/CXCR4+/EpCAM- lung cancer initiating cells sustain tumor dissemination and correlate with poor prognosis

Metastasis is the main reason for lung cancer-related mortality, but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here, we show that CD133(+)/CXCR4(+) cancer-initiating cells (CIC) directly isolated from patient-derived xenografts (PDX) of non-small cell lung cancer are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133(+)/CXCR4(+) cells in residual tumors and their metastatization. Immunophenotypic analysis of lung tumor cells intravenously injected or spontaneously disseminated to murine lungs demonstrated the survival advantage and increased colonization ability of a specific subset of CD133(+)/CXCR4(+) with reduced expression of epithelial cell adhesion molecule (EpCAM(-)), which also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cells from lungs of PDX-bearing mice enriched for CD133(+)/CXCR4(+)/EpCAM(-) CICs are highly tumorigenic and metastatic. Importantly, microenvironment stimuli eliciting epithelial-to-mesenchymal transition, including signals from cancer-associated fibroblasts, are able to increase the dissemination potential of lung cancer cells through the generation of the CD133(+)/CXCR4(+)/EpCAM(-) subset. These findings also have correlates in patient samples where disseminating CICs are enriched in metastatic lymph nodes (20-fold, P = 0.006) and their detection in primary tumors is correlated with poor clinical outcome (disease-free survival: P = 0.03; overall survival: P = 0.05). Overall, these results highlight the importance of specific cellular subsets in the metastatic process, the need for in-depth characterization of disseminating tumor cells, and the potential of therapeutic strategies targeting both primary tumor and tumor-microenvironment interactions.

Bone invading NSCLC cells produce IL-7: mice model and human histologic data

BackgroundBone metastases are a common and dismal consequence of lung cancer that is a leading cause of death. The role of IL-7 in promoting bone metastases has been previously investigated in NSCLC, but many aspects remain to be disclosed. To further study IL-7 function in bone metastasis, we developed a human-in-mice model of bone aggression by NSCLC and analyzed human bone metastasis biopsies.MethodsWe used NOD/SCID mice implanted with human bone. After bone engraftment, two groups of mice were injected subcutaneously with A549, a human NSCLC cell line, either close or at the contralateral flank to the human bone implant, while a third control group did not receive cancer cells. Tumor and bone vitality and IL-7 expression were assessed in implanted bone, affected or not by A549. Serum IL-7 levels were evaluated by ELISA. IL-7 immunohistochemistry was performed on 10 human bone NSCLC metastasis biopsies for comparison.ResultsAt 12 weeks after bone implant, we observed osteogenic activity and neovascularization, confirming bone vitality. Tumor aggressive cells implanted close to human bone invaded the bone tissue. The bone-aggressive cancer cells were positive for IL-7 staining both in the mice model and in human biopsies. Higher IL-7 serum levels were found in mice injected with A549 cells close to the bone implant compared to mice injected with A549 cells in the flank opposite to the bone implant.ConclusionsWe demonstrated that bone-invading cells express and produce IL-7, which is known to promote osteoclast activation and osteolytic lesions. Tumor-bone interaction increases IL-7 production, with an increase in IL-7 serum levels. The presented mice model of bone invasion by contiguous tumor is suitable to study bone-tumor cell interaction. IL-7 plays a role in the first steps of metastatic process.

Cross-talk between T cells and osteoclasts in bone resorption

Osteoclasts (OCs) are the exclusive bone resorptive cell, they derive from monocyte/macrophage precursors, which can circulate within the hematopoietic cell pool or be resident in a number of tissues. The maintenance of an adequate bone mass depends on the controlled and timely removal of old, damaged bone. The increase of OC activity is observed in many pathologies characterised by bone loss, such as osteoporosis, rheumatoid arthritis, bone metastasis, periprosthetic osteolysis in aseptic loosening of arthroplasty and also in pediatric diseases, such as phenilketonuria and 21-hydroxylase deficiency. During the bone resorption process there is an intense cross-talk between immune system cells and OCs. In particular, T cells release factors and cytokines, which rule osteoclastogenesis, and on the other hand, OCs produce factors that act on T cells. A primary mediator of osteoclastogenesis is the receptor activator of nuclear factor-κβ-RANK ligand-osteoprotegerin system, but also other cytokines promote OC activation according to the different pathologies. This review summarizes the main mechanisms promoting osteoclastogenesis in diseases characterised by bone loss, focusing on factors and cytokines involved in this process and on the interaction between OCs and T cells.

The EGFR family members sustain the neoplastic phenotype of ALK+ lung adenocarcinoma via EGR1

In non-small cell lung cancer (NSCLC), receptor tyrosine kinases (RTKs) stand out among causal dominant oncogenes, and the ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nonetheless, long-term RTK inhibition leads invariably to acquired resistance, tumor recurrence and metastatic dissemination. In ALK+ cell lines, inhibition of ALK signaling was associated with coactivation of several RTKs, whose pharmacological suppression reverted the partial resistance to ALK blockade. Remarkably, ERBB2 signaling synergized with ALK and contributed to the neoplastic phenotype. Moreover, the engagement of wild-type epidermal growth factor receptor or MET receptors could sustain cell viability through early growth response 1 (EGR1) and/or Erk1/2; Akt activation and EGR1 overexpression prevented cell death induced by combined ALK/RTK inhibition. Membrane expression of ERBB2 in a subset of primary naive ALK+ NSCLC could be relevant in the clinical arena. Our data demonstrate that the neoplastic phenotype of ALK-driven NSCLC relays ‘ab initio’ on the concomitant activation of multiple RTK signals via autocrine/paracrine regulatory loops. These findings suggest that molecular and functional signatures are required in de novo lung cancer patients for the design of efficacious and multi-targeted ‘patient-specific’ therapies.

Efficacy of a Cancer Vaccine against ALK-Rearranged Lung Tumors

Lung cancers harboring ALK translocations are treated with protein kinase inhibitors, which can extend survival. A cancer vaccine against ALK induced strong immune responses and enhanced survival when used alone, or in combination with kinase inhibitors or checkpoint inhibitors. Non–small cell lung cancer (NSCLC) harboring chromosomal rearrangements of the anaplastic lymphoma kinase (ALK) gene is treated with ALK tyrosine kinase inhibitors (TKI), but the treatment is successful for only a limited amount of time; most patients experience a relapse due to the development of drug resistance. Here, we show that a vaccine against ALK induced a strong and specific immune response that both prophylactically and therapeutically impaired the growth of ALK-positive lung tumors in mouse models. The ALK vaccine was efficacious also in combination with ALK TKI treatment and significantly delayed tumor relapses after TKI suspension. We found that lung tumors containing ALK rearrangements induced an immunosuppressive microenvironment, regulating the expression of PD-L1 on the surface of lung tumor cells. High PD-L1 expression reduced ALK vaccine efficacy, which could be restored by administration of anti–PD-1 immunotherapy. Thus, combinations of ALK vaccine with TKIs and immune checkpoint blockade therapies might represent a powerful strategy for the treatment of ALK-driven NSCLC. Cancer Immunol Res; 3(12); 1333–43. ©2015 AACR.

Iloprost modulates the immune response in systemic sclerosis

BackgroundIloprost has been suggested to possess anti-inflammatory and immunomodulating actions and it is widely use as a vasodilatator in systemic sclerosis (SSc). In this study we evaluate the effect of iloprost on immune response in SSc patients. To this extend we enrolled 15 women affected by SSc and infused iloprost for 5 days. The effect of iloprost on T cells and monocytes was measured by flow cytometry, Real time PCR and measuring cytokines production in vivo and in vitro by ELISA.ResultsOur results demonstrate that Iloprost reduces T cell and TNF alpha production both in vivo and in vitro. It reduces T regulatory cells number, but increases their activity after immune stimulation. It increases serum IL-2 and this increase persists 28 days after the last infusion, also RANKL was increased both in vivo and in vitro. We observed no effect on IFN gamma production.ConclusionsThese results suggest that iloprost has anti-inflammatory and immunomodulating effects, reducing TNF alpha production by T cells and the number of T regulatory cells and increasing IL-2 and RANKL.

C-met inhibition blocks bone metastasis development induced by renal cancer stem cells.

Cancer stem cells (CSCs) are key players in bone metastasis. In some renal tumors CSCs overexpress the HGF receptor c-MET, speculating that c-MET targeting could lead to bone metastasis inhibition. To address this hypothesis we isolated renal CD105+/CD24−CSCs, expressing c-MET receptor from a primary renal carcinoma. Then, to study their ability to metastasize to bone, we injected renal CSCs in NOD/SCID mice implanted with a human bone and we tested the effect of a c-MET inhibitor (JNJ-38877605) on bone metastasis development. JNJ-38877605 inhibited the formation of metastases at bone implant site. We showed that JNJ-38877605 inhibited the activation of osteoclasts induced by RCC stem cells and it stimulated osteoblast activity, finally resulting in a reduction of bone turnover consistent with the inhibition of bone metastases. We measured the circulating levels of osteotropic factors induced by RCC stem cells in the sera of mice treated with c-Met inhibitor, showing that IL-11 and CCL20 were reduced in mice treated with JNJ-38877605, strongly supporting the involvement of c-MET in the regulation of this process. To address the clinical relevance of c-MET upregulation during tumor progression, we analysed c-MET in renal cancer patients detecting an increased expression in the bone metastatic lesions by IHC. Then, we dosed CCL20 serum levels resulting significantly increased in patients with bone metastases compared to non-metastatic ones. Collectively, our data highlight the importance of the c-MET pathway in the pathogenesis of bone metastases induced by RCC stem cells in mice and humans.

The Impact of Immune System in Regulating Bone Metastasis Formation by Osteotropic Tumors

Bone metastases are frequent and debilitating consequence for many tumors, such as breast, lung, prostate, and kidney cancer. Many studies report the importance of the immune system in the pathogenesis of bone metastasis. Indeed, bone and immune system are strictly linked to each other because bone regulates the hematopoietic stem cells from which all cells of the immune system derive, and many immunoregulatory cytokines influence the fate of bone cells. Furthermore, both cytokines and factors produced by immune and bone cells promote the growth of tumor cells in bone, contributing to supporting the vicious cycle of bone metastasis. This review summarizes the current knowledge on the interactions among bone, immune, and tumor cells aiming to provide an overview of the osteoimmunology field in bone metastasis from solid tumors.

Abstract LB-309: EML4-ALK signaling is required for the maintenance of neoplastic phenotype of non-small cell lung cancer cells: Novel strategy for lung cancer-tailored therapies

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Lung cancer is the most common cancer in the world, and is lethal in 90% of the cases. In non-small cell lung cancer (NSCLC), deregulated receptor tyrosine kinases (RTKs) stand out among the causal dominant oncogenes. Consequently, the genomic and/or pharmacological ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nevertheless, this approach has serious constrains, probably due to the frequent co-activation of multiple RTKs in a considerable subsets of solid tumors. It has become also evident that during TK inhibitor-based regimens, RTK switching and/or the selection of resistant clones can frequently occur representing a serious and problematic drawback. In a subset of NSCLC, the Anaplastic Lymphoma Kinase (ALK) gene has been described to be translocated and fused to EML4. This determines the ALK constitutive dimerization and autophosphorylation leading to cellular transformation. Here, we investigated the ALK oncogenic addiction of human NSCLC and studied the putative co-operative role of other kinases. We first demonstrated that the ectopic expression of EML4-ALK in ALK positive NSCLC cell lines (H2228 and H3122) resulted in the activation of multiple signaling pathways, in manner similar to that described for other known ALK fusions. Although EML4-ALK can induce transformation in lung in vitro and in vivo, ALK inhibition via shRNA or small molecule inhibitors (CEPs, Cephalon) induced only the apoptosis of H3122 cells, whereas in H2228 it caused cell growth arrest. Moreover, the treatment with ALK inhibitors led to tumor regression, but not tumor eradication, in vivo. Based on Phosphoproteomic analyses we demonstrated that the phosphorylation status of several tyrosine kinases (such as, EGFR, Met, FGFR, Jak1 or IGFR) was affected by the ALK inhibition only in H2228 cell line, but not in H3122. Notably, the combined treatment with anti-ALK ([CEP14083][1], CEP2550, [CEP28122][2]) and -EGFR inhibitors resulted in an increased cell death of H2228 cells to values similar to those observed for ALK treated H3122 cells. Finally, gene expression analyses showed that known EGFR substrates were specifically down regulated upon the combined treatment in ALK positive H2228 cells. Our findings suggest that ALK signaling is required for the maintenance of the neoplastic phenotype of some ALK positive NSCLC cells and that its abrogation could represent a novel strategy for the treatment of a well-defined subset of human lung cancer (complete ALK addiction). More importantly, we showed that the tumor survival and maintenance of ALK positive neoplastic cells might relay on the concomitant activation of multiple RTKs. These findings further support the notion that molecular and functional signatures are required for designing molecular-based “patient specific” therapeutic protocols in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-309. [1]: /lookup/external-ref?link_type=GENPEPT&access_num=CEP14083&atom=%2Fcanres%2F70%2F8_Supplement%2FLB-309.atom [2]: /lookup/external-ref?link_type=GENPEPT&access_num=CEP28122&atom=%2Fcanres%2F70%2F8_Supplement%2FLB-309.atom