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Nucleotide sequence of the Smo gene around the triplet coding for the D477 position

Supplementary Material and Methods and Figure Legends

Chemical Structure and Molecular Data for MK-4101

Gene expression intensity data. Values shown represent average values for the various vehicle or treatment groups.

List of genes differentially regulated relative to untreated control at the various time points and treatment conditions and summary of pathway enrichment analysis using GSEA.

Background Clear cell RCC (ccRCC) accounts for approximately 75% of the renal cancer cases. Surgery treatment seems to be the best efficacious approach for the majority of patients. However, a consistent fraction (30%) of cases progress after surgery with curative intent. It is currently largely debated the use of adjuvant therapy for high-risk patients and the clinical and molecular parameters for stratifying beneficiary categories. In addition, the treatment of advanced forms lacks reliable driver biomarkers for the appropriated therapeutic choice. Thus, renal cancer patient management urges predictive molecular indicators and models for therapy-decision making. Methods Here, we developed and optimized new models and tools for ameliorating renal cancer patient management. We isolated from fresh tumor specimens heterogeneous multi-clonal populations showing epithelial and mesenchymal characteristics coupled to stem cell phenotype. These cells retained long lasting-tumor-propagating capacity provided a therapy monitoring approach in vitro and in vivo while being able to form parental tumors when orthotopically injected and serially transplanted in immunocompromised murine hosts. Results In line with recent evidence of multiclonal cancer composition, we optimized in vitro cultures enriched of multiple tumor-propagating populations. Orthotopic xenograft masses recapitulated morphology, grading and malignancy of parental cancers. High-grade but not the low-grade neoplasias, resulted in efficient serial transplantation in mice. Engraftment capacity paralleled grading and recurrence frequency advocating for a prognostic value of our developed model system. Therefore, in search of novel molecular indicators for therapy decision-making, we used Reverse-Phase Protein Arrays (RPPA) to analyze a panel of total and phosphorylated proteins in the isolated populations. Tumor-propagating cells showed several deregulated kinase cascades associated with grading, including angiogenesis and m-TOR pathways. Conclusions In the era of personalized therapy, the analysis of tumor propagating cells may help improve prediction of disease progression and therapy assignment. The possibility to test pharmacological response of ccRCC stem-like cells in vitro and in orthotopic models may help define a pharmacological profiling for future development of more effective therapies. Likewise, RPPA screening on patient-derived populations offers innovative approach for possible prediction of therapy response. Electronic supplementary material The online version of this article (10.1186/s13046-018-0874-4) contains supplementary material, which is available to authorized users.

Aberrant activation of the Hedgehog (Hh) pathway is responsible for the onset and progression of several malignancies. Small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector Gli1 have thus emerged recently as valuable anticancer agents. Here, we have designed, synthesized, and tested new Hh inhibitors taking advantage by the highly versatile and privileged isoflavone scaffold. The introduction of specific substitutions on the isoflavone's ring B allowed the identification of molecules targeting preferentially Smo or Gli1. Biological assays coupled with molecular modeling corroborated the design strategy, and provided new insights into the mechanism of action of these molecules. The combined administration of two different isoflavones behaving as Smo and Gli antagonists, respectively, in primary medulloblastoma (MB) cells highlighted the synergistic effects of these agents, thus paving the way to further and innovative strategies for the pharmacological inhibition of Hh signaling.

We synthesized 3-aroyl-1-arylpyrrole (ARAP) derivatives as potential anticancer agents having different substituents at the pendant 1-phenyl ring. Both the 1-phenyl ring and 3-(3,4,5-trimethoxyphenyl)carbonyl moieties were mandatory to achieve potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARAP 22 showed strong inhibition of the P-glycoprotein-overexpressing NCI-ADR-RES and Messa/Dx5MDR cell lines. Compounds 22 and 27 suppressed in vitro the Hedgehog signaling pathway, strongly reducing luciferase activity in SAG treated NIH3T3 Shh-Light II cells, and inhibited the growth of medulloblastoma D283 cells at nanomolar concentrations. ARAPs 22 and 27 represent a new potent class of tubulin polymerization and cancer cell growth inhibitors with the potential to inhibit the Hedgehog signaling pathway.

The Hedgehog (Hh) signaling pathway governs cell growth and tissue development. Malfunctioning of several Hh pathway components, including the key transcriptional effector Gli proteins, is responsible for the onset of several tumors. Gli proteins activity is finely controlled by multilayered regulatory mechanisms, the most prominent of which is their proteasome-dependent proteolytic cleavage or massive ubiquitin-mediated proteolysis. Here, we described multiple procedures to determine whether a Gli protein is ubiquitylated both in a cellular context and in vitro, in basal conditions or by different E3 ubiquitin ligases and whether these processes are associated to Gli proteasome degradation.

Aberrant activation of the Hedgehog (Hh) signaling pathway is implicated in the pathogenesis of many cancers, including medulloblastoma and basal cell carcinoma (BCC). In this study, using neonatally irradiated Ptch1+/− mice as a model of Hh-dependent tumors, we investigated the in vivo effects of MK-4101, a novel SMO antagonist, for the treatment of medulloblastoma and BCC. Results clearly demonstrated a robust antitumor activity of MK-4101, achieved through the inhibition of proliferation and induction of extensive apoptosis in tumor cells. Of note, beside antitumor activity on transplanted tumors, MK-4101 was highly efficacious against primary medulloblastoma and BCC developing in the cerebellum and skin of Ptch1+/− mice. By identifying the changes induced by MK-4101 in gene expression profiles in tumors, we also elucidated the mechanism of action of this novel, orally administrable compound. MK-4101 targets the Hh pathway in tumor cells, showing the maximum inhibitory effect on Gli1. MK-4101 also induced deregulation of cell cycle and block of DNA replication in tumors. Members of the IGF and Wnt signaling pathways were among the most highly deregulated genes by MK-4101, suggesting that the interplay among Hh, IGF, and Wnt is crucial in Hh-dependent tumorigenesis. Altogether, the results of this preclinical study support a therapeutic opportunity for MK-4101 in the treatment of Hh-driven cancers, also providing useful information for combination therapy with drugs targeting pathways cooperating with Hh oncogenic activity. Mol Cancer Ther; 15(6); 1177–89. ©2016 AACR.

Hedgehog (Hh) signaling has emerged in recent years as an attractive target for anticancer therapy because its aberrant activation is implicated in several cancers. Major progress has been made in the development of SMOOTHENED (SMO) antagonists, although they have shown several limitations due to downstream SMO pathway activation or the occurrence of drug-resistant SMO mutations. Recently, particular interest has been elicited by the identification of molecules able to hit glioma-associated oncogene (GLI) factors, the final effectors of the Hh pathway, which provide a valid tool to overcome anti-SMO resistance. Here, we review results achieved in developing GLI antagonists, explaining their mechanisms of action and highlighting their therapeutic potential. We also underline the relevance of structural details in their discovery and optimization.

Hedgehog (Hh) inhibitors have emerged as valid tools in the treatment of a wide range of cancers. Indeed, aberrant activation of the Hh pathway occurring either by ligand-dependent or -independent mechanisms is a key driver in tumorigenesis. The smoothened (Smo) receptor is one of the main upstream transducers of the Hh signaling and is a validated target for the development of anticancer compounds, as underlined by the FDA-approved Smo antagonist Vismodegib (GDC-0449/Erivedge) for the treatment of basal cell carcinoma. However, Smo mutations that confer constitutive activity and drug resistance have emerged during treatment with Vismodegib. For this reason, the development of new effective Hh inhibitors represents a major challenge for cancer therapy. Natural products have always represented a unique source of lead structures in drug discovery, and in recent years have been used to modulate the Hh pathway at multiple levels. Here, starting from an in house library of natural compounds and their derivatives, we discovered novel chemotypes of Hh inhibitors by mean of virtual screening against the crystallographic structure of Smo. Hh functional based assay identified the chalcone derivative 12 as the most effective Hh inhibitor within the test set. The chalcone 12 binds the Smo receptor and promotes the displacement of Bodipy-Cyclopamine in both Smo WT and drug-resistant Smo mutant. Our molecule stands as a promising Smo antagonist able to specifically impair the growth of Hh-dependent tumor cells in vitro and in vivo and medulloblastoma stem-like cells and potentially overcome the associated drug resistance.