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Supplementary Table 1 from Dual-Specificity Phosphatase 1 Ubiquitination in Extracellular Signal-Regulated Kinase–Mediated Control of Growth in Human Hepatocellular Carcinoma

Supplementary Figure 5 from Dual-Specificity Phosphatase 1 Ubiquitination in Extracellular Signal-Regulated Kinase–Mediated Control of Growth in Human Hepatocellular Carcinoma

Supplementary Figure 3 from Dual-Specificity Phosphatase 1 Ubiquitination in Extracellular Signal-Regulated Kinase–Mediated Control of Growth in Human Hepatocellular Carcinoma

Supplementary Figure 4 from Dual-Specificity Phosphatase 1 Ubiquitination in Extracellular Signal-Regulated Kinase–Mediated Control of Growth in Human Hepatocellular Carcinoma

Supplementary Table 3 from Dual-Specificity Phosphatase 1 Ubiquitination in Extracellular Signal-Regulated Kinase–Mediated Control of Growth in Human Hepatocellular Carcinoma

Supplementary Table 2 from Dual-Specificity Phosphatase 1 Ubiquitination in Extracellular Signal-Regulated Kinase–Mediated Control of Growth in Human Hepatocellular Carcinoma

Sustained activation of extracellular signal-regulated kinase (ERK) has been detected previously in numerous tumors in the absence of RAS-activating mutations. However, the molecular mechanisms responsible for ERK-unrestrained activity independent of RAS mutations remain unknown. Here, we evaluated the effects of the functional interactions of ERK proteins with dual-specificity phosphatase 1 (DUSP1), a specific inhibitor of ERK, and S-phase kinase-associated protein 2 (SKP2)/CDC28 protein kinase 1b (CKS1) ubiquitin ligase complex in human hepatocellular carcinoma (HCC). Levels of DUSP1, as assessed by real-time reverse transcription–PCR and Western blot analysis, were significantly higher in tumors with better prognosis (as defined by the length of patients' survival) when compared with both normal and nontumorous surrounding livers, whereas DUSP1 protein expression sharply declined in all HCC with poorer prognosis. In the latter HCC subtype, DUSP1 inactivation was due to either ERK/SKP2/CKS1-dependent ubiquitination or promoter hypermethylation associated with loss of heterozygosity at the DUSP1 locus. Noticeably, expression levels of DUSP1 inversely correlated with those of activated ERK, as well as with proliferation index and microvessel density, and directly with apoptosis and survival rate. Subsequent functional studies revealed that DUSP1 reactivation led to suppression of ERK, CKS1, and SKP2 activity, inhibition of proliferation and induction of apoptosis in human hepatoma cell lines. Taken together, the present data indicate that ERK achieves unrestrained activity during HCC progression by triggering ubiquitin-mediated proteolysis of its specific inhibitor DUSP1. Thus, DUSP1 may represent a valuable prognostic marker and ERK, CKS1, or SKP2 potential therapeutic targets for human HCC. [Cancer Res 2008;68(11):4192–200]

Supplementary Figure 2 from Dual-Specificity Phosphatase 1 Ubiquitination in Extracellular Signal-Regulated Kinase–Mediated Control of Growth in Human Hepatocellular Carcinoma

Supplementary Figure 1 from Dual-Specificity Phosphatase 1 Ubiquitination in Extracellular Signal-Regulated Kinase–Mediated Control of Growth in Human Hepatocellular Carcinoma

Breast Cancer (BC) is the leading cause of death due to cancer in women. Ensuring equitable, quality-assured and effective care has increased the complexity of BC management. This systematic review reports on the state-of-the art of available literature investigating the enactment of personalized treatment and patient-centered care models in BC clinical practice, building a framework for the delivery of personalized BC care within a Patient-Centered model. Databases were searched for articles (from the inception to December 2020) reporting on Patient-Centered or Personalized Medicine BC management models, assessing success factors or limits. Out of 1885 records, 25 studies were included in our analysis. The main success factors include clearly defined roles and responsibilities within a multi-professional collaboration, appropriate training programs and adequate communication strategies and adopting a universal genomic language to improve patients’ involvement in the decision-making process. Among detected barriers, delays in the use of genetic testing were linked to the lack of public reimbursement schemes and of clear indications in timing and appropriateness. Overall, both care approaches are complementary and necessary to effectively improve BC patient management. Our framework attempts to bridge the gap in assigning a central role played by shared decision-making, still scarcely investigated in literature.

Hepatocellular carcinoma (HCC) represents the predominant primary liver malignancy and the second most common cause of cancer-related death worldwide. Although several advances have been made in HCC screening and diagnosis, the therapeutic treatment of advanced disease at present relies mainly on sorafenib and regorafenib, two multi-kinase inhibitors, which demonstrate limited clinical efficacy. Recently, several genomic studies have unveiled the underlying genomic changes governing HCC development, consisting of mutations in prominent driver genes, as well as copy-number variations (CNVs), such as amplifications and deletions of whole chromosome regions, which exceed by far the frequency of individual mutations. Interestingly, CNVs are considered to mediate clonal expansion due to the presence of oncogenic driver genes, but generally comprise also neutral bystander genes with no implication in neoplastic transformation. While current therapeutic approaches largely aim at targeting mutation-induced dysregulated pathways, strategies targeting CNVs are not thoroughly pursued. We hypothesized that passenger aberrations in genomic amplifications of tumor cells could be therapeutically exploited by providing actionable molecules on the cell surface. To this end, we performed bioinformatic analyses of TCGA CNV data and uncovered a list of cell surface protein (CSP) genes which are frequently amplified in HCC and could be used as potential candidates for a “Trojan-horse” approach in this tumor entity. More specifically, a set of CSP genes was found to be amplified in more than 60% of HCCs, as well as other solid tumors. Genetic perturbations of their expression levels had no effect on the growth and proliferation of HCC cell lines, suggesting their passenger status in the context of this malignancy. Furthermore, amplification of CSP-genes resulted in increased protein expression, which was localized primarily at the plasma membrane. Interestingly, a wide range of normal tissues displayed absent to low cytoplasmic levels of these CSPs as compared to several solid tumors, which exhibited diverse cell surface-associated expression. In addition, pharmacological inhibition of various oncogenic signaling pathways in cancer cells led to significant CSP upregulation at the protein level in the absence of respective genomic amplifications, raising the therapeutic prospect of targeting these proteins as second hits in combination drug regimens. In summary, we have identified several CSPs as potential targets in HCC, which could achieve high tumor specificity while sparing normal tissue when targeted by anti-CSP radioisotope-conjugated antibodies or CSP-specific CAR-T cells. Citation Format: Christos Patsis, Joan Crous-Masó, Ilaria Salvato, Luise Butthof, Rossella Pellegrino, Thomas Longerich, Matthias Gaida, Petros Christopoulos, Darjus Tschaharganeh. Therapeutic exploitation of passenger amplifications in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4476.

Background & Aims: The Mouse Double Minute homolog 4 (MDM4) is one of the main negative p53 regulators in mammalian cells and mutational inactivation of p53 is a rare event in Western hepatocellular carcinomas (HCCs). 1q gains promote the upregulation of MDM4 in HCC, leading to a negative regulation of p53 activity. However, additional mechanisms might be involved that explain the increased MDM4 activity observed in HCCs with balanced MDM4 gene locus. Here we aimed at the identification of post-transcriptional mechanisms involved in the upregulation of MDM4 in HCC. Methods: To investigate a potential role of PI3K-AKT-mTOR signaling on MDM4 activity, HCC cell lines were treated with small molecule inhibitors and siRNAs and expression changes as well as functional analyses were recorded. Expression changes of central pathway components were investigated in human liver samples (normal liver, peritumorous liver tissue, and HCC). An AKT transgenic mouse model was used to show the involvement of AKT pathway in the regulation of MDM4 in vivo. Results: The inhibition of both PI3K-AKT and mTOR signaling pathways resulted in reduced MDM4 protein levels in HCC cell lines, which was associated with the transcriptional activation of p53-target genes. Biochemical assays revealed that both AKT-mediated phosphorylation and ubiquitin-specific protease 2a (USP2a)-mediated deubiquitination protected MDM4 from proteasomal in human HCC cell lines. In addition, AKT transgenic mice showed increased MDM4 protein levels indicating that AKT signaling is involved in the stabilization of MDM4 protein in vivo. Furthermore, the Eukaryotic translational Elongation Factor 1 alpha 2 (EEF1A2), which is frequently upregulated in human HCC, sustained the PI3K-AKT-mTOR cascade both in vitro and in vivo. In human HCCs, a strong positive correlation between the overexpression of EEF1A2, pAKT, USP2a, and MDM4 was observed, which was associated with shorter survival of HCC patients. Conclusions: Our data demonstrate that the EEF1A2/PI3K/AKT/mTOR cascade inactivates wild-type p53 in human HCC through the stabilization of the MDM4 protooncogene via a post-transcriptional mechanism involving an AKT-mediated phosphorylation of MDM4 and USP2a-mediated de-ubiquitination. Since the sustained activation of the EEF1A2/PI3K/AKT/mTOR/MDM4 axis has impact on the survival probability of HCC patients, it may thus represent a promising therapeutic target. Citation Format: Rossella Pellegrino, Diego F. Calvisi, Olaf Neumann, Xin Chen, Chunmei Wang, Bernhard Radlwimmer, Sara Ladu, Frank Dombrowski, Matthias Evert, Peter Schirmacher, Thomas Longerich. The EEF1A2-PI3K-AKT-mTOR axis supports the protumorigenic function of MDM4 in human hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4300. doi:10.1158/1538-7445.AM2013-4300