Corosolic Acid Inhibits Cancer Progress Through Inactivating YAP in Hepatocellular Carcinoma

Chemotherapy is critical for the treatment of hepatocellular carcinoma (HCC). Despite the proapoptotic effects of corosolic acid (CA) treatment, its underlying mechanism is not completely clear. The aim of this study was to determine the molecular mechanism of CA in HCC treatment. MTT assay was used to determine the IC50 of CA. Immunoprecipitation and immunofluorescence were used to detect the interaction and subcellular localization of Yes-associated protein (YAP) and mouse double minute 2 (MDM2). In addition, in vivo xenotransplantation was performed to assess the effects of CA, YAP, and MDM2 on tumorigenesis. The IC50 of CA was about 40 M in different HCC cell lines, and CA decreased YAP expression by reducing its stability and increasing its ubiquitination. CA treatment and MDM2 overexpression significantly decreased the crosstalk between YAP and cAMP-responsive element-binding protein (CREB), TEA domain transcription factor (TEAD), and Runt-related transcription factor 2 (Runx2). CA stimulation promoted the translocation of YAP and MDM2 from the nucleus to the cytoplasm and increased their binding. In addition, CA treatment obviously reduced tumorigenesis, whereas this effect was abolished when cells were transfected with sh-MDM2 or Vector-YAP. The present study uncovered that CA induced cancer progress repression through translocating YAP from the nucleus in HCC, which might provide a new therapeutic target for HCC.

Downregulation of MDM2 Leads to Anti-Proliferative Effects through Activation of p53-Associated Pathway Mediated By Both Dual Inhibitor MX69 and Mir-548c-3p in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by abnormal proliferation of clonal plasma cells in the bone marrow. MM remains an incurable disease with a high rate of relapse and development of drug resistance. Mouse double minute 2 homolog (MDM2) has been characterized as an oncogene that is associated with cancer development and radio/chemotherapy resistance in cancer. However, the mechanism(s) underlying MDM2 overexpression and its association with drug resistance in MM have not been fully explored. In addition, the effect of the newly discovered dual inhibitor MX69, which targets MDM2 and XIAP, is unknown in MM. To assess the effects of MDM2 overexpression in MM patients, we performed GEO data (GSE6477, GSE31161) differential expression analysis on bone marrow samples from three patient groups- normal donors, newly diagnosed MM and relapsed MM. We found that relapsed MM patients expressed high MDM2 compared to newly diagnosed MM patients, and both groups expressed higher MDM2 compared to normal donors (p<0.001). Furthermore, immunoblotting and qRT-PCR showed upregulated MDM2 expression in drug resistant MM cell lines (MM1R, 8226R5) as compared to their parental drug sensitive cells (MM1S, 8226S). XIAP is an important inhibitor of apoptotic proteins caspases 3, 7 and 9. Elevated expression of XIAP is involved in chemotherapy resistance in cancer. To determine whether MDM2 and XIAP downregulation by dual inhibitor MX69 could induce apoptosis in MM cells, we treated two drug resistant MM cell lines with MX69. Cell viability assay showed that MX69 reduced viability of MM cells in a time- and dose-dependent manner. Since MDM2 is an antagonist of tumor suppressor p53, we measured the expression of p53 following MX69 treatment and found that MX69-mediated downregulation of MDM2 significantly enhanced p53, PUMA and p21 expression in MM cell lines harboring wild-type p53. In MM p53 null cells, we found that p53 family pro-apoptotic factors p63 and p73 were increased upon MX69 treatment. To investigate the downstream mechanism of MDM2 targeting in MM drug resistance, we knocked down MDM2 in MM cells via siRNA-mediated silencing. Cell viability assay indicated that siMDM2 reduced cell proliferation compared to negative control (P=0.009). Following MDM2 silencing, XIAP expression was decreased and p53 expression was increased in p53 positive MM cell lines, whereas p63, p73 and p21 were increased in both p53 positive and p53 null MM cells. Furthermore, combined treatment of MX69 with dexamethasone (Dex), doxorubicin (Dox) or lenalidomide (Len) displayed synergistic toxic effects (combination index ranging from 0.437 to 0.8) in drug resistant MM cell lines. MX69 alone or in combination with Dex, Dox and Len significantly increased apoptosis and decreased proliferation of MM cells. To explore whether miRNAs play a role in regulating MDM2 overexpression and MM drug resistance, several miRNA target prediction algorithms were exploited to generate a selective miRNA library for subsequent screening. Four miRNAs (miR-890, miR-211-5p, miR-429 and miR-548c-3p) were common among GSE49261, GSE39571, GSE16558 and GSE17498 GEO datasets. These four miRNAs were downregulated in MM patients and negatively correlated with MDM2. Furthermore, analyzing prediction score revealed that miR-548c-3p had the highest binding score with MDM2 3'UTR. Moreover, MiR-548c-3p showed a significant downregulation in drug resistant cells in comparison with their parental sensitive cells (p<0.0001). To determine whether MDM2 expression is selectively regulated by miR-548c-3p, we transfected two MM drug resistant cells with miR-548c-3p mimics and found that MDM2 mRNA and protein levels were suppressed and the cell viability was significantly reduced compared to the scrambled controls. In conclusion, our results indicate that MDM2 overexpression is associated with disease progression and drug resistance in MM. Treatment of drug resistant MM cells with MDM2/XIAP dual inhibitor MX69 sensitizes the cells to anti-myeloma drugs and induces apoptosis. Our finding of the regulatory interaction between miR-548c-3p and MDM2 reveals key insights into the dysregulation of MDM2 in MM and provides a new therapeutic approach to target MDM2 by restoration of miR-548c-3p. Disclosures No relevant conflicts of interest to declare.

Bcor Mutations Cooperate with Tet2 Deficiency to Promote RAS Transformation of Mouse Hematopoietic Progenitors

Somatic mutations can have highly stereotyped positions in the myeloid clonal hierarchy and distinct patterns of co-occurring mutations. Gene mutations that cause aberrant activation of RAS/MAPK signaling are typically late events in myeloid disease progression and are closely associated with leukemic transformation. We hypothesized that the phenotypic output of oncogenic RAS signaling is dynamically reprogrammed during leukemogenesis based on evolving genetic and epigenetic context. To identify genetic alterations that may modulate RAS-mediated transformation, we evaluated 1273 adults with myelodysplastic syndrome, including 150 with mutations in NRAS, KRAS, PTPN11, CBL, RIT1, NF1, or FLT3. Somatic mutations in ASXL1 (q<0.0001), RUNX1 (q<0.0001), EZH2 (q<0.0001), BCOR (q=0.0002), and STAG2 (q=0.001) were most significantly associated with co-occurring RAS pathway mutations, compared to those without RAS pathway mutations, while TP53 mutations were less frequent (q=0.059). We validated these observations in an independent cohort of 6343 unselected patients, including 1081 patients harboring either RAS pathway mutations (n=651),TP53 mutations (n=494), or both (n=57). To define the effects of sequential acquisition of driver mutations, we developed a mouse serial transplantation model of somatic myeloid transformation. First, we used in vivo pI:pC treatment to induce biallelic inactivation of Tet2 in adult Mx1-Cre/Tet2flox/floxmice. After 12 weeks, we purified Tet2-/-or control hematopoietic stem and progenitor cells (HSPCs) and used CRISPR/Cas9 to separately introduce inactivating mutations in Ezh2, Asxl1-exon12, Stag2, or Bcor, then evaluated their functional effects using ex vivo serial replating or in vivo competitive transplantation. Tet2-/-HSPCs with control sgRNA showed a modest enhancement of serial replating compared to Tet2-wild type HSPCs, while Tet2-/-HSPCs Asxl1, Stag2, and Bcor, but not Ezh2 sgRNA had markedly enhanced serial replating capacity (>6 platings in all replicates). In primary transplantation, secondary mutations caused in vivo clonal advantage after 16 weeks, but never resulted in histologic transformation to acute leukemia. We next evaluated the impact of tertiary NRASG12Dmutations in each pairwise Tet2-/-CRISPR combination (Asxl1, Bcor, Ezh2, Stag2, control). We purified HSPCs from recipient mice 16 weeks after primary transplantation, transduced with a lentiviral NRASG12Dexpression vector and transplanted into secondary recipients. Recipients of Tet2/Bcor/NRAS, Tet2/Asxl1/NRAS, or Tet2/Ezh2/NRAS cells succumbed to CD11b+myeloid disease with variable latency in Bcor (14 days), Ezh2 (50 days), and Asxl1 (120 days) cells, suggesting that combined Tet2 and PRC1/2 alterations may modify the effects of oncogenic RAS signaling. To determine whether pre-existing epigenetic mutations cooperate to alter the transcriptional response to acute oncogenic stress compared to wild type cells, weperformed RNA-seq 12 and 24 hours after induced expression of NRASG12D in isogenic immortalized mouse progenitor cells deficient for Tet2, Bcor, or both Tet2 and Bcor. We observed rapid activation of inflammatory and cellular senescence programs in all conditions, suggesting a genotype-independent immediate early response to oncogenic signaling. However, we also identified genotype-specific regulation of tumor suppressor and cell cycle checkpoint pathways. While Cdnk1a expression was strongly induced in all conditions, Cdnk2a expression (and p16Ink4a and p19ARF protein levels) was preferentially upregulated in the context of Bcor deficiency. Moreover, expression of the p53 negative regulator Mdm2 was increased 11-fold in Tet2/Bcor-deficient cells, but only 4 to 5-fold in wild type, Tet2-, or Bcor-deficient cells. Tet2/Bcor-deficient cells were significantly more sensitive to treatment with the Mdm2 antogonist, Nutlin, upon induction of NRAS expression than were wild-type cells, suggesting that Mdm2 overexpression directly mediates acquired tolerance of oncogene stress. These human genetic data and mouse models suggest that epigenetic alterations occurring during early myeloid leukemogenesis may enable evasion of oncogene protection mechanism. Bcor mutations can pair with initiating Tet2 mutations to facilitate RAS mediated transformation while incurring a dependency on Mdm2 overexpression. Disclosures No relevant conflicts of interest to declare.

Advanced Glycation End Products Increase MDM2 Expression via Transcription Factor KLF5

Type 2 diabetes increases the risk for all-site cancers including colon cancer. Diabetic patients present typical pathophysiological features including an increased level of advanced glycation end products (AGEs), which comes from a series of nonenzymatic reactions between sugars and biological macromolecules, positively associated with the occurrence of diabetic complications. MDM2 is an oncogene implicated in cancer development. The present study investigated whether diabetes promoted MDM2 expression in colon cells and the underlying mechanisms. Our results showed that AGE increased the protein level of MDM2 in a cell model and promoted binding between MDM2 and Rb as well as p53, which led to degradation of Rb and p53. KLF5 was able to bind to the regulatory sequence of the MDM2 gene, and knockdown of the KLF5 protein level inhibited the AGE-triggered MDM2 overexpression, which indicated that KLF5 was the transcription factor for MDM2. In a mouse model of diabetes, we found that AGE level was increased in serum. The protein levels of both KLF5 and MDM2 were increased. KLF5 was able to bind to the regulatory sequence of the MDM2 gene. In conclusion, our results suggest that diabetes increases the level of AGE which enhances the expression of MDM2 via transcription factor KLF5 in colon cells. MDM2 overexpression is a candidate biological link between type 2 diabetes and colon cancer development.

Inhibition of MDM2 via Nutlin-3A: A Potential Therapeutic Approach for Pleural Mesotheliomas with MDM2-Induced Inactivation of Wild-Type P53

Previously, our group demonstrated that nuclear expression of E3 ubiquitin ligase (MDM2) in malignant pleural mesothelioma (MPM) is significantly associated with decreased overall survival. A possible explanation may be that overexpression of MDM2 leads to a proteasomal degradation of TP53 that eventually results in a loss of TP53-induced apoptosis and senescence. It is well known from other tumor entities that restoration of TP53 activity, e.g., by MDM2 inhibition, results in an instant TP53-induced stress and/or DNA damage response of cancer cells. Nutlin-3A (acis-imidazoline analogue) has been described as a potent and selective MDM2 inhibitor preventing MDM2-TP53-interaction by specific binding to the hydrophobic TP53-binding pocket of MDM2. In the present study, the effects of MDM2 inhibition in MPM via Nutlin-3A and standard platinum based chemotherapeutic agents were comparatively tested in three MPM cell lines (NCI-H2052, MSTO-211H, and NCI-H2452) showing different expression profiles of TP53, MDM2, and its physiological inhibitor of MDM2—P14/ARF. Ourin vitroexperiments on MPM cell lines revealed that Nutlin-3A in combination with cisplatin resulted in up to 9.75 times higher induction of senescence (p=0.0050) and up to 5 times higher apoptosis rate (p=0.0067) compared to the commonly applied cisplatin and pemetrexed regimens. Thus Nutlin-3A, a potent inhibitor of MDM2, is associated with a significant induction of senescence and apoptosis in MPM cell lines, making Nutlin-3A a promising substance for a targeted therapy in the subgroup of MPM showing MDM2 overexpression.

Pedunculated, well differentiated liposarcoma of the oesophagus mimicking giant fibrovascular polyp

We present a rare case of a big oesophageal liposarcoma causing dysphagia and weight loss in a 75-year-old patient. Endoscopically, a pedunculated lesion with subtotal obstruction of the oesophageal lumen had been detected and thoracoabdominal oesophageal resection with gastric sleeve reconstruction was performed. Surprisingly, a liposarcoma of the oesophagus was revealed on histopathological analysis, showing MDM2 overexpression. Oncological follow-up has been uneventful and the patient remains in good clinical shape at 15 months after surgery.

Dedifferentiated Liposarcoma of the Retroperitoneum With Heterologous Osteosarcomatous Differentiation and a Striking Aneurysmal Bone Cyst–Like Morphology

A 69-year-old woman with a 10-year medical history of recurrent retroperitoneal dedifferentiated liposarcoma presented with a 3-cm large hemorrhagic and multicystic left-sided retroperitoneal mass. Histopathological examination of the resected specimen showed a heterogeneous, high-grade mesenchymal nonlipogenic tumor with areas of osteoblastic/osteosarcomatous differentiation and aneurysmal bone cyst–like features. Based on the clinical presentation, the morphology, and the supportive immunohistochemical and molecular findings (MDM2 overexpression and amplification of the MDM2 gene, respectively), a diagnosis of a dedifferentiated liposarcoma with heterologous osteosarcomatous differentiation and an aneurysmal bone cyst–like morphology was made. To the best of our knowledge, this is the first description of aneurysmal bone cyst–like morphology in dedifferentiated liposarcoma, further expanding the broad morphological spectrum of dedifferentiated liposarcoma.