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Supplemental Table 1 shows detailed MYD88 L265P mutation frequency in B cell neoplasms. Data are summarized from all publications in PUBMED containing "MYD88 L265P".

The supplementary documents include: (1) Supplementary methods for detection of MYC mutations and rearrangements, assessment of Myc expression, and functional studies. (2) Supplementary Tables S1-S8. Supplementary Table S1. Numbers of MYC mutation present in the coding sequence and the untranslated regions. Supplementary Table S2. Clinical and molecular characteristics of the DLBCL cohort with wild-type or mutated Myc or N11S single nucleotide polymorphism. Supplementary Table S3. List of Myc mutations and corresponding patient survival. Supplementary Table S4. Multivariate survival analysis. Supplementary Table S5. Molecular characteristics of patients with wild-type or mutated MYC untranslated regions. Supplementary Table S6. List of MYC 3Ã,'UTR mutations and corresponding patient survival. Supplementary Table S7. Gene profiling comparisons between wild-type and mutated MYC. Supplementary Table S8. Brief summary of major findings by this study. (3) Legends for Supplementary Figures S1-S5. Supplementary Figure S1. Survival analysis for MYC mutations in DLBCL respective to GCB/ABC subtypes, homo/heterozygosity, and MYC translocations. Supplementary Figure S2. Comparison of Myc levels and gene expression profiles between DLBCL groups. Supplementary Figure S3. Differential expression of genes between the WT-Myc and MUT-Myc groups. Supplementary Figure S4. Comparison of gene expression between the WT-Myc and MUT-Myc groups. Supplementary Figure S5. Differential expression of proteins between the WT-Myc and MUT-Myc groups.

Next-generation sequencing has revealed cancer genomic landscapes, in which over 100 driver genes that, when altered by intragenic mutations, can promote oncogenesis. MYD88 is a driver gene found in hematologic B-cell malignancies. A missense mutation (L265P) changing leucine at position 265 to proline in MYD88 is found in ∼90% of Waldenström macroglobulinemia (WM) cases and in significant portions of activated B-cell diffuse large B-cell lymphomas and IgM monoclonal gammopathy of undetermined significance. Few cancers such as WM have a single amino acid substitution in one gene like MYD88 L265P that occurs in ∼90% of cases, making WM paradigmatic for study of a single causative mutation in oncogenesis. In this review, we summarize the frequency and cancer spectrum of MYD88 L265P and its downstream effects in lymphoid cancers. Malignant B cells with MYD88 L265P are likely transformed from IgM-producing B cells either in response to T-cell–independent antigens or in response to protein antigens before class switching. We also discuss therapeutic strategies that include targeting Bruton tyrosine kinase and other kinases, interfering with the assembly of MYD88 and its interacting partners, and MYD88 L265P-specific peptide-based immunotherapy. Cancer Res; 78(10); 2457–62. ©2018 AACR.

Purpose: MYC is a critical driver oncogene in many cancers, and its deregulation in the forms of translocation and overexpression has been implicated in lymphomagenesis and progression of diffuse large B-cell lymphoma (DLBCL). The MYC mutational profile and its roles in DLBCL are unknown. This study aims to determine the spectrum of MYC mutations in a large group of patients with DLBCL, and to evaluate the clinical significance of MYC mutations in patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) immunochemotherapy.Experimental Design: We identified MYC mutations in 750 patients with DLBCL using Sanger sequencing and evaluated the prognostic significance in 602 R-CHOP–treated patients.Results: The frequency of MYC mutations was 33.3% at the DNA level (mutations in either the coding sequence or the untranslated regions) and 16.1% at the protein level (nonsynonymous mutations). Most of the nonsynonymous mutations correlated with better survival outcomes; in contrast, T58 and F138 mutations (which were associated with MYC rearrangements), as well as several mutations occurred at the 3′ untranslated region, correlated with significantly worse survival outcomes. However, these mutations occurred infrequently (only in approximately 2% of DLBCL). A germline SNP encoding the Myc-N11S variant (observed in 6.5% of the study cohort) was associated with significantly better patient survival, and resulted in reduced tumorigenecity in mouse xenografts.Conclusions: Various types of MYC gene mutations are present in DLBCL and show different impact on Myc function and clinical outcomes. Unlike MYC gene translocations and overexpression, most MYC gene mutations may not have a role in driving lymphomagenesis. Clin Cancer Res; 22(14); 3593–605. ©2016 AACR.

Myeloid differentiation primary response protein 88 (MYD88) is a critical universal adapter that transduces signaling from Toll-like and interleukin receptors to downstream nuclear factor-κB (NF-κB). MYD88L265P (leucine changed to proline at position 265) is a gain-of-function mutation that occurs frequently in B-cell malignancies such as Waldenstrom macroglobulinemia. In this study, E3 ligase RING finger protein family 138 (RNF138) catalyzed K63-linked nonproteolytic polyubiquitination of MYD88L265P, resulting in enhanced recruitment of interleukin-1 receptor–associated kinases and elevated NF-κB activation. However, RNF138 had little effect on wild-type MYD88 (MYD88WT). With either RNF138 knockdown or mutation on MYD88 ubiquitination sites, MYD88L265P did not constitutively activate NF-κB. A20, a negative regulator of NF-κB signaling, mediated K48-linked polyubiquitination of RNF138 for proteasomal degradation. Depletion of A20 further augmented MYD88L265P-mediated NF-κB activation and lymphoma growth. Furthermore, A20 expression correlated negatively with RNF138 expression and NF-κB activation in lymphomas with MYD88L265P and in those without. Strikingly, RNF138 expression correlated positively with NF-κB activation in lymphomas with MYD88L265P, but not in those without it. Our study revealed a novel mutation-specific biochemical reaction that drives B-cell oncogenesis, providing a therapeutic opportunity for targeting oncogenic MYD88L265P, while sparing MYD88WT, which is critical to innate immunity.

Although epidemiologic studies have suggested a possible association between occupational exposures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the risk of development of multiple myeloma, definitive evidence in support of this association is lacking. In the present study, we employed the Vk*Myc mouse model of multiple myeloma to assess the impact of TCDD exposure on multiple myeloma pathogenesis. TCDD induced splenomegaly and multiple peripheral blood abnormalities, including anemia and high serum IgG levels. In addition, TCDD triggered bone lytic lesions, as well as renal protein deposition, a phenomenon associated with human myeloma kidney disease. Even in wild-type C57BL/6 mice, TCDD increased serum IgG levels, induced anemia, and increased plasma cell presence in the spleen and bone marrow, hallmarks of benign monoclonal gammopathy. Lastly, TCDD induced AKT activation and the DNA damage response, key pathogenic events in myeloma pathogenesis, in animal spleen and/or bone marrow. These data indicate that TCDD accelerates monoclonal gammopathy development and promotes progression to multiple myeloma in genetically-predisposed mice. This work offers the first direct experimental evidence establishing TCDD as an environmental risk factor for monoclonal gammopathy of undetermined significance and multiple myeloma.

Next-generation sequencing has revealed cancer genomic landscapes, in which over 100 driver genes that, when altered by intragenic mutations, can promote oncogenesis. MYD88 is a driver gene found in hematologic B-cell malignancies. A missense mutation (L265P) changing leucine at position 265 to proline in MYD88 is found in ∼90% of Waldenström macroglobulinemia (WM) cases and in significant portions of activated B-cell diffuse large B-cell lymphomas and IgM monoclonal gammopathy of undetermined significance. Few cancers such as WM have a single amino acid substitution in one gene like MYD88 L265P that occurs in ∼90% of cases, making WM paradigmatic for study of a single causative mutation in oncogenesis. In this review, we summarize the frequency and cancer spectrum of MYD88 L265P and its downstream effects in lymphoid cancers. Malignant B cells with MYD88 L265P are likely transformed from IgM-producing B cells either in response to T-cell–independent antigens or in response to protein antigens before class switching. We also discuss therapeutic strategies that include targeting Bruton tyrosine kinase and other kinases, interfering with the assembly of MYD88 and its interacting partners, and MYD88 L265P-specific peptide-based immunotherapy. Cancer Res; 78(10); 2457–62. ©2018 AACR.

MicroRNA-21 (miR-21) is one of the most abundant microRNAs in mammalian cells. It has been intensively studied for its role in regulating apoptosis and oncogenic transformation. However, the impact of miR-21 on host anti-tumor immunity remains unknown. Tumor-associated macrophages are a major leukocyte type that infiltrates tumors and predominantly develops into immunosuppressive, tumor-promoting M2-like macrophages. In contrast, the pro-inflammatory M1-like macrophages have tumoricidal activity. In this study, we show that genetic deficiency of miR-21 promotes the polarization of macrophages toward an M1-like phenotype in vivo and in vitro in the presence of tumor cells; thus it confers host mice with enhanced anti-tumor immunity. By downregulating JAK2 and STAT1, miR-21 inhibits the IFN-γ-induced STAT1 signaling pathway, which is required for macrophage M1 polarization. We also show that the expression of miR-21 in macrophages is regulated upon polarization stimuli as well as upon macrophages co-culturing with tumor cells. Thus, tumor cells may stimulate miR-21 expression in tumor-associated macrophages to prevent tumoricidal M1 polarization. However, augmented STAT1 signaling mediated by miR-21 deficiency upregulates PD-L1 expression in macrophages, which is known to inhibit phagocytic anti-tumor activity. This adverse effect can be alleviated by PD-1 blockade; indeed, miR-21 depletion in macrophages and PD-1 antibody treatment offer superior anti-tumor activity than either agent alone. These studies shed lights on potential application of the combination of miR-21 inhibition and immune checkpoint blockade to target the tumor microenvironment.

Neoalbaconol, derived from Albatrellus confluens, shows anti-cancer activities in the previously study, but its role in angiogenesis is unknown. Here, we determined whether neoalbaconol could attenuate angiogenesis and how does it occur. Data demonstrated that neoalbaconol could inhibit the proliferation of breast cancer cells and induce apoptosis. Also, neoalbaconol suppressed vascular endothelial growth factor (VEGF)-induced human umbilical vascular endothelial cells (HUVECs) proliferation, migration, invasion, and capillary-like tube formation in vitro and reduced tumor angiogenesis in vivo. VEGF receptor activation and the downstream signal transduction cascades activation were inhibited by neoalbaconol. Additionally, neoalbaconol blocked EGFR-mediated VEGF production. EGFR overexpression reversed the neoalbaconol-induced VEGF reduction, confirming the importance of the EGFR inhibition in anti-angiogenesis of neoalbaconol. Furthermore, neoalbaconol inhibited tumor growth and tumor angiogenesis in a breast cancer xenograft model in vivo. Taken together, these results indicate that neoalbaconol could inhibit tumor angiogenesis and growth through direct suppression effects on vascular endothelial cells and reduction of proangiogenic factors in cancer cells.

Additional file 2. Supplementary Tables

Additional file 1. Supplementary Figures

Background Medulloblastoma is the most frequently occurring malignant brain tumor in children. Current treatment strategies for medulloblastoma include aggressive surgery, cranio-spinal irradiation and adjuvant chemotherapy. Because current treatments can cause severe long-term side effects and are not curative, successful treatment remains a challenge. Methods In this study, we employed a high-throughput cell viability assay to screen 12,800 compounds and to identify drug candidates with anti-proliferative properties for medulloblastoma cells. We also tested these compounds for attenuating medulloblastoma tumor development using mouse xenografts. Results We identified two histone deacetylase inhibitors (dacinostat and quisinostat) with anti-proliferative properties for medulloblastoma cells. We showed that both compounds induce cytotoxicity, trigger cell apoptosis, and block cell cycle progression at the G2/M phase. In addition, dacinostat and quisinostat attenuated xenograft medulloblastoma growth in mice. Conclusions Our findings suggest that histone deacetylase inhibitors are potent therapeutic agents against medulloblastoma.

Patients carryingTP53germline mutations develop Li-Fraumeni syndrome (LFS), a rare genetic disorder with high risk of several cancers, most notably breast cancer, sarcoma, and brain tumors. A noncoding polymorphism (rs78378222) inTP53, carried by scores of millions of people, was associated with moderate risk of brain, colon, and prostate tumors, and other neoplasms. We found a positive association between this variant and soft tissue sarcoma (odds ratio [OR] = 4.55,P= 3.3 x 10−5). In sharp contrast, this variant was protective against breast cancer (OR = 0.573,P= 0.0078). We generated a mouse line carrying this variant and found that this variant accelerated spontaneous tumorigenesis and tumor development at the brain, prostate, colon, and skeletal muscle, but strikingly, it significantly delayed mammary tumorigenesis. The variant created a miR-382 targeting site and compromised a miR-325 targeting site. Their differential expression resulted in p53 downregulation in the brain and several other tissues, but p53 upregulation in the mammary gland of the mutant mice compared to that of wild-type littermates. Thus, thisTP53variant is at odds with LFS mutants in breast cancer predisposition yet consistent with LFS mutants in susceptibility to soft tissue sarcoma and glioma. Our findings elucidate an underlying mechanism of cancer susceptibility that is conferred by genetic variation and yet altered by microRNA expression.

The WHO clearly identifies tumors as a curable or a chronic disease. The use of natural agents in cancer prevention and therapy is currently playing an important role. Our laboratory has been investigating various natural phenolic compounds, including grifolin, neoalbaconol and epigallocatechin-3-gallate (EGCG). In the present review, we focus on the anticancer activities and the molecular mechanisms of these compounds. Grifolin, a secondary metabolite isolated from the mushroom Albatrellus confluens, has been shown to inhibit cell growth and induce cell cycle arrest in multiple cancer cell lines by targeting extracellular signal-regulated kinase 1 or by upregulating death-associated protein kinase 1 (DAPK1) via p53. We also demonstrated that neoalbaconol, a novel small-molecular compound with a drimane-type sesquiterpenoid structure obtained from Albatrellus confluens, regulates cell metabolism by targeting 3-phosphoinositide-dependent protein kinase 1 (PDK1) and inhibits cancer cell growth. EGCG, a well known catechin found in tea, has gained much attention for its anticancer effects. Previously, we found that it regulates EBV lytic infection through the phosphoinositide-3 kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase (MAPK) pathways in EBV-positive cancer cells. Therefore, these natural agents could be used as potential leading compounds in the prevention of tumor progression and/or EBV-related cancer.

Figure S1. IDO1 was induced by IFN-γ. IDO1 was induced by IFN-γ. (A) IDO1 expression of A549 and DLD-1 stimulated by 1-300 ng/ml IFN-γ for 24 h. IDO1 expression was measured using flow cytometry. (B) IDO1 expression in DLD-1 cells treated with 30 ng/ml IFN-γ for 3-48h. (C) IDO1 expression curves plotted from data in (A) and (B). (PDF 368 kb)

Figure S2. IDO2 and TDO expression in relationship to survival of patients with colorectal cancer. (A) Overall survival of patients with colon cancer (n = 382) in the TCGA database. Patients were classified as having high or low IDO2 (left) or TDO (right) expression according the median level. (B) Survival in days according to IDO2 expression level of patients in Panel A. (PDF 266Â kb)

Figure S5. The activation of T cells exhibits no difference when co-cultured with tumor cells with or without miR-153. T cells were co-cultured in DLD-1+miR-153 (orange) or DLD-1+NC cells (green) for 24 hours. The expression of the designated T cell activation markers was measured by flow cytometry. (PDF 206Â kb)

Colorectal cancer is a major cancer type worldwide. 5-fluorouracil, often given with leucovorin, is the most commonly used drug in colorectal cancer chemotherapy, yet development of drug resistance to 5-fluorouracil in colorectal cancer cells is the primary cause of chemotherapy failure. Most patients receiving intravenous 5-fluorouracil develop side effects. Leucovorin, due to its vitamin-like profile, has few side-effects. Drug repurposing is the application of approved drugs to treat new indications. In this study, we performed a novel drug-repurposing screening to identify Food and Drug Administration-approved chemotherapeutic compounds possessing synergistic activity with leucovorin against colorectal cancer cells. We found that the combination of bortezomib and leucovorin enhanced caspase activation and increased apoptosis in colorectal cancer cells better than either agent alone. Further, the synergistic induction of apoptosis and inhibition of tumor growth were also observed in mouse colorectal cancer xenografts. These data support leucovorin enhances the anti-cancer effect of bortezomib and present this novel combinatorial treatment against colorectal cancer.

The p53 tumor suppressor gene is the most frequently mutated gene in cancer. Significant progress has been made to discern the importance of p53 in coordinating cellular responses to DNA damage, oncogene activation, and other stresses. Noncoding RNAs are RNA molecules functioning without being translated into proteins. In this work, we discuss the dichotomy of p53 regulation by noncoding RNAs with four unconventional questions. First, is overexpression of microRNAs responsible for p53 inactivation in the absence of p53 mutation? Second, are there somatic mutations in the noncoding regions of the p53 gene? Third, is there a germline mutant in the noncoding regions of the p53 gene that predisposes carriers to cancer? Fourth, can p53 activation mediated by a noncoding RNA mutation cause cancer? This work highlights the prominence of noncoding RNAs in p53 dysregulation and tumorigenesis.

Programmed cell death plays an important role in animal development, tissue homeostasis and eliminating harmful or virally infected cells. Necroptosis, a novel form of programmed cell death, is caspase independent but RIPK and RIPK3 dependent. Moreover, it is suggested that necroptosis can be specifically inhibited by small molecular inhibitors such as necrostatin-1. Its signaling pathways have something in common with apoptosis, although the molecular mechanisms of necroptosis need to be further elucidated. Previous evidences suggest that necroptosis has significant effects in regulating various physiological processes and disease, such as ischemic brain injury, immune system disorders and cancer. In this review, the molecular mechanism of necroptosis is described and how it could be manipulated in the treatment of cancer is summarized.

Grifolin, a secondary metabolite isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens, has been shown to inhibit the growth of some cancer cell lines in vitro by induction of apoptosis in previous studies of our group. However, the mechanisms of action are not completely understood. An apoptosis-related gene expression profiling analysis provided a clue that death-associated protein kinase 1 (dapk1) gene was upregulated at least twofold in response to grifolin treatment in nasopharyngeal carcinoma cell CNE1. Here, we further investigated the role of DAPK1 in apoptotic effect induced by grifolin. We observed that protein as well as mRNA level of DAPK1 was induced by grifolin in a dose-dependent manner in nasopharyngeal carcinoma cell CNE1. We found that grifolin increased both Ser392 and Ser20 phosphorylation levels of transcription factor p53 protein, which could promote its transcriptional activity. Moreover, induced by grifolin, the recruitment of p53 to dapk1 gene promoter was confirmed to enhance markedly using EMSA and ChIP assays analysis. The involvement of DAPK1 in grifolin-induced apoptosis was supported by the studies that introducing siRNA targeting DAPK1 to CNE1 cells remarkably interfered grifolin-caused apoptotic effect as well as the activation of caspase-3. Grifolin induced upregulation of DAPK1 via p53 was also observed in tumour cells derived from human breast cancer and human colon cancer. The findings suggest that upregulation of DAPK1 via p53-DAPK1 pathway is an important mechanism of grifolin contributing to its ability to induce apoptotic effect. Since growing evidence found a significant loss of DAPK1 expression in a large variety of tumour types, grifolin may represent a promising candidate in the intervention of cancer via targeting DAPK1.

Necroptosis/regulated necrosis is a caspase-independent, but receptor interacting protein kinase (RIPK)-dependent form of cell death. In previous studies, neoalbaconol (NA), a constituent extracted from Albatrellus confluens, was demonstrated to induce necroptosis in some cancer cell lines. The molecular mechanism of NA-induced necroptosis is described in this research study. We determined that NA-induced cell death is partly dependent on tumor necrosis factor α (TNFα) feed-forward signaling. More importantly, NA abolished the ubiquitination of RIPK1 by down-regulating E3 ubiquitin ligases, cellular inhibitors of apoptosis protein 1/2 (cIAP1/2) and TNFα receptor-associated factors (TRAFs). The suppression of RIPK1 ubiquitination induced the activation of the non-canonical nuclear factor-κB (NF-κB) pathway and stimulated the transcription of TNFα. Moreover, we also found that NA caused RIPK3-mediated reactive oxygen species (ROS) production and contribution to cell death. Taken together, these results suggested that two distinct mechanisms are involved in NA-induced necroptosis and include RIPK1/NF-κB-dependent expression of TNFα and RIPK3-dependent generation of ROS.

Purpose: MYC is a critical driver oncogene in many cancers, and its deregulation in the forms of translocation and overexpression has been implicated in lymphomagenesis and progression of diffuse large B-cell lymphoma (DLBCL). The MYC mutational profile and its roles in DLBCL are unknown. This study aims to determine the spectrum of MYC mutations in a large group of patients with DLBCL, and to evaluate the clinical significance of MYC mutations in patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) immunochemotherapy. Experimental Design: We identified MYC mutations in 750 patients with DLBCL using Sanger sequencing and evaluated the prognostic significance in 602 R-CHOP–treated patients. Results: The frequency of MYC mutations was 33.3% at the DNA level (mutations in either the coding sequence or the untranslated regions) and 16.1% at the protein level (nonsynonymous mutations). Most of the nonsynonymous mutations correlated with better survival outcomes; in contrast, T58 and F138 mutations (which were associated with MYC rearrangements), as well as several mutations occurred at the 3′ untranslated region, correlated with significantly worse survival outcomes. However, these mutations occurred infrequently (only in approximately 2% of DLBCL). A germline SNP encoding the Myc-N11S variant (observed in 6.5% of the study cohort) was associated with significantly better patient survival, and resulted in reduced tumorigenecity in mouse xenografts. Conclusions: Various types of MYC gene mutations are present in DLBCL and show different impact on Myc function and clinical outcomes. Unlike MYC gene translocations and overexpression, most MYC gene mutations may not have a role in driving lymphomagenesis. Clin Cancer Res; 22(14); 3593–605. ©2016 AACR.

Lung cancer and colorectal cancer account for over one-third of all cancer deaths in the United States. MicroRNA-301a (miR-301a) is an activator of both nuclear factor-κB (NF-κB) and Stat3, and is overexpressed in both deadly malignancies. In this work, we show that genetic ablation of miR-301a reduces Kras-driven lung tumorigenesis in mice. And miR-301a deficiency protects animals from dextran sodium sulfate-induced colon inflammation and colitis-associated colon carcinogenesis. We also demonstrate that miR-301a deletion in bone marrow-derived cells attenuates tumor growth in the colon carcinogenesis model. Our findings ascertain that one microRNA-miR-301a-activates two major inflammatory pathways (NF-κB and Stat3) in vivo, generating a pro-inflammatory microenvironment that facilitates tumorigenesis.

Grifolin, a secondary metabolite isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens, has been shown to induce Cl phase cell-cycle arrest in tumor cells in previous studies of our group. However, the mechanisms of action are not completely understood. Our group further demonstrated that grifolin upregulates death-associated protein kinase 1 (DAPK1) in nasopharyngeal carcinoma cells (NPCs). Here, we found that grifolin induced dephosphorylation of DAPK1 (Ser308) to activate DAPK1 and subsequent phosphorylation of its potential downstream effector p21 (Thr145) in nasopharyngeal carcinoma cell CNE1. Inhibition of DAPK1 by introducing siRNA targeting DAPK1 reversed the grifolin- induced phosphorylation of p21. Furthermore, we confirmed that grifolin increased the half-life of p21 and promoted its stability. Flow cytometry analysis demonstrated that DAPK1 was involved in grifolin-induced Cl phase arrest in CNE1 cells. The similar effects induced by grifolin and mechanism beneath were identified in another nasopharyngeal carcinoma cell HONE1. In addition, we observed that grifolin promoted the protein-protein interaction of DAPK1 and ERK1/2 to prevent ERK1/2 nucleolus translocation. Our findings indicate that DAPK1 plays a crucial role in the induction of cell-cycle arrest at Cl phase by grifolin. Grifolin might represent a promising candidate in the prevention and intervention of cancer by targeting DAPK1 signaling to induce cell cycle Cl phase arrest. (C) 2011 Elsevier B.V. All rights reserved.

The present study is to generate a mouse model of a p53 noncoding germline polymorphism that predisposes human carriers to multiple types of cancer. p53, encoded by TP53, is recognized as the guardian of the human genome. Three distinct sets of alterations occur at the TP53 locus, which spans ∼20kb in the human genome: tumor-associated somatic mutations, germline mutations causing Li-Fraumeni syndrome, and germline polymorphisms. The first two types of TP53 mutations occur in the coding sequence (CDS) and produce mutant p53 proteins that lack most or all of the normal tumor suppressive functions and often confer oncogenic properties. By contrast, only a few of the >200 naturally occurring germline variants of TP53 in human populations cause measurable perturbation of p53 function. More than 90% of TP53 germline variants occur in 10 introns alleged to have no cancer-related biological consequences. None of the 20 variants in the TP53 CDS have been reproducibly linked to cancer predisposition. Genome-wide association studies (GWAS) reported no significant association between any germline TP53 polymorphism and any cancer with a P value ≤ 10-7 (a threshold for genome-wide significance) until a single nucleotide polymorphism (SNP) rs78378222 was found to be associated strongly with skin basal cell carcinoma. rs78378222 is located in the fifth nucleotide of the TP53 polyadenylation signal (PAS) that is required for polyadenylation machinery recognition and subsequent cleavage and polyadenylation of mRNA. The risk-associated minor allele is a “C”, resulting in an alternative PAS (AATACA) instead of the canonical PAS (AATAAA). This variant is also associated with an increased risk of colorectal adenoma, prostate cancer, glioma, and esophageal squamous cell carcinoma. It is notable that cancer susceptibility of this p53 noncoding variant does not mirror that of p53 coding germline mutations (patients with Li-Fraumeni syndrome develop predominantly breast cancer, brain tumors, acute leukemia, sarcoma, and adrenal cortical carcinoma). Specifically, carriers with the p53 PAS variant are not predisposed to breast cancer. The p53 PAS variant is uniquely positioned among cancer-susceptibility alleles: (i) low frequency (∼2% in the general populations), (ii) intermediate cancer risk (odds ratio 1.39-3.54), and (iii) predisposing carriers to multiple types of cancer. To mimic this naturally occurring human variant, we have generated a mouse line with a p53 gene carrying the alternative PAS (AATACA) using zinc-finger nuclease technology. The resultant mutant p53 allele is termed p531755C as the 1755th nucleotide of mouse p53 reference mRNA is the ortholog of human rs78378222. We demonstrate that heterozygote p53+/1755C and homozygote p531755C/1755C mice treated with azoxymethane (AOM) and dextran sodium sulfate (DSS) developed more colon tumors than the p53+/+ controls and homozygous mice developed more advanced tumors than heterozygous mice. With data from murine tumorigenesis and human epidemiology, we propose that this p53 noncoding germline variant is a cancer susceptibility allele associated with Lynch syndrome rather than Li-Fraumeni syndrome. Citation Format: Qipan Deng, Yong Li. TP53 coding and noncoding variants in cancer susceptibility: From Li-Fraumeni syndrome to Lynch syndrome. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 37. doi:10.1158/1538-7445.CANSUSC14-37

Many natural compounds derived from plants or microbes show promising potential for anticancer treatment, but few have been found to target energy-relevant regulators. In this study, we report that neoalbaconol (NA), a novel small-molecular compound isolated from the fungus, Albatrellus confluens, could target 3-phosphoinositide-dependent protein kinase 1 (PDK1) and inhibit its downstream phosphoinositide-3 kinase (PI3-K)/Akt-hexokinase 2 (HK2) pathway, which eventually resulted in energy depletion. By targeting PDK1, NA reduced the consumption of glucose and ATP generation, activated autophagy and caused apoptotic and necroptotic death of cancer cells through independent pathway. Necroptosis was remarkably induced, which was confirmed by several necroptosis-specific markers: the activation of autophagy, presence of necrotic morphology, increase of receptor-interacting protein 1 (RIP1)/RIP3 colocalization and interaction and rescued by necroptosis inhibitor necrostatin-1. The possibility that Akt overexpression reversed the NA-induced energy crisis confirmed the importance of the PDK1-Akt-energy pathway in NA-mediated cell death. Moreover, NA shows the capability to inhibit PI3-K/Akt signaling and suppress tumor growth in the nasopharyngeal carcinoma (NPC) nude mouse model. These results supported the feasibility of NA in anticancer treatments.

Additional file 2: Figure S1. Compound activity and plate Z′-factor for the primary screening. (A) Compound activity distribution. Blue dots (Inhibitor Control): positive control group (10 μM staurosporine, 100% inhibition); black dots (Neutral Control): negative control group (DMSO group, 0% inhibition); yellow dots (Hit Compound): primary hits (hit compound) (125 compounds that displayed ≥ 90% inhibition chosen for a dose-response analysis); grey dots (Compound): compounds not chosen for further confirmation (% inhibition

Figure S3. miRNAs are predicted to target the IDO1 3' UTR. (A) Schematic representation of the pmirGLO vector carrying two dual luciferase genes and the IDO1 3' UTR. (B and C) Dual luciferase assays from 293T cells (B) or Hela cells (C) transfected with miR-153 and the pmirGLO construct. (PDF 189Â kb)

Table S1. Supplementary table. (PDF 37 kb)

Additional file 2: Figure S1. Compound activity and plate Z′-factor for the primary screening. (A) Compound activity distribution. Blue dots (Inhibitor Control): positive control group (10 μM staurosporine, 100% inhibition); black dots (Neutral Control): negative control group (DMSO group, 0% inhibition); yellow dots (Hit Compound): primary hits (hit compound) (125 compounds that displayed ≥ 90% inhibition chosen for a dose-response analysis); grey dots (Compound): compounds not chosen for further confirmation (% inhibition

Figure S4. IDO1 downregulation by miR-153 is inducer and time independence. (A and B) IDO1 expression in DLD-1 cells treated by Cp-G DNA or LPS was down-regulated by miR-153 as measured by flow cytometry (A) or by western blotting (B). DLD-1 cells were treated with LPS or cp-G DNA for 12 h. (C and D) The IDO1 protein (C) or mRNA (D) levels in DLD-1 and HCT-116 cells transfected with or without miR-153 before treated with LPS for 6 to 24 h. (PDF 309Â kb)

Table S1. Supplementary table. (PDF 37 kb)

Figures S1 and S2. Supplementary figures. (PDF 1473 kb)

Figure S3. miRNAs are predicted to target the IDO1 3' UTR. (A) Schematic representation of the pmirGLO vector carrying two dual luciferase genes and the IDO1 3' UTR. (B and C) Dual luciferase assays from 293T cells (B) or Hela cells (C) transfected with miR-153 and the pmirGLO construct. (PDF 189Â kb)

Figure S4. IDO1 downregulation by miR-153 is inducer and time independence. (A and B) IDO1 expression in DLD-1 cells treated by Cp-G DNA or LPS was down-regulated by miR-153 as measured by flow cytometry (A) or by western blotting (B). DLD-1 cells were treated with LPS or cp-G DNA for 12 h. (C and D) The IDO1 protein (C) or mRNA (D) levels in DLD-1 and HCT-116 cells transfected with or without miR-153 before treated with LPS for 6 to 24 h. (PDF 309Â kb)