De-Chen Lin

Genomic and molecular characterization of esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide and particularly common in certain regions of Asia. Here we report the whole-exome or targeted deep sequencing of 139 paired ESCC cases, and analysis of somatic copy number variations (SCNV) of over 180 ESCCs. We identified previously uncharacterized mutated genes such as FAT1, FAT2, ZNF750 and KMT2D, in addition to those already known (TP53, PIK3CA and NOTCH1). Further SCNV evaluation, immunohistochemistry and biological analysis suggested their functional relevance in ESCC. Notably, RTK-MAPK-PI3K pathways, cell cycle and epigenetic regulation are frequently dysregulated by multiple molecular mechanisms in this cancer. Our approaches also uncovered many druggable candidates, and XPO1 was further explored as a therapeutic target because it showed both gene mutation and protein overexpression. Our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets.

The genomic landscape of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) has extremely skewed ethnic and geographic distributions, is poorly understood at the genetic level and is in need of effective therapeutic approaches. Here we determined the mutational landscape of 128 cases with NPC using whole-exome and targeted deep sequencing, as well as SNP array analysis. These approaches revealed a distinct mutational signature and nine significantly mutated genes, many of which have not been implicated previously in NPC. Notably, integrated analysis showed enrichment of genetic lesions affecting several important cellular processes and pathways, including chromatin modification, ERBB-PI3K signaling and autophagy machinery. Further functional studies suggested the biological relevance of these lesions to the NPC malignant phenotype. In addition, we uncovered a number of new druggable candidates because of their genomic alterations. Together our study provides a molecular basis for a comprehensive understanding of, and exploring new therapies for, NPC.

Overexpression of PLK1 is associated with poor survival by inhibiting apoptosis via enhancement of survivin level in esophageal squamous cell carcinoma.

PLK1 is essential for the maintenance of genomic stability during mitosis. In our study, we found that overexpression of PLK1 was an independent prognostic factor (RR = 4.253, p = 0.020) and significantly correlated with survivin, an antiapoptotic protein, in esophageal squamous cell carcinoma (ESCC). Reverse transcription‐polymerase chain reaction and fluorescence in situ hybridization (FISH) revealed upregulation of PLK1 mRNA and amplification of PLK1 gene, respectively. Depletion of PLK1 activated the intrinsic apoptotic pathway, which was substantiated by loss of mitochondrial membrane potential, reduction of Mcl‐1 and Bcl‐2 as well as activation of caspase‐9. Coimmunoprecipitation and confocal microscopy displayed that PLK1 was associated with survivin and PLK1 depletion led to downregulation of survivin. Cotransfection of survivin constructs could partially reverse PLK1‐depletion‐induced apoptosis. These data suggest that PLK1 might be a useful prognostic marker and a potential therapeutic target for ESCC. Survivin is probably involved in antiapoptotic function of PLK1.

Protein alterations in Escc and clinical implications: a review

Esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype of esophageal cancer in Asia, characterized by high incidence and mortality rate. Although significant progress has been made in surgery and adjuvant chemoradiotherapy, the prognosis of the patients with this cancer still remains poor. Investigation into protein alterations that occurred in tumors can provide clues to discover new biomarkers for improving diagnosis and guiding targeted therapy. Hundreds of papers have appeared over the past several decades concerning protein alterations in ESCC. This review summarizes all the dysregulated proteins investigated in the disease from 187 published papers and analyzes their contributions to tumor development and progression. We document protein alterations associated with tumor metastasis and the transition from normal esophageal epithelia to dysplasia in order to reveal the most useful markers for prediction of clinical outcome, early detection, and identification of high-risk patients for targeted therapies. In particular, we discuss the largest and most rigorous studies on prognostic implications of proteins in ESCC, in which cyclin D1, p53, E-cadherin and VEGF appeared to have the strongest evidence as independent predictors of patient outcome.

Calreticulin promotes cell motility and enhances resistance to anoikis through STAT3-CTTN-Akt pathway in esophageal squamous cell carcinoma.

We have shown earlier that overexpression of Calreticulin (CRT) contributed to a poor prognosis for patients with esophageal squamous cell carcinoma (ESCC). Here, we have shown an important role of CRT in tumorigenesis through enhancing cell motility and anoikis resistance. SiRNA-mediated knockdown of CRT caused impaired cell migration, invasion and resistance to anoikis. Notably, CRT downregulation decreased the expression of Cortactin (CTTN), which has been previously reported as a candidate oncogene associated with anoikis through the PI3K–Akt pathway. In addition, Akt phosphorylation was abolished after CRT downregulation and its activation can be refreshed by CRT upregulation, suggesting that CRT-enhanced cell resistance to anoikis through the CRT–CTTN–PI3K–Akt pathway. Moreover, the CTTN mRNA level was decreased in CRT–siRNA cells, coupled with the inactivation of STAT3. Expression of both CTTN and p-STAT3 was reduced in tumor cells following incubation with the JAK-specific inhibitor, AG490. Chromatin immunoprecipitation assay showed direct binding of p-STAT3 to the conservative STAT3-binding sequences in CTTN promoter. Furthermore, overexpression of CTTN in CRT-downregulated ESCC cells restored its motility and resistance to anoikis. This study not only reveals a role of CRT in motility promotion and anoikis resistance in ESCC cells, but also identifies CRT as an upstream regulator in the CRT–STAT3–CTTN–Akt pathway.

An emerging role of PARK2 in cancer

PARK2 (PARKIN) is an E3 ubiquitin ligase involved in multiple signaling pathways and cellular processes. Activity of PARK2 is tightly regulated through inter- and intra-molecular interactions. Dysfunction of PARK2 is associated with the progression of parkinsonism. Notably, frequent PARK2 inactivation has been identified in various human cancers. Park2-deficient mice are more susceptible to tumorigenesis, indicating its crucial role as a tumor suppressor. However, biological studies also show that PARK2 possesses both pro-survival and growth suppressive functions. Here, we summarize the genetic lesions of PARK2 in human cancers and discuss the current knowledge of PARK2 in cancer progression. We further highlight future efforts for the study of PARK2 in cancer.

Reciprocal activation between PLK1 and Stat3 contributes to survival and proliferation of esophageal cancer cells.

BACKGROUND & AIMS Aberrant activation of the signal transducer and activator of transcription (Stat)3 and overexpression of polo-like kinase (PLK)1 each have been associated with cancer pathogenesis. The mechanisms and significance of dysregulation of Stat3 and PLK1 in carcinogenesis and cancer progression are unclear. We investigated the relationship between Stat3 and PLK1 and the effects of their dysregulation in esophageal squamous cell carcinoma (ESCC) cells. METHODS We used immunoblot, quantitative reverse-transcription polymerase chain reaction, immunochemistry, chromatin immunoprecipitation, mobility shift, and reporter assays to investigate the relationship between Stat3 and PLK1. We used colony formation, fluorescence-activated cell sorting, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and xenograft tumor assays to determine the effects of increased activation of Stat3 and PLK1 in proliferation and survival of ESCC cells. RESULTS Stat3 directly activated transcription of PLK1 in esophageal cancer cells and mouse embryonic fibroblast cell NIH3T3. PLK1 then potentiated the expression of Stat3; β-catenin was involved in PLK1-dependent transcriptional activation of Stat3. This mutual regulation between Stat3 and PLK1 was required for proliferation of esophageal cancer cells and resistance to apoptosis in culture and as tumor xenografts in mice. Furthermore, phosphorylation of Stat3 and overexpression of PLK1 were correlated in a subset of ESCC. CONCLUSIONS Stat3 and PLK1 control each others transcription in a positive feedback loop that contributes to the development of ESCC. Increased activity of Stat3 and overexpression of PLK1 promote survival and proliferation of ESCC cells in culture and in mice.

Profiling of somatic mutations in acute myeloid leukemia with FLT3-ITD at diagnosis and relapse

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

Spatial intratumoral heterogeneity and temporal clonal evolution in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is among the most common malignancies, but little is known about its spatial intratumoral heterogeneity (ITH) and temporal clonal evolutionary processes. To address this, we performed multiregion whole-exome sequencing on 51 tumor regions from 13 ESCC cases and multiregion global methylation profiling for 3 of these 13 cases. We found an average of 35.8% heterogeneous somatic mutations with strong evidence of ITH. Half of the driver mutations located on the branches of tumor phylogenetic trees targeted oncogenes, including PIK3CA, NFE2L2 and MTOR, among others. By contrast, the majority of truncal and clonal driver mutations occurred in tumor-suppressor genes, including TP53, KMT2D and ZNF750, among others. Interestingly, phyloepigenetic trees robustly recapitulated the topological structures of the phylogenetic trees, indicating a possible relationship between genetic and epigenetic alterations. Our integrated investigations of spatial ITH and clonal evolution provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ESCC.

Suppression of Anoikis by SKP2 Amplification and Overexpression Promotes Metastasis of Esophageal Squamous Cell Carcinoma

The gene of SKP2, located on chromosome 5p13, plays a critical role in cell cycle progression, especially at the G1-S transition, putatively through its control of several cell cycle regulator proteins including p27kip1, p21cip1, p57kip2, p130, cyclin E, and c-Myc. Previous studies in this laboratory revealed that gain of chromosome 5p was often seen in esophageal squamous cell carcinoma (ESCC). In the present study, we examined the amplification status and expression level of SKP2 in ESCC and investigated its clinicopathologic significance. Amplification and elevated expression of SKP2 correlated significantly with tumor stage and positive lymph node metastasis (P < 0.05). The SKP2 protein expression level as determined by immunohistochemical staining showed a significant inverse correlation with p27 protein. In vivo assay showed that inhibition of SKP2 expression also decreased tumor growth and lung metastasis of ESCC cells. At the molecular level, knockdown of SKP2 by RNA interference inhibited cell migration and invasion ability. Knockdown of SKP2 expression sensitized cancer cells to anoikis, and a wobble mutant of SKP2 that is resistant to SKP2 small interfering RNA can rescue this effect. Expression level of pAkt decreased after SKP2 knockdown. Treatment of cells with phosphoinositidyl 3-kinase inhibitor (LY294002) and constitutively activator (insulin-like growth factor I) had significant effects on the anoikis of SKP2 RNA interference cells. These results show for the first time that SKP2 is amplified and overexpressed in ESCC. Elevated expression of SKP2 protected cancer cells from anoikis, and this effect was mediated, at least in part, by the phosphoinositidyl 3-kinase-Akt pathway. (Mol Cancer Res 2009;7(1):12–22)