Proteogenomics of Non-smoking Lung Cancer in East Asia Delineates Molecular Signatures of Pathogenesis and Progression.

Lung cancer in East Asia is characterized by a high percentage of never-smokers, early onset and predominant EGFR mutations. To illuminate the molecular phenotype of this demographically distinct disease, we performed a deep comprehensive proteogenomic study on a prospectively collected cohort in Taiwan, representing early stage, predominantly female, non-smoking lung adenocarcinoma. Integrated genomic, proteomic, and phosphoproteomic analysis delineated the demographically distinct molecular attributes and hallmarks of tumor progression. Mutational signature analysis revealed age- and gender-related mutagenesis mechanisms, characterized by high prevalence of APOBEC mutational signature in younger females and over-representation of environmental carcinogen-like mutational signatures in older females. A proteomics-informed classification distinguished the clinical characteristics of early stage patients with EGFR mutations. Furthermore, integrated protein network analysis revealed the cellular remodeling underpinning clinical trajectories and nominated candidate biomarkers for patient stratification and therapeutic intervention. This multi-omic molecular architecture may help develop strategies for management of early stage never-smoker lung adenocarcinoma.

Overexpressed-eIF3I interacted and activated oncogenic Akt1 is a theranostic target in human hepatocellular carcinoma.

UNLABELLED: Eukaryotic translation initiation factor 3 subunit I (eIF3I) with transforming capability is often overexpressed in human hepatocellular carcinoma (HCC) but its oncogenic mechanisms remain unknown. We demonstrate that eIF3I is overexpressed in various cancers along with activated Akt1 phosphorylation and kinase activity in an eIF3I dose-dependent manner. A novel eIF3I and Akt1 protein interaction was identified in HCC cell lines and tissues and was required for eIF3I-mediated activation of Akt1 signaling. Expression of either antisense eIF3I or dominant negative Akt1 mutant suppressed eIF3I-mediated Akt1 oncogenic signaling and various other tumorigenic effects. Oncogenic domain mapping of the eIF3I and Akt1 interaction suggested that the C-terminal eIF3I interacted with the Akt1 kinase domain and conferred the majority of oncogenic functions. In addition, eIF3I interaction with Akt1 prevented PP2A dephosphorylation of Akt1 and resulted in constitutively active Akt1 oncogenic signaling. Importantly, concordant expression of endogenous eIF3I and phospho-Akt1 was detected in HCC cell lines and tissues. Treatment of eIF3I overexpressing HCC cells with the Akt1 specific inhibitor API-2 suppressed eIF3I-mediated tumorigenesis in vitro and in vivo. CONCLUSION: We describe a constitutive Akt1 oncogenic mechanism resulting from interaction of overexpressed eIF3I with Akt1 that prevents PP2A-mediated dephosphorylation. Overexpression of eIF3I in HCC is oncogenic and is a surrogate marker and therapeutic target for treatment with Akt1 inhibitors.

Prognostic comparative genes predict targets for sorafenib combination therapies in hepatocellular carcinoma.

With the increasing incidence and mortality of human hepatocellular carcinoma (HCC) worldwide, revealing innovative targets to improve therapeutic strategies is crucial for prolonging the lives of patients. To identify innovative targets, we conducted a comprehensive comparative transcriptome analysis of 5,410 human HCCs and 974 mouse liver cancers to identify concordantly expressed genes associated with patient survival. Among the 664 identified prognostic comparative HCC (pcHCC) genes, upregulated pcHCC genes were associated with prognostic clinical features, including large tumor size, vascular invasion and late HCC stages. Interestingly, after validating HCC patient prognoses in multiple independent datasets, we matched the 664 aberrant pcHCC genes with the sorafenib-altered genes in TCGA_LIHC patients and found these 664 pcHCC genes were enriched in sorafenib-related functions, such as downregulated xenobiotic and lipid metabolism and upregulated cell proliferation. Therapeutic agents targeting aberrant pcHCC genes presented divergent molecular mechanisms, including suppression of sorafenib-unrelated oncogenic pathways, induction of sorafenib-unrelated ferroptosis, and modulation of sorafenib transportation and metabolism, to potentiate sorafenib therapeutic effects in HCC combination therapy. Moreover, the pcHCC genes NCAPG and CENPW, which have not been targeted in combination with sorafenib treatment, were knocked down and combined with sorafenib treatment, which reduced HCC cell viability based on disruption to the p38/STAT3 axis, thereby hypersensitizing HCC cells. Together, our results provide important resources and reveal that 664 pcHCC genes represent innovative targets suitable for developing therapeutic strategies in combination with sorafenib based on the divergent synergistic mechanisms for HCC tumor suppression.

Phosphoproteomics Reveals the Role of Constitutive KAP1 Phosphorylation by B-cell Receptor Signaling in Chronic Lymphocytic Leukemia.

Application of B-cell receptor (BCR) pathway inhibitor ibrutinib for chronic lymphocytic leukemia (CLL) is a major breakthrough, yet the downstream effects following inhibition of BCR signaling and during relapse await further clarification. By comparative phosphoproteomic profiling of B cells from patients with CLL and healthy donors, as well as CLL B cells collected at multiple time points during the course of ibrutinib treatment, we provided the landscape of dysregulated phosphoproteome in CLL and its dynamic alterations associated with ibrutinib treatment. Particularly, differential phosphorylation events associated with several signaling pathways, including BCR pathway, were enriched in patient CLL cells. A constitutively elevated phosphorylation level of KAP1 at serine 473 (S473) was found in the majority of CLL samples prior to treatment. Further verification showed that BCR activation promoted KAP1 S473 phosphorylation, whereas ibrutinib treatment abolished it. Depletion of KAP1 in primary CLL cells decelerated cell-cycle progression and ectopic expression of a KAP1 S473 phospho-mimicking mutant accelerated G2-M cell-cycle transition of CLL cells. Moreover, temporal phosphoproteomic profiles using a series of CLL cells isolated from one patient during the ibrutinib treatment revealed the dynamic changes of several molecules associated with BCR signaling in the ibrutinib responsive and recurrent stages. IMPLICATIONS: This phosphoproteomic analysis and functional validation illuminated the phosphorylation of KAP1 at S473 as an important downstream BCR signaling event and a potential indicator for the success of ibrutinib treatment in CLL.

Overlapping high-resolution copy number alterations in cancer genomes identified putative cancer genes in hepatocellular carcinoma.

UNLABELLED: Recurrent cancer genome aberrations are indicators of residing crucial cancer genes. Although recent advances in genomic technologies have led to a global view of cancer genome aberrations, the identification of target genes and biomarkers from the aberrant loci remains difficult. To facilitate searches of cancer genes in human hepatocellular carcinoma (HCC), we established a comprehensive protocol to analyze copy number alterations (CNAs) in cancer genomes using high-density single nucleotide polymorphism arrays with unpaired reference genomes. We identified common HCC genes by overlapping the shared aberrant loci in multiple cell lines with functional validation and clinical implications. A total of 653 amplicons and 57 homozygous deletions (HDs) were revealed in 23 cell lines. To search for novel HCC genes, we overlapped aberrant loci to uncover 6 HDs and 126 amplicons shared by at least two cell lines. We selected two novel genes, fibronectin type III domain containing 3B (FNDC3B) at the 3q26.3 overlapped amplicon and solute carrier family 29 member 2 (SLC29A2) at the 11q13.2 overlapped amplicon, to investigate their aberrations in HCC tumorigenesis. Aberrant up-regulation of FNDC3B and SLC29A2 occurred in multiple HCC data sets. Knockdown of these genes in amplified cells decreased cell proliferation, anchorage-independent growth, and tumor formation in xenograft models. Importantly, up-regulation of SLC29A2 in HCC tissues was significantly associated with advanced stages (P = 0.0031), vascular invasion (P = 0.0353), and poor patient survival (P = 0.0325). Overexpression of FNDC3B or SLC29A2 in unamplified HCC cells promoted cell proliferation through activation of the signal transducer and activator of transcription 3 signaling pathway. CONCLUSION: A standardized genome-wide CNA analysis protocol using data from user-generated or public domains normalized with unpaired reference genomes has been established to facilitate high-throughput detection of cancer genes as significant target genes and biomarkers for cancer diagnosis and therapy.

Mutant KRAS promotes liver metastasis of colorectal cancer, in part, by upregulating the MEK-Sp1-DNMT1-miR-137-YB-1-IGF-IR signaling pathway.

Although the role of insulin-like growth factor-I receptor (IGF-IR) in promoting colorectal liver metastasis is known, the mechanism by which IGF-IR is upregulated in colorectal cancer (CRC) is not defined. In this study, we obtained evidence that mutant KRAS transcriptionally activates IGF-IR gene expression through Y-box-binding protein (YB)-1 upregulation via a novel MEK-Sp1-DNMT1-miR-137 pathway in CRC cells. The mechanistic link between the tumor suppressive miR-137 and the translational regulation of YB-1 is intriguing because epigenetic silencing of miR-137 represents an early event in colorectal carcinogenesis due to promoter hypermethylation. This proposed signaling axis was further verified by the immunohistochemical evaluations of liver metastases from a cohort of 46 KRAS mutant CRC patients, which showed a significant correlation in the expression levels among Sp1, miR-137, YB-1, and IGF-1R. Moreover, suppression of the expression of YB-1 and IGF-IR via genetic knockdown or the pharmacological inhibition of MEK hampers KRAS-driven colorectal liver metastasis in our animal model studies. From a translational perspective, the identification of this KRAS-driven pathway might provide a mechanistic rationale for the use of a MEK inhibitor as an adjuvant, in combination with standard of care, to prevent the recurrence of colorectal liver metastasis in KRAS mutant CRC patients after receiving liver resection, which warrants further investigation.

HDAC inhibitors augmented cell migration and metastasis through induction of PKCs leading to identification of low toxicity modalities for combination cancer therapy.

PURPOSE: Histone deacetylase inhibitors (HDACi) are actively explored as new-generation epigenetic drugs but have low efficacy in cancer monotherapy. To reveal new mechanism for combination therapy, we show that HDACi induce cell death but simultaneously activate tumor-progressive genes to ruin therapeutic efficacy. Combined treatments to target tumorigenesis and HDACi-activated metastasis with low toxic modalities could develop new strategies for long-term cancer therapy. EXPERIMENTAL DESIGN: Because metastasis is the major cause of cancer mortality, we measured cell migration activity and profiled metastasis-related gene expressions in HDACi-treated cancer cells. We developed low toxic combination modalities targeting tumorigenesis and HDACi-activated metastasis for preclinical therapies in mice. RESULTS: We showed that cell migration activity was dramatically and dose dependently enhanced by various classes of HDACi treatments in 13 of 30 examined human breast, gastric, liver, and lung cancer cell lines. Tumor metastasis was also enhanced in HDACi-treated mice. HDACi treatments activated multiple PKCs and downstream substrates along with upregulated proapoptotic p21. For targeting tumorigenesis and metastasis with immediate clinical impact, we showed that new modalities of HDACi combined drugs with PKC inhibitory agent, curcumin or tamoxifen, not only suppressed HDACi-activated tumor progressive proteins and cell migration in vitro but also inhibited tumor growth and metastasis in vivo. CONCLUSION: Treatments of different structural classes of HDACi simultaneously induced cell death and promoted cell migration and metastasis in multiple cancer cell types. Suppression of HDACi-induced PKCs leads to development of low toxic and long-term therapeutic strategies to potentially treat cancer as a chronic disease.

Identification of oncogenic point mutations and hyperphosphorylation of anaplastic lymphoma kinase in lung cancer.

The oncogenic property of anaplastic lymphoma kinase (ALK) plays an essential role in the pathogenesis of various cancers and serves as an important therapeutic target. In this study, we identified frequent intragenic loss of heterozygosity and six novel driver mutations within ALK in lung adenocarcinomas. Overexpression of H694R or E1384K mutant ALK leads to hyperphosphorylation of ALK, and activation of its downstream mediators STAT3, AKT, and ERK resulted in enhanced cell proliferation, colony formation, cell migration, and tumor growth in xenograft models. Furthermore, the activated phospho-Y1604 ALK was increasingly detected in 13 human lung cancer cell lines and 263 lung cancer specimens regardless of tumor stages and types. Treatment of two different ALK inhibitors, WHI-P154 and NVP-TAE684, resulted in the down-regulation of aberrant ALK signaling, shrinkage of tumor, and suppression of metastasis and significantly improved survival of ALK mutant-bearing mice. Together, we identified that novel ALK point mutations possessed tumorigenic effects mainly through hyperphosphorylation of Y1604 and activation of downstream oncogenic signaling. The upregulated phospho-Y1604 ALK could serve as a diagnostic biomarker for lung cancer. Furthermore, targeting oncogenic mutant ALKs with inhibitors could be a promising strategy to improve the therapeutic efficacy of fatal lung cancers.

Epigenetic activation of alpha4, beta2 and beta6 integrins involved in cell migration in trichostatin A-treated Hep3B cells.

The epigenetic modulation by histone deacetylase (HDAC) inhibitors including trichostatin A (TSA) has been known to block cell proliferation, induce apoptosis and inhibit cell migration in human cancer cells that represents the potential therapeutic agents for cancers and fibrosis. However, more than 55% of Hep3B cells remained alive after our initial study of 100 nM TSA treatment. To further study the epigenetic modulation and the biological function of newly activated genes by HDAC inhibitor involved in HCC progression and metastasis, we profiled 23 integrin genes including 15alpha and 8beta in TSA-treated Hep3B cells. Six integrins including three down-regulated alpha6, alpha10, beta8 and three significant up-regulated alpha4, beta2, beta6 integrins were revealed after semi-quantitative RT-PCR. To confirm the epigenetic modulation and explore their biological functions, we selected the three significantly up-regulated integrins for confirmation of protein up-regulation, hyperacetylated-histones by ChIP assays, and functional inhibition by specific neutralizing antibodies of integrins. Our results indicated that epigenetic modulation in TSA-treated Hep3B cells up-regulated new integrins including alpha4, beta2 and beta6 and reduced migration activities by specific neutralizing antibodies to 61.3%, 42.4% and 34.5%, respectively. Our novel findings provided a better understanding of the epigenetic modulation of integrins and suggested that targeting the epigenetic up-regulated integrins to abrogate the migration activity might be a promising strategy to prevent HCC progression.