Zaozao Wang

Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients

Significance In a few milliliters of blood from a cancer patient, one can isolate a few circulating tumor cells (CTCs). Originating from the primary tumor, CTCs seed metastases, which account for the majority of cancer-related deaths. We demonstrate the analyses of the whole genome of single CTCs, which are highly needed for personalized treatment. We discovered that copy number variations (CNVs), one of the major genomic variations, are specific to cancer types, reproducible from cell to cell, and even from patient to patient. We hypothesize that CNVs at certain genomic loci are selected for and lead to metastasis. Our work shows the prospect of noninvasive CTC-based cancer diagnostics. Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of small-cell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics.

microRNAs and ceRNAs: RNA networks in pathogenesis of cancer

microRNAs (miRNAs) are a class of endogenous, single-stranded non-coding RNAs of 20-23 nucleotides in length, functioning as negative regulators of gene expression at the post-transcriptional level. The dysregulation of miRNAs has been demonstrated to play critical roles in tumorigenesis, either through inhibiting tumor suppressor genes or activating oncogenes inappropriately. Besides their promising clinical applications in cancer diagnosis and treatment, recent studies have uncovered that miRNAs could act as a regulatory language, through which messenger RNAs, transcribed pseudogenes, and long noncoding RNAs crosstalk with each other and form a novel regulatory network. RNA transcripts involved in this network have been termed as competing endogenous RNAs (ceRNAs), since they influence each others level by competing for the same pool of miRNAs through miRNA response elements (MREs) on their target transcripts. The discovery of miRNA-ceRNA network not only provides the possibility of an additional level of post-transcriptional regulation, but also dictates a reassessment of the existing regulatory pathways involved in cancer initiation and progression.

Validation of the Memorial Sloan-Kettering Cancer Center Nomogram to Predict Disease-Specific Survival after R0 Resection in a Chinese Gastric Cancer Population

Background Prediction of disease-specific survival (DSS) for individual patient with gastric cancer after R0 resection remains a clinical concern. Since the clinicopathologic characteristics of gastric cancer vary widely between China and western countries, this study is to evaluate a nomogram from Memorial Sloan-Kettering Cancer Center (MSKCC) for predicting the probability of DSS in patients with gastric cancer from a Chinese cohort. Methods From 1998 to 2007, clinical data of 979 patients with gastric cancer who underwent R0 resection were retrospectively collected from Peking University Cancer Hospital & Institute and used for external validation. The performance of the MSKCC nomogram in our population was assessed using concordance index (C-index) and calibration plot. Results The C-index for the MSKCC predictive nomogram was 0.74 in the Chinese cohort, compared with 0.69 for American Joint Committee on Cancer (AJCC) staging system (P<0.0001). This suggests that the discriminating value of MSKCC nomogram is superior to AJCC staging system for prognostic prediction in the Chinese population. Calibration plots showed that the actual survival of Chinese patients corresponded closely to the MSKCC nonogram-predicted survival probabilities. Moreover, MSKCC nomogram predictions demonstrated the heterogeneity of survival in stage IIA/IIB/IIIA/IIIB disease of the Chinese patients. Conclusion In this study, we externally validated MSKCC nomogram for predicting the probability of 5- and 9-year DSS after R0 resection for gastric cancer in a Chinese population. The MSKCC nomogram performed well with good discrimination and calibration. The MSKCC nomogram improved individualized predictions of survival, and may assist Chinese clinicians and patients in individual follow-up scheduling, and decision making with regard to various treatment options.

GOLPH3 predicts survival of colorectal cancer patients treated with 5-fluorouracil-based adjuvant chemotherapy

BackgroundGolgi phosphoprotein 3 (GOLPH3) has been validated as a potent oncogene involved in the progression of many types of solid tumors, and its overexpression is associated with poor clinical outcome in many cancers. However, it is still unknown the association of GOLPH3 expression with the prognosis of colorectal cancer (CRC) patients who received 5-fluorouracil (5-FU)-based adjuvant chemotherapy.MethodsThe expression of GOLPH3 was determined by qRT-PCR and immunohistochemistry in colorectal tissues from CRC patients treated with 5-FU based adjuvant chemotherapy after surgery. The association of GOLPH3 with clinicopathologic features and prognosis was analysed. The effects of GOLPH3 on 5-FU sensitivity were examined in CRC cell lines.ResultsGOLPH3 expression was elevated in CRC tissues compared with matched adjacent noncancerous tissues. Kaplan-Meier survival curves indicated that high GOLPH3 expression was significantly associated with prolonged disease-free survival (DFS, P = 0.002) and overall survival (OS, P = 0.011) in patients who received 5-FU-based adjuvant chemotherapy. Moreover, multivariate analysis showed that GOLPH3 expression was an independent prognostic factor for DFS in CRC patients treated with 5-FU-based chemotherapy (HR, 0.468; 95%CI, 0.222-0.987; P = 0.046). In vitro, overexpression of GOLPH3 facilitated the 5-FU chemosensitivity in CRC cells; while siRNA-mediated knockdown of GOLPH3 reduced the sensitivity of CRC cells to 5-FU-induced apoptosis.ConclusionsOur results suggest that GOLPH3 is associated with prognosis in CRC patients treated with postoperative 5-FU-based adjuvant chemotherapy, and may serve as a potential indicator to predict 5-FU chemosensitivity.

Downregulated USP3 mRNA functions as a competitive endogenous RNA of SMAD4 by sponging miR-224 and promotes metastasis in colorectal cancer

Increasing evidence shows that competitive endogenous RNAs (ceRNAs) can affect the expression of other transcripts by sequestering common microRNAs (miRNAs), and participate in tumourigenesis. As a potent tumour suppressor in colorectal cancer (CRC), SMAD4 is regulated by many miRNAs. However, the regulation of SMAD4 by ceRNAs has never been examined. In the present study, we found that USP3 modulated SMAD4 expression in a miRNA dependent, and protein-coding gene independent manner. USP3 and SMAD4 were directly targeted by miR-224, and overexpression of the USP3 3′UTR could inhibit metastasis caused by the loss of USP3. The correlation of USP3, SMAD4 and miR-224 expression was further verified in CRC specimens. Additionally, the loss of USP3 was associated with distal metastasis and a poor prognosis. Altogether, our study demonstrates USP3 as a bona fide SMAD4 ceRNA. The results from this study may provide new insights into the prevention and treatment of CRC.

Evaluation of immune responses of gastric cancer patients treated by laparoscopic and open gastrectomy

Laparoscopic surgery has been shown to offer more advantages than open surgery for the treatment of gastric cancer. However, the perioperative immune responses after laparoscopic surgery remain largely unexplored. Here, we analyzed the immunity of gastric cancer patients treated by laparoscopy-assisted gastrectomy (LAG) or open gastrectomy (OG) with D2 dissection. Seventy patients were randomized into LAG and OG groups. The operative details and postoperative outcomes of the two groups were compared. The immune factors were measured preoperatively as well as on the first, third, and seventh postoperative days (POD). Our results indicate that there was no significant difference between two groups in clinical characteristics. The profiles of white blood cells, neutrophils, lymphocytes, as well as the lymphocyte subpopulations, including T lymphocytes, B lymphocytes, CD4+ T cells, and CD8+ T cells were similar in the LAG and the OG groups. Only the natural killer cell counts were significantly higher in LAG-treated patients than in OG-treated ones on POD7. Moreover, no statistical differences were found between the two groups with respect to the levels of perioperative cytokines, including IFN-γ, IL-4, IL-6, and TNF-α. However, compared with the OG group, most of the immune factors in the LAG group had trends to return to preoperative levels on POD7. Our study demonstrates that the immune function of LAG-treated patients showed patterns similar to that of OG-treated patients, although larger prospective multicenter trials are needed to further evaluate the immunological status of LAG.

Whole exome sequencing reveals intertumor heterogeneity and distinct genetic origins of sporadic synchronous colorectal cancer

Sporadic synchronous colorectal cancer (CRC) refers to more than one primary tumor detected in a single patient at the time of the first diagnosis without predisposition of cancer development. Given the same genetic and microenvironment they raise, sporadic synchronous CRC is a unique model to study CRC tumorigenesis. We performed whole exome sequencing in 32 fresh frozen tumor lesions from 15 patients with sporadic synchronous CRC to compare their genetic alterations. This approach identified ubiquitously mutated genes in the range from 0.34% to 4.22% and from 0.8% to 7.0% in non‐hypermutated tumors and hypermutated tumors, respectively, in a single patient. We show that both ubiquitously mutated genes and candidate cancer genes from different tumors in the same patient mutated at different sites. Consistently, obvious differences in somatic copy number variations (SCNV) were found in most patients with non‐hypermutated tumor lesions, which had ubiquitous copy number amplification rates ranging from 0% to 8.8% and ubiquitous copy number deletion rates ranging from 0% to 8.2%. Hypermutated lesions were nearly diploid with 0% to 18.8% common copy number aberrations. Accordingly, clonal structures, altered signaling pathways and druggable genes in a single patient with synchronous CRC varied significantly. Taken together, the disparate SCNVs and mutations in synchronous CRC supported the field effect theory of tumorigenesis. Moreover, the intertumor heterogeneity of synchronous CRCs implies that analysis of all tumor lesions from the same patient is necessary for appropriate clinical treatment decisions.

CENPH Inhibits Rapamycin Sensitivity by Regulating GOLPH3-dependent mTOR Signaling Pathway in Colorectal Cancer

Background: Centromere protein H (CENPH) is known as a fundamental component of the active centromere complex, and its overexpression is correlated with poor prognosis in various solid tumors. mTOR inhibitor rapamycin has been shown to possess antitumor activity, as well as prevent intestinal tumorigenesis. However, the prognostic value of CENPH in colorectal cancer (CRC) and the role of CENPH in rapamycin sensitivity remain unknown. Materials and methods: The effect of CENPH on the cell proliferation, clonogenicity, and cell response to rapamycin in CRC were evaluated by MTT and/or colony formation assays. For the underlying mechanisms, the interaction between CENPH and GOLPH3 were detected by co-immunoprecipitation, GST pull-down, and His-tag pull-down assays, as well as the laser scanning confocal microscopy. The status of kinases in mTOR signaling was determined by Western blot. Finally, the clinical significance of CENPH was analyzed using public CRC datasets with CENPH transcripts and clinical information. Results: CENPH inhibited CRC malignant phenotypes, conferred reduced sensitivity to rapamycin, and attenuated both mTORC1 and mTORC2 in mTOR signaling pathway through the interaction with golgi phosphoprotein 3 (GOLPH3), which has been identified as a potential oncogene and modulates the response to rapamycin. Moreover, elevated levels of CENPH were detected in CRC tissues, compared with normal colorectal tissues. High levels of CENPH expression gradually decreased according to CRC tumor stages. Patients with high CENPH expression had favorable survival. Conclusions: Our results suggest that CENPH inhibits rapamycin sensitivity by regulating GOLPH3 dependent mTOR pathway. High CENPH expression is associated with better prognosis in CRC patients. Taken together, CENPH may serve as a potential predictor for rapamycin sensitivity and therapeutic target for CRC patients.

STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer

BackgroundSerine/threonine protein kinase 25 (STK25) is critical in regulating whole-body glucose and insulin homeostasis and the accumulation of ectopic lipids. The Warburg effect, also known as aerobic glycolysis, is an essential metabolic characteristic of cancer cells. However, the effects of STK25 on aerobic glycolysis of cancer cells remain unexplored. The aim of this study is to investigate the role of STK25 in colorectal cancer (CRC) and to elucidate the underlying mechanisms.MethodsThe influences of STK25 on the cell proliferation were evaluated by MTT and colony formation assays. The roles of STK25 in aerobic glycolysis were determined by glucose uptake and lactate production assays. The interaction between STK25 and GOLPH3 was detected by co-immunoprecipitation, GST pull-down, and His-tag pull-down assays. Western blot was used to measure the expression of glycolytic genes, and the status of kinases in mTOR pathway. Moreover, a xenograft mouse model was used to investigate the effects of STK25 in vivo. The prognostic significance of STK25 was analyzed using public CRC datasets by a log-rank test.ResultsSTK25 suppressed proliferation, glycolysis and glycolytic gene expression in CRC cells. STK25 interacted with GOLPH3 and mediated glycolysis through GOLPH3-regulated mTOR signaling. Consistent with these observations, silencing of STK25 promoted tumor growth and glycolytic gene expression in an in vivo xenograft mouse model. Moreover, high levels of STK25 correlated with favorable prognosis in patients with CRC.ConclusionsOur results demonstrated that STK25 negatively regulates the proliferation and glycolysis via GOLPH3-dependent mTOR signaling. Accordingly, STK25 could be a potential therapeutic target for the treatment of CRC.

KIF14 promotes cell proliferation via activation of Akt and is directly targeted by miR-200c in colorectal cancer

As a mitotic kinesin, kinesin family member 14 (KIF14) has been reported to serve oncogenic roles in a variety of malignancies; however, its functional role and regulatory mechanisms in colorectal cancer (CRC) remain unclear. In the present study, KIF14 was observed to be markedly overexpressed in CRC, and this upregulation was associated with tumor size and marker of proliferation Ki-67 immunostaining scores. Gain- and loss-of-function experiments were applied to identify the function of KIF14 in CRC progression. In vitro and in vivo assays revealed that KIF14 promoted CRC cell proliferation and accelerated the cell cycle via activation of protein kinase B. In addition, the present study investigated the potential mechanisms underlying KIF14 overexpression in CRC. Bioinformatics analyses and validation experiments, including reverse transcription-quantitative polymerase chain reaction, western blotting and a Dual-Luciferase reporter assay, demonstrated that, in addition to genomic amplification and transcriptional activation, KIF14 was regulated by microRNA (miR)-200c at the post-transcriptional level. Rescue experiments further demonstrated that decreased miR-200c expression could facilitate KIF14 to exert its pro-proliferative role. The expression of miR-200c was negatively correlated with KIF14 in CRC specimens. Collectively, the findings of the present study demonstrated the oncogenic role of KIF14 in colorectal tumorigenesis, and also revealed a complexity of regulatory mechanisms mediating KIF14 overexpression, which may provide insight for developing novel treatments for patients with CRC.