Chit Chow

Constitutive activation of distinct NF-κB signals in EBV-associated nasopharyngeal carcinoma.

As a distinct type of head and neck cancer, non‐keratinizing nasopharyngeal carcinoma (NPC) is closely associated with EBV infection and massive lymphoid infiltration. The unique histological features suggest that local inflammation plays an important role in NPC tumourigenesis. We comprehensively characterized NF‐κB signalling, a key inflammatory pathway which might contribute to the tumourigenesis of this EBV‐associated cancer. By EMSA, western blotting, and immunohistochemical staining, constitutive activation of distinct NF‐κB complexes, either p50/p50/Bcl3 or p50/RelB, was found in almost all EBV‐positive NPC tumours. siRNA or chemical inhibition of NF‐κB signalling significantly inhibited the growth of EBV‐positive NPC cells C666‐1. Gene expression profiling identified a number of NF‐κB target genes involved in cell proliferation, apoptosis, immune response, and transcription. We further confirmed that p50 signals modulate the expression of multiple oncogenes (MYB, BCL2), chemokines, and chemokine receptors (CXCL9, CXCL10, CX3CL1, and CCL20). The findings support a crucial role of these constitutively activated NF‐κB signals in NPC tumourigenesis and local inflammation. In addition to expression of the viral oncoprotein LMP1, genetic alteration of several NF‐κB regulators (eg TRAF3, TRAF2, NFKBIA, A20) also contributes to the aberrant NF‐κB activation in EBV‐associated NPC. Except for LMP1‐expressing C15 cells, all NPC tumour lines harbour at least one of these genetic alterations. Importantly, missense mutations of TRAF3, TRAF2, and A20 were also detected in 3/33 (9.1%) primary tumours. Taken together with the reported LTBR amplification in 7.3% of primary NPCs, genetic alterations in NF‐κB pathways occurred in at least 16% of cases of this cancer. The findings indicate that distinct NF‐κB signals are constitutively activated in EBV‐positive NPC cells by either multiple genetic changes or EBV latent genes. Copyright

MET Amplification and Exon 14 Splice Site Mutation Define Unique Molecular Subgroups of Non–Small Cell Lung Carcinoma with Poor Prognosis

Purpose: Activation of MET oncogene as the result of amplification or activation mutation represents an emerging molecular target for cancer treatment. We comprehensively studied MET alterations and the clinicopathologic correlations in a large cohort of treatment-naïve non–small cell lung carcinoma (NSCLC). Experimental Design: Six hundred eighty-seven NSCLCs were tested for MET exon 14 splicing site mutation (METΔ14), DNA copy number alterations, and protein expression by Sanger sequencing, FISH, and IHC, respectively. Results: METΔ14 mutation was detected in 2.62% (18/687) of NSCLC. The mutation rates were 2.6% in adenocarcinoma, 4.8% in adenosquamous carcinoma, and 31.8% in sarcomatoid carcinoma. METΔ14 mutation was not detected in squamous cell carcinoma, large cell carcinoma, and lymphoepithelioma-like carcinoma but significantly enriched in sarcomatoid carcinoma (P < 0.001). METΔ14 occurred mutually exclusively with known driver mutations but tended to coexist with MET amplification or copy number gain (P < 0.001). Low-level MET amplification and polysomy might occur in the background of EGFR or KRAS mutation whereas high-level amplification (MET/CEP7 ratio ≥5) was mutually exclusive to the major driver genes except METΔ14. Oncogenic METΔ14 mutation and/or high-level amplification occurred in a total of 3.3% (23/687) of NSCLC and associated with higher MET protein expression. METΔ14 occurred more frequently in older patients whereas amplification was more common in ever-smokers. Both METΔ14 and high-level amplification were independent prognostic factors that predicted poorer survival by multivariable analysis. Conclusions: The high incidence of METΔ14 mutation in sarcomatoid carcinoma suggested that MET inhibition might benefit this specific subgroup of patients. Clin Cancer Res; 22(12); 3048–56. ©2016 AACR. See related commentary by Drilon, p. 2832

Inhibition of NOTCH3 signalling significantly enhances sensitivity to cisplatin in EBV-associated nasopharyngeal carcinoma†

Nasopharyngeal carcinoma (NPC) is an EBV‐associated epithelial malignancy which is prevalent in south‐east Asia and southern China. Despite the multiple genetic and epigenetic changes reported, the contribution of dysregulated signalling pathways to this distinct type of head and neck cancer is not well understood. Here we demonstrate the up‐regulation of NOTCH ligands (JAG1 or DLL4) and effector (HEY1) in the majority of EBV‐positive tumour lines and primary tumours. Among the NOTCH receptors, NOTCH3 was over‐expressed in all EBV‐positive tumour lines and 92.5% of primary tumours. Aberrant activation of NOTCH3 signalling was consistently detected in all these samples. These findings imply that NOTCH3 may play an crucial role in the development of NPC. By NOTCH3 specific siRNA, NOTCH3 signalling was suppressed and thereby significant growth inhibition and apoptosis induction occurred in NPC cells. Down‐regulation of a number of targets involved in cell proliferation, eg CCND1, C‐MYC,NFKB1, and survival, eg BCL2, BCL‐XL, SURVIVIN, was confirmed in the NOTCH3 knockdown NPC cells. Importantly, NOTCH3 knockdown highly enhanced the sensitivity of NPC cells to cisplatin treatment. Furthermore, we revealed that the ability of NPC cells to form spheroids in vitro and tumours in nude mice was also significantly decreased after knockdown of NICD3 expression. These findings indicate that activation of NOTCH3 pathway is a critical oncogenic event in NPC tumourigenesis. Targeting NOTCH3 signalling may serve as a potential therapeutic approach for treating patients suffering from EBV‐associated NPC. Copyright

Identification of a novel 12p13.3 amplicon in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer commonly occurring in southern China. To decipher the molecular basis of this cancer, we performed high‐resolution array CGH analysis on eight tumour lines and 10 primary tumours to identify the genes involved in NPC tumorigenesis. In this study, multiple regions of gain were consistently found at 1q21‐q24, 7q11‐12, 7q21‐22., 11q13, 12p13, 12q13, 19p13 and 19q13. Importantly, a 2.1 Mb region at 12p13.31 was highly amplified in a NPC xenograft, xeno‐2117. By FISH mapping, we have further delineated the amplicon to a 1.24 region flanked by RP11‐319E16 and RP11‐433J6. Copy number gains of this amplicon were confirmed in 21/41 (51%) primary tumours, while three cases (7.3%) showed high copy number amplification. Among the 13 genes within this amplicon, three candidate genes, lymphotoxin beta receptor (LTβR), tumour necrosis factor receptor superfamily memeber 1A (TNFRSF1R) and FLJ10665, were specifically over‐expressed in the NPC xenograft with 12p13.3 amplification. However, only LTβR was frequently over‐expressed in primary tumours. LTβR is a member of the TNF family of receptors, which can modulate NF‐κB signalling pathways. Over‐expression of LTβR in nasopharyngeal epithelial cells resulted in an increase of NF‐κB activity and cell proliferation. In vivo study showed that suppression of LTβR by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification. These findings implied that LTβR is a potential NPC‐associated oncogene within the 12p13.3 amplicon and that its alteration is important in NPC tumorigenesis. Copyright

Detection of methicillin-resistant Staphylococcus aureus using a gold nanoparticle-based colourimetric polymerase chain reaction assay.

We report the use of gold nanoparticles (Au NPs) for direct colourimetric polymerase chain reaction (PCR) detection of methicillin-resistant Staphylococcus aureus (MRSA) in clinical specimens. The colourimetric assay comprised of 2 Au NP probes functionalized with Staphylococcus aureus 23S rRNA- and mecA-specific oligonucleotides. In this study, 72 clinical samples were tested, which included positive blood culture (n=23), urine (n=8), respiratory samples (n=23), as well as wound swabs, pus and body fluid (n=18). Results were recorded qualitatively by direct visual examination and quantitatively by UV-vis spectrophotometry. Using conventional bacterial culture as the gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of this colourimetric assay were 97.14%, 91.89%, 91.89% and 97.14%, respectively, which were comparable to that of commercial real-time PCR assays with a lower cost per reaction. Our assay also showed good agreement with bacterial culture (κ=0.889). The overall detection limit was 500 ng target amplicon, which was comparable to or better than other similar Au NP biosensors. Interestingly, our data revealed the possible relationship between Au NP probe-target hybridization site and assay performance, which might provide hints for design of the Au NP biosensors for nucleic acid detection. To conclude, our study was the first report on the use of Au NP colourimetric assay for direct detection of MRSA in various types of clinical specimens. Further evaluation of the assay is needed in large-scale trials which can also allow for some modifications to streamline the procedures for routine use.

Allelic Loss of Chromosome 6q in Gastric Carcinoma

Loss of the long arm of chromosome 6 (6q) has frequently been reported in gastric carcinoma, and most gastric cancer patients have evidence of intestinal metaplasia in the stomach. However, the relationship between loss of chromosome 6q and intestinal metaplasia has not been studied. In the first part of the study, we define the critical deletion region of chromosome 6q using loss of heterozygosity technique (LOH). Seventeen microsatellite markers were used to detect loss of heterozygosity (LOH) in 37 microdissected gastric tumors. We also examined intestinal metaplasia (IM) foci of the stomach in the same cancer patient (17 cases). Losses on chromosome 6q were detected in high frequency (51%) by LOH. Two distinct regions of common allelic loss were identified: one centered on the marker D6S300 (at 6q16.1) and the second on D6S446 (at 6q27), with LOH frequency of 36% and 31.3%, respectively. The deletions fall into 2 discrete regions, suggesting the existence of at least 2 tumor suppressor genes in 6q. The losses at 6q27 were confirmed by fluorescence in situ hybridization study (FISH). In the cases with LOH in the tumor, no LOH were detected in the autologous IM areas, but losses were detected by FISH. In some cases, these genetic changes may be acquired in the transition from normal gastric mucosa to intestinal metaplasia.

Gastric cancer cell detection in peritoneal washing: cytology versus RT-PCR for CEA transcripts.

This study investigates the sensitivity and specificity of cytology, qualitative, and real-time RT-PCR methods in free cancer cell detection of peritoneal washing from gastric cancer patients. Peritoneal washings were collected from 65 gastric cancer patients for routine cytology and total RNA extraction for qualitative and real-time RT-PCR for CEA. The sensitivity and false-positive rate was 51.1%, 0% for cytology, 48.9% and 5% for qualitative RT-PCR for CEA, and 42.5% and 5% for real-time RT-PCR for CEA. The qualitative and real time RT-PCR results show high concordance rate (89.7%). The highest sensitivity was obtained by the combination of cytology with qualitative RT-PCR for CEA (70.2%). RT-PCR results were positive in 63.6% of cytologic “atypia” cases. Combination of cytology and either of the RT-PCR methods resulted in significantly higher sensitivity than any one of the three methods alone (P < 0.05). There was no definite advantage of the real-time RT-PCR over the conventional RT-PCR.

Regulation of APC/CCdc20 activity by RASSF1A-APC/CCdc20 circuitry.

RASSF1A is a key tumor-suppressor gene that is often inactivated in a wide variety of solid tumors. Studies have illustrated that RASSF1A plays vital roles in the regulation of cell-cycle progression and functions as a guardian of mitosis. Nevertheless, the precise mechanism of RASSF1A-dependent regulation of mitosis remains largely unclear. APC/CCdc20 is the master switch and regulator of mitosis. The activity of APC/CCdc20 is tightly controlled by phosphorylation and specific inhibitors to ensure the sequential ubiquitination of downstream targets. Here, we report on the novel finding of a regulated circuitry that controls the timely expression and hence activity of APC/CCdc20 during mitosis. Our study showed that RASSF1A and APC/CCdc20 form a molecular relay that regulates the APC/CCdc20 activity at early mitosis. We found that RASSF1A inhibits APC/CCdc20 function through its D-box motifs. Paradoxically, RASSF1A was also demonstrated to be ubiquitinated by APC/CCdc20 in vitro and degraded at prometaphase despite of active spindle checkpoint presence. The first two unique D-boxes at the N-terminal of RASSF1A served as specific degron recognized by APC/CCdc20. Importantly, we found that Aurora A and Aurora B directly phosphorylate RASSF1A, a critical step by which RASSF1A switches from being an inhibitor to a substrate of APC/CCdc20 during the course of mitotic progression. As a result of RASSF1A degradation, APC/CCdc20 can then partially activate the ubiquitination of Cyclin A in the presence of spindle checkpoint. This circuitry is essential for the timely degradation of Cyclin A. To conclude, our results propose a new model for RASSF1A–APC/CCdc20 interaction in ensuring the sequential progression of mitosis.

Regulation of APC/C Cdc20 activity by RASSF1A–APC/C Cdc20 circuitry

RASSF1A is a key tumor-suppressor gene that is often inactivated in a wide variety of solid tumors. Studies have illustrated that RASSF1A plays vital roles in the regulation of cell-cycle progression and functions as a guardian of mitosis. Nevertheless, the precise mechanism of RASSF1A-dependent regulation of mitosis remains largely unclear. APC/CCdc20 is the master switch and regulator of mitosis. The activity of APC/CCdc20 is tightly controlled by phosphorylation and specific inhibitors to ensure the sequential ubiquitination of downstream targets. Here, we report on the novel finding of a regulated circuitry that controls the timely expression and hence activity of APC/CCdc20 during mitosis. Our study showed that RASSF1A and APC/CCdc20 form a molecular relay that regulates the APC/CCdc20 activity at early mitosis. We found that RASSF1A inhibits APC/CCdc20 function through its D-box motifs. Paradoxically, RASSF1A was also demonstrated to be ubiquitinated by APC/CCdc20 in vitro and degraded at prometaphase despite of active spindle checkpoint presence. The first two unique D-boxes at the N-terminal of RASSF1A served as specific degron recognized by APC/CCdc20. Importantly, we found that Aurora A and Aurora B directly phosphorylate RASSF1A, a critical step by which RASSF1A switches from being an inhibitor to a substrate of APC/CCdc20 during the course of mitotic progression. As a result of RASSF1A degradation, APC/CCdc20 can then partially activate the ubiquitination of Cyclin A in the presence of spindle checkpoint. This circuitry is essential for the timely degradation of Cyclin A. To conclude, our results propose a new model for RASSF1A–APC/CCdc20 interaction in ensuring the sequential progression of mitosis.

Overexpression of PIN1 Enhances Cancer Growth and Aggressiveness with Cyclin D1 Induction in EBV-Associated Nasopharyngeal Carcinoma

Background Nasopharyngeal carcinoma (NPC) is a peculiar Epstein Barr virus (EBV)-associated malignancy that is prevalent in South-East Asia. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) isomerizes specific phosphorylated amino acid residues, which makes it an important regulator in cell survival and apoptosis. In this study, we investigated the contribution made by PIN1 in NPC tumorigenesis and PIN1’s potential role as a therapeutic target. Methods The expression of PIN1 was examined in a panel of NPC cell lines, xenografts and primary tumors. The functional roles of PIN1 in NPC cells were elucidated by the knockdown and overexpression of PIN1 in in vitro and in vivo nude mice models by siRNA and lenti-viral transfection, respectively. The antitumor effects of the PIN1 inhibitor Juglone in NPC cells were also evaluated. Results We revealed the consistent overexpression of PIN1 in almost all EBV-associated NPC cell lines, xenografts and primary tumors. PIN1 suppression was capable of inhibiting cyclin D1 expression and activating caspase-3 in NPC cells. It positively regulated NPC cell proliferation, colony formation and anchorage-independent growth. The inhibition of PIN1 suppressed tumor growth in vitro and in vivo. Conclusions This study demonstrates the oncogenic role of PIN1 in NPC tumorigenesis, and shows that its overexpression can enhance tumor cell growth via the upregulation of cyclinD1. Our findings inform the development of novel treatments targeting PIN1 for NPC patients.