Predicting osimertinib‐treatment outcomes through EGFR mutant‐fraction monitoring in the circulating tumor DNA of EGFR T790M‐positive patients with non‐small cell lung cancer (WJOG8815L)

The WJOG8815L phase II clinical study involves patients with non‐small cell lung cancer (NSCLC) that harbored the EGFR T790M mutation, which confers resistance to EGFR tyrosine kinase inhibitors (TKIs). The purpose of this study was to assess the predictive value of monitoring EGFR genomic alterations in circulating tumor DNA (ctDNA) from patients with NSCLC that undergo treatment with the third‐generation EGFR‐TKI osimertinib. Plasma samples of 52 patients harboring the EGFR T790M mutation were obtained pretreatment (Pre), on day 1 of treatment cycle 4 (C4) or cycle 9 (C9), and at diagnosis of disease progression or treatment discontinuation (PD/stop). CtDNA was screened for EGFR‐TKI‐sensitizing mutations, the EGFR T790M mutation, and other genomic alterations using the cobas EGFR Mutation Test v2 (cobas), droplet digital PCR (ddPCR), and targeted deep sequencing. Analysis of the sensitizing—and T790M—EGFR mutant fractions (MFs) was used to determine tumor mutational burden. Both MFs were found to decrease during treatment, whereas rebound of the sensitizing EGFR MF was observed at PD/stop, suggesting that osimertinib targeted both T790M mutation‐positive tumors and tumors with sensitizing EGFR mutations. Significant differences in the response rates and progression‐free survival were observed between the sensitizing EGFR MF‐high and sensitizing EGFR MF‐low groups (cutoff: median) at C4. In conclusion, ctDNA monitoring for sensitizing EGFR mutations at C4 is suitable for predicting the treatment outcomes in NSCLC patients receiving osimertinib (Clinical Trial Registration No.: UMIN000022076).

Abbreviations

CIs

confidence intervals

ctDNA

circulating tumor DNA

ddPCR

droplet digital PCR

EGFR

epidermal growth factor receptor

MFs

mutant fractions

NGS

next‐generation sequencing

NSCLC

non‐small cell lung cancer

ORR

overall response rate

OS

overall survival

PD

progressive disease

PFS

progression‐free survival

PR

partial response

SD

stable disease

TKI

tyrosine kinase inhibitor

     

Metabolic enzyme ACSL3 is a prognostic biomarker and correlates with anticancer effectiveness of statins in non‐small cell lung cancer

Lung cancer is one of the most common cancers, still characterized by high mortality rates. As lipid metabolism contributes to cancer metabolic reprogramming, several lipid metabolism genes are considered prognostic biomarkers of cancer. Statins are a class of lipid‐lowering compounds used in treatment of cardiovascular disease that are currently studied for their antitumor effects. However, their exact mechanism of action and specific conditions in which they should be administered remains unclear. Here, we found that simvastatin treatment effectively promoted antiproliferative effects and modulated lipid metabolism‐related pathways in non‐small cell lung cancer (NSCLC) cells and that the antiproliferative effects of statins were potentiated by overexpression of acyl‐CoA synthetase long‐chain family member 3 (ACSL3). Moreover, ACSL3 overexpression was associated with worse clinical outcome in patients with high‐grade NSCLC. Finally, we found that patients with high expression levels of ACSL3 displayed a clinical benefit of statins treatment. Therefore, our study highlights ACSL3 as a prognostic biomarker for NSCLC, useful to select patients who would obtain a clinical benefit from statin administration.

Abbreviations

3‐HMGCR

3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase

95% CI

95% confidence intervals

ACSL3

acyl‐CoA synthetase long‐chain family member 3

ACSLs

long‐chain acyl‐CoA synthetases

ALP

alkaline phosphatase

APOA1

apolipoprotein A1

ATCC

American Type Culture Collection

CASP9

caspase 9

ECAR

extracellular acidification rate

ECOG

Eastern Cooperative Oncology Group

EMT

epithelial‐to‐mesenchymal transition

ER

endoplasmic reticulum

FAs

fatty acids

FFPE

formalin‐fixed, paraffin‐embedded

GTEx

genotype‐tissue expression

HR

Hazard ratio

IC50

half‐maximal inhibitory concentration

LDH

lactate dehydrogenase

MTT

3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium

NID1

nidogen 1

No ORF

no open reading frame

NSCLC

non‐small cell lung cancer

OCR

oxygen consumption rate

OS

overall survival

PGE2

prostaglandins E2

RETN

resistin

TCGA

The Cancer Genome Atlas

TMA

tumor tissue microarray

     

Intratumoral immunosuppression profiles in 11q‐deleted neuroblastomas provide new potential therapeutic targets

High‐risk neuroblastoma (NB) patients with 11q deletion frequently undergo late but consecutive relapse cycles with fatal outcome. To date, no actionable targets to improve current multimodal treatment have been identified. We analyzed immune microenvironment and genetic profiles of high‐risk NB correlating with 11q immune status. We show in two independent cohorts that 11q‐deleted NB exhibits various immune inhibitory mechanisms, including increased CD4+ resting T cells and M2 macrophages, higher expression of programmed death‐ligand 1, interleukin‐10, transforming growth factor‐beta‐1, and indoleamine 2,3‐dioxygenase 1 (P < 0.05), and also higher chromosomal breakages (P ≤ 0.02) and hemizygosity of immunosuppressive miRNAs than MYCN‐amplified and other 11q‐nondeleted high‐risk NB. We also analyzed benefits of maintenance treatment in 83 high‐risk stage M NB patients focusing on 11q status, either with standard anti‐GD2 immunotherapy (n = 50) or previous retinoic acid‐based therapy alone (n = 33). Immunotherapy associated with higher EFS (50 vs. 30, P = 0.028) and OS (72 vs. 52, P = 0.047) at 3 years in the overall population. Despite benefits from standard anti‐GD2 immunotherapy in high‐risk NB patients, those with 11q deletion still face poor outcome. This NB subgroup displays intratumoral immune suppression profiles, revealing a potential therapeutic strategy with combination immunotherapy to circumvent this immune checkpoint blockade.

Abbreviations

11q‐del

11q‐deleted

ADCC

antibody‐dependent cellular cytotoxicity

CDC

complement‐dependent cytotoxicity

COJEC

chemotherapeutic agents cisplatin, vincristine, carboplatin, etoposide, and cyclophosphamide

CTLA‐4

cytotoxic T lymphocyte antigen 4

EFS

event‐free survival

FISH

fluorescence in situ hybridization

HR

hazard ratio

ICI

immune checkpoint inhibitor

IDO1

indoleamine 2,3‐dioxygenase 1

IFN‐γ

interferon‐γ

IL‐10

interleukin 10

INRG

International Neuroblastoma Risk Group

miR

microRNA

MLPA

multiplex ligation‐dependent probe amplification

MMR

mismatch repair

MNA

MYCN amplification

MS

metastatic special stage

MSI

microsatellite instability

NB

neuroblastoma

NCA

numerical chromosome aberrations

NOS

nitric oxide synthase

OS

overall survival

PD‐1

programmed cell death protein 1

PD‐L1

programmed death‐ligand 1

SCA

segmental chromosome aberrations

TAM

tumor‐associated macrophages

Tfh

follicular helper T cells

TGF‐β

tumor growth factor‐β

TMB

tumor mutational burden

TME

tumor microenvironment

TNF‐α

tumor necrosis factor‐α

Treg

regulatory T cells

     

Multiplex immune protein profiling of fine‐needle aspirates from patients with non‐small‐cell lung cancer reveals signatures associated with PD‐L1 expression and tumor stage

Biomarker signatures identified through minimally invasive procedures already at diagnosis of non‐small‐cell lung cancer (NSCLC) could help to guide treatment with immune checkpoint inhibitors (ICI). Here, we performed multiplex profiling of immune‐related proteins in fine‐needle aspirate (FNA) samples of thoracic lesions from patients with NSCLC to assess PD‐L1 expression and identify related protein signatures. Transthoracic FNA samples from 14 patients were subjected to multiplex antibody‐based profiling by proximity extension assay (PEA). PEA profiling employed protein panels relevant to immune and tumor signaling and was followed by Qlucore^® Omics Explorer analysis. All lesions analyzed were NSCLC adenocarcinomas, and PEA profiles could be used to monitor 163 proteins in all but one sample. Multiple key immune signaling components (including CD73, granzyme A, and chemokines CCL3 and CCL23) were identified and expression of several of these proteins (e.g., CCL3 and CCL23) correlated to PD‐L1 expression. We also found EphA2, a marker previously linked to inferior NSCLC prognosis, to correlate to PD‐L1 expression. Our identified protein signatures related to stage included, among others, CXCL10 and IL12RB1. We conclude that transthoracic FNA allows for extensive immune and tumor protein profiling with assessment of putative biomarkers of important for ICI treatment selection in NSCLC.     

Clinical significance of BIM deletion polymorphism in chemoradiotherapy for non‐small cell lung cancer

The standard treatment for locally advanced non‐small cell lung cancer (NSCLC) is chemoradiotherapy (CRT) followed by anti‐programmed cell death‐ligand 1 (anti‐PD‐L1) treatment. BIM deletion polymorphism induces the suppression of apoptosis resulting from epidermal growth factor (EGFR)‐tyrosine kinase inhibitors in EGFR‐mutated NSCLC patients. We aimed to examine the effects of BIM polymorphism on CRT and anti‐PD‐L1/PD‐1 treatment in NSCLC patients. In this retrospective study of 1312 patients with unresectable NSCLC treated at Higashi‐Hiroshima Medical Center and Hiroshima University Hospital between April 1994 and October 2019, we enrolled those who underwent CRT or chemotherapy using carboplatin + paclitaxel or cisplatin + vinorelbine, or anti‐PD‐L1/PD‐1 treatment. Of 1312 patients, 88, 80, and 74 underwent CRT, chemotherapy, and anti‐PD‐L1/PD‐1 treatment, respectively, and 17.0%, 15.2% and 17.6% of these patients showed BIM polymorphism. Among patients receiving CRT, the progression‐free survival was significantly shorter in those with BIM deletion than in those without. In the multivariate analyses, BIM polymorphism was an independent factor of poor anti‐tumor effects. These results were not observed in the chemotherapy and anti‐PD‐L1/PD‐1 treatment groups. In in vitro experiments, BIM expression suppression using small interfering RNA in NSCLC cell lines showed a significantly suppressed anti‐tumor effect and apoptosis after irradiation but not chemotherapy. In conclusion, we showed that BIM polymorphism was a poor‐predictive factor for anti‐tumor effects in NSCLC patients who underwent CRT, specifically radiotherapy. In the implementation of CRT in patients with BIM polymorphism, we should consider subsequent treatment, keeping in mind that CRT may be insufficient.     

The RNA‐binding protein MEX3A is a prognostic factor and regulator of resistance to gemcitabine in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer. Most patients present with advanced disease at diagnosis, which only permits palliative chemotherapeutic treatments. RNA dysregulation is a hallmark of most human cancers, including PDAC. To test the impact of RNA processing dysregulation on PDAC pathology, we performed a bioinformatics analysis to identify RNA‐binding proteins (RBPs) associated with prognosis. Among the 12 RBPs associated with progression‐free survival, we focused on MEX3A because it was recently shown to mark an intestinal stem cell population that is refractory to chemotherapeutic treatments, a typical feature of PDAC. Increased expression of MEX3A was correlated with higher disease stage in PDAC patients and with tumor development in a mouse model of PDAC. Depletion of MEX3A in PDAC cells enhanced sensitivity to chemotherapeutic treatment with gemcitabine, whereas its expression was increased in PDAC cells selected upon chronic exposure to the drug. RNA‐sequencing analyses highlighted hundreds of genes whose expression is sensitive to MEX3A expression, with significant enrichment in cell cycle genes. MEX3A binds to its target mRNAs, like cyclin‐dependent kinase 6 (CDK6), and promotes their stability. Accordingly, knockdown of MEX3A caused a significant reduction in PDAC cell proliferation and in progression to the S phase of the cell cycle. These findings uncover a novel role for MEX3A in the acquisition and maintenance of chemoresistance by PDAC cells, suggesting that it may represent a novel therapeutic target for PDAC.

Abbreviations

CLIP

UV‐crosslink and RNA immunoprecipitation

DFS

disease‐free survival

DR

drug resistant

EMT

mesenchymal transition

MC

MITO‐Cre

MKC

MITO‐Kras‐Cre

PARG

poly (ADP‐ribose) glycohydrolase

PDAC

pancreatic ductal adenocarcinoma

PI

propidium iodide

RBPs

RNA‐binding proteins

RNA‐seq

RNA sequencing

RNP

ribonucleoprotein

TGCA

The Cancer Genome Atlas

     

Circulating tumor cell detection and single‐cell analysis using an integrated workflow based on ChimeraX®‐i120 Platform: A prospective study

Circulating tumor cell (CTC) analysis holds great potential to be a noninvasive solution for clinical cancer management. A complete workflow that combined CTC detection and single‐cell molecular analysis is required. We developed the ChimeraX^®‐i120 platform to facilitate negative enrichment, immunofluorescent labeling, and machine learning‐based identification of CTCs. Analytical performances were evaluated, and a total of 477 participants were enrolled to validate the clinical feasibility of ChimeraX^®‐i120 CTC detection. We analyzed copy number alteration profiles of isolated single cells. The ChimeraX^®‐i120 platform had high sensitivity, accuracy, and reproducibility for CTC detection. In clinical samples, an average value of > 60% CTC‐positive rate was found for five cancer types (i.e., liver, biliary duct, breast, colorectal, and lung), while CTCs were rarely identified in blood from healthy donors. In hepatocellular carcinoma patients treated with curative resection, CTC status was significantly associated with tumor characteristics, prognosis, and treatment response (all P < 0.05). Single‐cell sequencing analysis revealed that heterogeneous genomic alteration patterns resided in different cells, patients, and cancers. Our results suggest that the use of this ChimeraX^®‐i120 platform and the integrated workflow has validity as a tool for CTC detection and downstream genomic profiling in the clinical setting.

Abbreviations

ADABOOST

AdaBoost classification trees

AFP

alpha‐fetoprotein

AUC

areas under the curve

BC

breast cancer

BCLC

barcelona clinic liver cancer

BHL

benign hepatic lesion

CCD

charge‐coupled device

CHB

chronic hepatitis B

CK

cytokeratin

CNA

copy number alteration

CNLC

Chinese staging for liver cancer

CRC

colorectal cancer

CTC

circulating tumor cell

CTM

circulating tumor microemboli

CV

coefficient of variation

DAPI

4’,6‐diamidine‐2’‐phenylindole dihydrochloride

EpCAM

epithelial cell adhesion molecule

FPR

false‐positive rate

GBM

stochastic gradient boosting

HCC

hepatocellular carcinoma

HD

healthy donor

ICC

intrahepatic cholangiocarcinoma

LC

liver cirrhosis

LCA

lung cancer

LOD

limit of detection

PBS

phosphate‐buffered saline

PCR

polymerase chain reaction

RF

random forest

ROC

receiver operating characteristic

SVM

support vector machines

TCGA

The Cancer Genome Atlas

TPR

true‐positive rate

TTR

time to recurrence

WBC

white blood cell

WGA

whole‐genome amplification

WGS

whole‐genome sequencing

XGB

extreme gradient boosting

     

5‐Hydroxymethylcytosine signature in circulating cell‐free DNA as a potential diagnostic factor for early‐stage colorectal cancer and precancerous adenoma

Approximately 85% colorectal cancers (CRCs) are thought to evolve through the adenoma‐to‐carcinoma sequence associated with specific molecular alterations, including the 5‐hydroxymethylcytosine (5hmC) signature in circulating cell‐free DNA (cfDNA). To explore colorectal disease progression and evaluate the use of cfDNA as a potential diagnostic factor for CRC screening, here, we performed genome‐wide 5hmC profiling in plasma cfDNA and tissue genomic DNA (gDNA) acquired from 101 samples (63 plasma and 38 tissues), collected from 21 early‐stage CRC patients, 21 AD patients, and 21 healthy controls (HC). The gDNA and cfDNA 5hmC signatures identified in gene bodies and promoter regions in CRC and AD groups were compared with those in HC group. All the differential 5hmC‐modified regions (DhMRs) were gathered into four clusters: Disease‐enriched, AD‐enriched, Disease‐lost, and AD‐lost, with no overlap. AD‐related clusters, AD‐enriched and AD‐lost, displayed the unique 5hmC signals in AD patients. Disease‐enriched and Disease‐lost clusters indicated the general 5hmC changes when colorectal lesions occurred. Cancer patients with a confirmable adenoma history segmentally gathered in AD‐enriched clusters. KEGG functional enrichment and GO analyses determined distinct differential 5hmC‐modified profiles in cfDNA of HC individuals, AD, and CRC patients. All patients had comprehensive 5hmC signatures where Disease‐enriched and Disease‐lost DhMR clusters demonstrated similar epigenetic modifications, while AD‐enriched and AD‐lost DhMR clusters indicated complicated subpopulations in adenoma. Analysis of CRC patients with adenoma history showed exclusive 5hmC‐gain characteristics, consistent with the ‘parallel’ evolution hypothesis in adenoma, either developed through the adenoma‐to‐carcinoma sequence or not. These findings deepen our understanding of colorectal disease and suggest that the 5hmC modifications of different pathological subtypes (cancer patients with or without adenoma history) could be used to screen early‐stage CRC and assess adenoma malignancy with large‐scale follow‐up studies in the future.

Abbreviations

5hmC

5‐hydroxymethylcytosine

AD

precancerous adenoma

cfDNA

cell‐free DNA

CRC

colorectal cancer

DhmR

differential 5hmC‐modified regions

gDNA

genomics DNA

HC

healthy control

hMRs

5hmC‐modified regions

     

Long non‐coding RNA RACGAP1P promotes breast cancer invasion and metastasis via miR‐345‐5p/RACGAP1‐mediated mitochondrial fission

Long non‐coding RNAs (lncRNAs) are emerging as key molecules in various cancers, yet their potential roles in the pathogenesis of breast cancer are not fully understood. Herein, using microarray analysis, we revealed that the lncRNA RACGAP1P, the pseudogene of Rac GTPase activating protein 1 (RACGAP1), was up‐regulated in breast cancer tissues. Its high expression was confirmed in 25 pairs of breast cancer tissues and 8 breast cell lines by qRT‐PCR. Subsequently, we found that RACGAP1P expression was positively correlated with lymph node metastasis, distant metastasis, TNM stage, and shorter survival time in 102 breast cancer patients. Then, in vitro and in vivo experiments were designed to investigate the biological function and regulatory mechanism of RACGAP1P in breast cancer cell lines. Overexpression of RACGAP1P in MDA‐MB‐231 and MCF7 breast cell lines increased their invasive ability and enhanced their mitochondrial fission. Conversely, inhibition of mitochondrial fission by Mdivi‐1 could reduce the invasive ability of RACGAP1P‐overexpressing cell lines. Furthermore, the promotion of mitochondrial fission by RACGAP1P depended on its competitive binding with miR‐345‐5p against its parental gene RACGAP1, leading to the activation of dynamin‐related protein 1 (Drp1). In conclusion, lncRNA RACGAP1P promotes breast cancer invasion and metastasis via miR‐345‐5p/RACGAP1 pathway‐mediated mitochondrial fission.

Abbreviations

CDS

coding sequence

ceRNAs

competitive endogenous RNAs

Drp1

dynamin‐related protein 1

FFPE

formalin‐fixed paraffin‐embedded

lncRNAs

long non‐coding RNAs

miRNAs

microRNAs

RACGAP1

Rac GTPase activating protein 1

TCGA

The Cancer Genome Atlas

     

Comprehensive cross‐platform comparison of methods for non‐invasive EGFR mutation testing: results of the RING observational trial

Several platforms for noninvasive EGFR testing are currently used in the clinical setting with sensitivities ranging from 30% to 100%. Prospective studies evaluating agreement and sources for discordant results remain lacking. Herein, seven methodologies including two next‐generation sequencing (NGS)‐based methods, three high‐sensitivity PCR‐based platforms, and two FDA‐approved methods were compared using 72 plasma samples, from EGFR‐mutant non‐small‐cell lung cancer (NSCLC) patients progressing on a first‐line tyrosine kinase inhibitor (TKI). NGS platforms as well as high‐sensitivity PCR‐based methodologies showed excellent agreement for EGFR‐sensitizing mutations (K = 0.80–0.89) and substantial agreement for T790M testing (K = 0.77 and 0.68, respectively). Mutant allele frequencies (MAFs) obtained by different quantitative methods showed an excellent reproducibility (intraclass correlation coefficients 0.86–0.98). Among other technical factors, discordant calls mostly occurred at mutant allele frequencies (MAFs) ≤ 0.5%. Agreement significantly improved when discarding samples with MAF ≤ 0.5%. EGFR mutations were detected at significantly lower MAFs in patients with brain metastases, suggesting that these patients risk for a false‐positive result. Our results support the use of liquid biopsies for noninvasive EGFR testing and highlight the need to systematically report MAFs.

Abbreviations

BEAMing

beads, emulsion, amplification, and magnetics

cfDNA

circulating free DNA, cell‐free DNA

cobas

cobas^® EGFR Mutation Test v2 (Roche Diagnostics)

ctDNA

circulating tumor DNA

CUSUM

cumulative sum

ddPCR

droplet digital polymerase chain reaction

dPCR

digital polymerase chain reaction

EGFR

epidermal growth factor receptor

FFPE

formalin‐fixed, paraffin‐embedded

ICC

intraclass correlation coefficient

MAF

mutant allele frequency

NGS platforms

Ion S5™ XL and GeneRead™

NGS

next‐generation sequencing

NSCLC

non‐small‐cell lung cancer

PNA‐Q‐PCR

peptic nucleic acid probe‐based real‐time polymerase chain reaction

Therascreen

Therascreen EGFR Plasma RGQ PCR Kit (QIAgen)

TKI

tyrosine kinase inhibitor

     

TGFβ promotes YAP‐dependent AXL induction in mesenchymal‐type lung cancer cells

The acquisition of chemoresistance remains a major cause of cancer mortality due to the limited accessibility of targeted or immune therapies. However, given that severe alterations of molecular features during epithelial‐to‐mesenchymal transition (EMT) lead to acquired chemoresistance, emerging studies have focused on identifying targetable drivers associated with acquired chemoresistance. Particularly, AXL, a key receptor tyrosine kinase that confers resistance against targets and chemotherapeutics, is highly expressed in mesenchymal cancer cells. However, the underlying mechanism of AXL induction in mesenchymal cancer cells is poorly understood. Our study revealed that the YAP signature, which was highly enriched in mesenchymal‐type lung cancer, was closely correlated to AXL expression in 181 lung cancer cell lines. Moreover, using isogenic lung cancer cell pairs, we also found that doxorubicin treatment induced YAP nuclear translocation in mesenchymal‐type lung cancer cells to induce AXL expression. Additionally, the concurrent activation of TGFβ signaling coordinated YAP‐dependent AXL expression through SMAD4. These data suggest that crosstalk between YAP and the TGFβ/SMAD axis upon treatment with chemotherapeutics might be a promising target to improve chemosensitivity in mesenchymal‐type lung cancer.

Abbreviations

AUC

area under the curve

AXL

AXL receptor tyrosine kinase

BCL2

B‐cell lymphoma 2

CTD2

cancer target discovery and development

CTGF

connective tissue growth factor

DEG

differentially expressed genes

DOXO

doxorubicin

EMT

epithelial–mesenchymal transition

Eto

etoposide

FDA

Food and Drug Administration

ITGB3

integrin beta‐3

MAPK

mitogen‐activated protein kinase

MMP2

matrix metalloproteinase‐2

MMP9

matrix metalloproteinase‐9

mRNA

messenger RNA

NF‐κB

nuclear factor kappa‐light‐chain‐enhancer of activated B cells

SBE

SMAD binding element

SERPINE1

serpin family E member 1

siRNA

small interfering RNA

ssGSEA

single‐sample gene set enrichment analysis

TCGA

The Cancer Genome Atlas

TGFβ

transforming growth factor beta

YAP

Yes‐associated protein

YAP8SA

mutants of inhibitory phosphorylation site at eight serine to Alanine of YAP

ZEB1

zinc finger E‐box binding homeobox 1

ZEB2

zinc finger E‐box‐binding homeobox 2

     

The FABP12/PPARγ pathway promotes metastatic transformation by inducing epithelial‐to‐mesenchymal transition and lipid‐derived energy production in prostate cancer cells

Early stage localized prostate cancer (PCa) has an excellent prognosis; however, patient survival drops dramatically when PCa metastasizes. The molecular mechanisms underlying PCa metastasis are complex and remain unclear. Here, we examine the role of a new member of the fatty acid‐binding protein (FABP) family, FABP12, in PCa progression. FABP12 is preferentially amplified and/or overexpressed in metastatic compared to primary tumors from both PCa patients and xenograft animal models. We show that FABP12 concurrently triggers metastatic phenotypes (induced epithelial‐to‐mesenchymal transition (EMT) leading to increased cell motility and invasion) and lipid bioenergetics (increased fatty acid uptake and accumulation, increased ATP production from fatty acid β‐oxidation) in PCa cells, supporting increased reliance on fatty acids for energy production. Mechanistically, we show that FABP12 is a driver of PPARγ activation which, in turn, regulates FABP12''s role in lipid metabolism and PCa progression. Our results point to a novel role for a FABP‐PPAR pathway in promoting PCa metastasis through induction of EMT and lipid bioenergetics.

Abbreviations

AR

androgen receptor

ATP

adenosine triphosphate

CN

copy number

CPT1

carnitine palmitoyltransferase I

CS

citrate synthase

EMT

epithelial–mesenchymal transition

ET

electron transfer‐state

FABP

fatty acid‐binding protein

LD

lipid droplet

OA

oleic acid

PCa

prostate cancer

PPAR

peroxisome proliferator‐activated receptor

PPRE

peroxisome proliferator‐activated receptor response element

TZD

thiazolidinediones

     

Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

Metastasis accounts for poor prognosis of cancers and related deaths. Accumulating evidence has shown that long noncoding RNAs (lncRNAs) play critical roles in several types of cancer. However, which lncRNAs contribute to metastasis of colon cancer is still largely unknown. In this study, we found that lncRNA LINC01578 was correlated with metastasis and poor prognosis of colon cancer. LINC01578 was upregulated in colon cancer, associated with metastasis, advanced clinical stages, poor overall survival, disease‐specific survival, and disease‐free survival. Gain‐of‐function and loss‐of‐function assays revealed that LINC01578 enhanced colon cancer cell viability and mobility in vitro and colon cancer liver metastasis in vivo. Mechanistically, nuclear factor kappa B (NF‐κB) and Yin Yang 1 (YY1) directly bound to the LINC01578 promoter, enhanced its activity, and activated LINC01578 expression. LINC01578 was shown to be a chromatin‐bound lncRNA, which directly bound NFKBIB promoter. Furthermore, LINC01578 interacted with and recruited EZH2 to NFKBIB promoter and further repressed NFKBIB expression, thereby activating NF‐κB signaling. Through activation of NF‐κB, LINC01578 further upregulated YY1 expression. Through activation of the NF‐κB/YY1 axis, LINC01578 in turn enhanced its own promoter activity, suggesting that LINC01578 and NF‐κB/YY1 formed a positive feedback loop. Blocking NF‐κB signaling abolished the oncogenic roles of LINC01578 in colon cancer. Furthermore, the expression levels of LINC01578, NFKBIB, and YY1 were correlated in clinical tissues. Collectively, this study demonstrated that LINC01578 promoted colon cancer metastasis via forming a positive feedback loop with NF‐κB/YY1 and suggested that LINC01578 represents a potential prognostic biomarker and therapeutic target for colon cancer metastasis.

Abbreviations

ChIP

chromatin immunoprecipitation

ChIRP

chromatin isolation by RNA purification

COAD

colon adenocarcinoma

CPAT

Coding‐Potential Assessment Tool

CPC

coding potential calculator

DFS

disease‐free survival

DSS

disease‐specific survival

EdU

5‐ethynyl‐2''‐deoxyuridine

H&E

hematoxylin and eosin

HR

hazard ratio

IHC

immunohistochemistry

IKK

IκB kinase

IκB

inhibitory κB

lncRNAs

long noncoding RNAs

NC

negative control

NCBI

National Center for Biotechnology Information

NF‐κB

nuclear factor kappa B

qRT‐PCR

quantitative real‐time polymerase chain reaction

RIP

RNA immunoprecipitation

RPISeq

RNA‐Protein Interaction Prediction

TCGA

The Cancer Genome Atlas

TNF

tumor necrosis factor

TUNEL

TdT‐mediated dUTP Nick‐End Labeling

YY1

Yin Yang 1

     

SMARCA4 mutations in KRAS‐mutant lung adenocarcinoma: a multi‐cohort analysis

KRAS is a key oncogenic driver in lung adenocarcinoma (LUAD). Chromatin‐remodeling gene SMARCA4 is comutated with KRAS in LUAD; however, the impact of SMARCA4 mutations on clinical outcome has not been adequately established. This study sought to shed light on the clinical significance of SMARCA4 mutations in LUAD. The association of SMARCA4 mutations with survival outcomes was interrogated in four independent cohorts totaling 564 patients: KRAS‐mutant patients with LUAD who received nonimmunotherapy treatment from (a) The Cancer Genome Atlas (TCGA) and (b) the MSK‐IMPACT Clinical Sequencing (MSK‐CT) cohorts; and KRAS‐mutant patients with LUAD who received immune checkpoint inhibitor‐based immunotherapy treatment from (c) the MSK‐IMPACT (MSK‐IO) and (d) the Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) immunotherapy cohorts. Of the patients receiving nonimmunotherapy treatment, in the TCGA cohort (n = 155), KRAS‐mutant patients harboring SMARCA4 mutations (KS) showed poorer clinical outcome [P = 6e‐04 for disease‐free survival (DFS) and 0.031 for overall survival (OS), respectively], compared to KRAS‐TP53 comutant (KP) and KRAS‐only mutant (K) patients; in the MSK‐CT cohort (n = 314), KS patients also exhibited shorter OS than KP (P = 0.03) or K (P = 0.022) patients. Of patients receiving immunotherapy, KS patients consistently exhibited the shortest progression‐free survival (PFS; P = 0.0091) in the MSK‐IO (n = 77), and the shortest PFS (P = 0.0026) and OS (P = 0.0014) in the WFBCCC (n = 18) cohorts, respectively. Therefore, mutations of SMARCA4 represent a genetic factor leading to adverse clinical outcome in lung adenocarcinoma treated by either nonimmunotherapy or immunotherapy.

Abbreviations

DCB

durable clinical benefit

DFS

disease‐free survival

K

KRAS‐only mutant

KL

KRAS‐STK11 comutant

KP

KRAS‐TP53 comutant

KS

KRAS‐SMARCA4 comutant

LUAD

lung adenocarcinoma

LUSC

lung squamous carcinoma

MSK‐CT

the MSK‐IMPACT clinical sequencing cohort

MSK‐IO

MSK‐IMPACT cohort

NSCLC

non‐small‐cell lung cancer

OS

overall survival

PFS

progression‐free survival

TCGA

The Cancer Genome Atlas

WFBCCC

the Wake Forest Baptist Comprehensive Cancer Center

     

First‐line pembrolizumab vs chemotherapy in metastatic non‐small‐cell lung cancer: KEYNOTE‐024 Japan subset

This prespecified subanalysis of the global, randomized controlled phase III KEYNOTE‐024 study of pembrolizumab vs chemotherapy in previously untreated metastatic non‐small‐cell lung cancer without EGFR/ALK alterations and a programmed death ligand 1 (PD‐L1) tumor proportion score of 50% or higher evaluated clinical outcomes among patients enrolled in Japan. Treatment consisted of pembrolizumab 200 mg every 3 weeks (35 cycles) or platinum‐based chemotherapy (four to six cycles). The primary end‐point was progression‐free survival; secondary end‐points included overall survival and safety. Of 305 patients randomized in KEYNOTE‐024 overall, 40 patients were enrolled in Japan (all received treatment: pembrolizumab, n = 21; chemotherapy, n = 19). Median progression‐free survival was 41.4 (95% confidence interval [CI], 4.2‐42.5) months with pembrolizumab and 4.1 (95% CI, 2.8‐8.3) months with chemotherapy (hazard ratio [HR], 0.27 [95% CI, 0.11‐0.65]; one‐sided, nominal P = .001). Median overall survival was not reached (NR) (95% CI, 22.9‒NR) and 21.5 (95% CI, 5.2‐35.0) months, respectively (HR, 0.39 [95% CI, 0.17‐0.91]; one‐sided, nominal P = .012). Treatment‐related adverse events occurred in 21/21 (100%) pembrolizumab‐treated and 18/19 (95%) chemotherapy‐treated patients; eight patients (38%) and nine patients (47%), respectively, had grade 3‐5 events. Immune‐mediated adverse events and infusion reactions occurred in 11 pembrolizumab‐treated patients (52%) and four chemotherapy‐treated patients (21%), respectively; four patients (19%) and one patient (5%), respectively, had grade 3‐5 events. Consistent with results from KEYNOTE‐024 overall, first‐line pembrolizumab improved progression‐free survival and overall survival vs chemotherapy with manageable safety among Japanese patients with metastatic non‐small‐cell lung cancer without EGFR/ALK alterations and a PD‐L1 tumor proportion score of 50% or higher. The trial is registered with Clinicaltrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT02142738","term_id":"NCT02142738"}}NCT02142738.     

Circ_0008039 supports breast cancer cell proliferation,migration, invasion,and glycolysis by regulating the miR‐140‐3p/SKA2 axis

Circular RNAs (circRNAs) have been shown to modulate gene expression and participate in the development of multiple malignancies. The purpose of this study was to investigate the role of circ_0008039 in breast cancer (BC). The expression of circ_0008039, miR‐140‐3p, and spindle and kinetochore‐associated protein 2 (SKA2) was detected by qRT‐PCR. Cell viability, colony formation, migration, and invasion were evaluated using methylthiazolyldiphenyl‐tetrazolium bromide (MTT) assay, colony formation assay, and transwell assay, respectively. Glucose consumption and lactate production were measured using commercial kits. Protein levels of hexokinase II (HK2) and SKA2 were determined by western blot. The interaction between miR‐140‐3p and circ_0008039 or SKA2 was verified by dual‐luciferase reporter assay. Finally, a mouse xenograft model was established to investigate the roles of circ_0008039 in BC in vivo. We found that circ_0008039 and SKA2 were upregulated in BC tissues and cells, while miR‐140‐3p was downregulated. Knockdown of circ_0008039 suppressed BC cell proliferation, migration, invasion, and glycolysis. Moreover, miR‐140‐3p could bind to circ_0008039 and its inhibition reversed the inhibitory effect of circ_0008039 interference on proliferation, migration, invasion, and glycolysis in BC cells. SKA2 was verified as a direct target of miR‐140‐3p and its overexpression partially inhibited the suppressive effect of miR‐140‐3p restoration in BC cells. Additionally, circ_0008039 positively regulated SKA2 expression by sponging miR‐140‐3p. Consistently, silencing circ_0008039 restrained tumor growth via increasing miR‐140‐3p and decreasing SKA2. In conclusion, circ_0008039 downregulation suppressed BC cell proliferation, migration, invasion, and glycolysis partially through regulating the miR‐140‐3p/SKA2 axis, providing an important theoretical basis for treatment of BC.

Abbreviations

ANOVA

analysis of variance

BC

breast cancer

circRNAs

circular RNAs

DMSO

dimethyl sulfoxide

ECAR

extracellular acidification rate

ECL

enhanced chemiluminescence

FBS

fetal bovine serum

HK2

hexokinase II

MEGM

mammary epithelial growth medium

miR‐140‐3p

microRNA‐140‐3p

MTT

methylthiazolyldiphenyl‐tetrazolium bromide

PBS

phosphate‐buffered saline

PRKAR1B

protein kinase A regulatory subunit R1‐beta

SD

standard ± deviation

SKA2

spindle and kinetochore‐associated protein 2

     

Cancer diagnostics based on plasma protein biomarkers: hard times but great expectations

Cancer diagnostics based on the detection of protein biomarkers in blood has promising potential for early detection and continuous monitoring of disease. However, the currently available protein biomarkers and assay formats largely fail to live up to expectations, mainly due to insufficient diagnostic specificity. Here, we discuss what kinds of plasma proteins might prove useful as biomarkers of malignant processes in specific organs. We consider the need to search for biomarkers deep down in the lowest reaches of the proteome, below current detection levels. In this regard, we comment on the poor molecular detection sensitivity of current protein assays compared to nucleic acid detection reactions, and we discuss requirements for achieving detection of vanishingly small amounts of proteins, to ensure detection of early stages of malignant growth through liquid biopsy.

Abbreviations

ACPP

acid phosphatase

AFP

alpha‐fetoprotein

CEA

carcinoembryonic antigen

CTLA4

cytotoxic T lymphocyte‐associated protein 4

FDA

(US) Food and Drug Administration

GTEx project

Genotype‐Tissue Expression project

HCA

human cell Atlas

IL‐6

interleukin 6

PD‐1

programmed cell death 1 receptor

PD‐L1

programmed cell death ligand 1

PSA

prostate‐specific antigen

TCGA

The Cancer Genome Atlas