Defining the dimensions of circulating tumor cells in a large series of breast,prostate, colon,and bladder cancer patients

Circulating tumor cells (CTCs) in the blood of cancer patients are of high clinical relevance. Since detection and isolation of CTCs often rely on cell dimensions, knowledge of their size is key. We analyzed the median CTC size in a large cohort of breast (BC), prostate (PC), colorectal (CRC), and bladder (BLC) cancer patients. Images of patient‐derived CTCs acquired on cartridges of the FDA‐cleared CellSearch^® method were retrospectively collected and automatically re‐analyzed using the accept software package. The median CTC diameter (μm) was computed per tumor type. The size differences between the different tumor types and references (tumor cell lines and leukocytes) were nonparametrically tested. A total of 1962 CellSearch^® cartridges containing 71 612 CTCs were included. In BC, the median computed diameter (CD) of patient‐derived CTCs was 12.4 μm vs 18.4 μm for cultured cell line cells. For PC, CDs were 10.3 μm for CTCs vs 20.7 μm for cultured cell line cells. CDs for CTCs of CRC and BLC were 7.5 μm and 8.6 μm, respectively. Finally, leukocytes were 9.4 μm. CTC size differed statistically significantly between the four tumor types and between CTCs and the reference data. CTC size differences between tumor types are striking and CTCs are smaller than cell line tumor cells, whose size is often used as reference when developing CTC analysis methods. Based on our data, we suggest that the size of CTCs matters and should be kept in mind when designing and optimizing size‐based isolation methods.

Abbreviations

ACCEPT

Automated CTC Classification, Enumeration, and PhenoTyping software

BC

breast cancer

BLC

bladder cancer

CD

computed diameter

CEL

cultured tumor cell (cell line)

CK

cytokeratin

CRC

colorectal cancer

CTC‐L

circulating tumor cells derived from cerebrospinal fluid (liquor)

CTCs

circulating tumor cells

DAPI

4′6‐diamidino‐2‐phenylindole

EMT

epithelial–mesenchymal transition

EpCAM

epithelial cell adhesion molecule

IQR

interquartile range

KW test

Kruskal–Wallis test

MWU test

Mann–Whitney U test

NCR

nucleus/cytoplasm ratio

P2A

perimeter to area

PC

prostate cancer

TIF

tagged Image Format files

TXT

text file

μm

micrometer

µm²

square micrometers

     

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

     

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

     

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

     

Downregulation of miR‐326 and its host gene β‐arrestin1 induces pro‐survival activity of E2F1 and promotes medulloblastoma growth

Persistent mortality rates of medulloblastoma (MB) and severe side effects of the current therapies require the definition of the molecular mechanisms that contribute to tumor progression. Using cultured MB cancer stem cells and xenograft tumors generated in mice, we show that low expression of miR‐326 and its host gene β‐arrestin1 (ARRB1) promotes tumor growth enhancing the E2F1 pro‐survival function. Our models revealed that miR‐326 and ARRB1 are controlled by a bivalent domain, since the H3K27me3 repressive mark is found at their regulatory region together with the activation‐associated H3K4me3 mark. High levels of EZH2, a feature of MB, are responsible for the presence of H3K27me3. Ectopic expression of miR‐326 and ARRB1 provides hints into how their low levels regulate E2F1 activity. MiR‐326 targets E2F1 mRNA, thereby reducing its protein levels; ARRB1, triggering E2F1 acetylation, reverses its function into pro‐apoptotic activity. Similar to miR‐326 and ARRB1 overexpression, we also show that EZH2 inhibition restores miR‐326/ARRB1 expression, limiting E2F1 pro‐proliferative activity. Our results reveal a new regulatory molecular axis critical for MB progression.

Abbreviations

ARRB1

β‐arrestin1

BTC

bulk tumor cell

CSCs

cancer stem cells

EZH2

enhancer of zeste homolog 2

GCP

granule cell progenitors

MB

medulloblastoma

OFC

oncosphere‐forming cell

     

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

     

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

     

Downregulation of Glutamine Synthetase,not glutaminolysis,is responsible for glutamine addiction in Notch1‐driven acute lymphoblastic leukemia

The cellular receptor Notch1 is a central regulator of T‐cell development, and as a consequence, Notch1 pathway appears upregulated in > 65% of the cases of T‐cell acute lymphoblastic leukemia (T‐ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T‐ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti‐Notch therapies in T‐ALL models. In this work, we report that Notch1 upregulation in T‐ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1‐driven T‐ALL both in vitro and in vivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1‐induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1‐driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1‐driven leukemia.

Abbreviations

7‐AAD

7‐Aminoactinomycin D

BPTES

bis‐2‐(5‐phenylacetamido‐1,2,4‐thiadiazol‐2‐yl)ethyl sulfide

DON

diazo‐5‐oxo‐L‐norleucine

ECAR

extracellular acidification rate

GDH

glutamate dehydrogenase

GLS

glutaminase

GS

glutamine synthetase

GSI

γ‐secretase inhibitor

MSO

L‐methionine sulfoximine

mTORC1

mammalian target of rapamycin complex 1

NICD

Notch intracellular domain

PI

propidium iodide

RAP

rapamycin

T‐ALL

T‐cell acute lymphoblastic leukemia

TCA

tricarboxylic acid

αKG

α‐ketoglutarate

     

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

     

Periostin+cancer‐associated fibroblasts promote lymph node metastasis by impairing the lymphatic endothelial barriers in cervical squamous cell carcinoma

Lymph node metastasis (LNM), a critical prognostic determinant in cancer patients, is critically influenced by the presence of numerous heterogeneous cancer‐associated fibroblasts (CAFs) in the tumor microenvironment. However, the phenotypes and characteristics of the various pro‐metastatic CAF subsets in cervical squamous cell carcinoma (CSCC) remain unknown. Here, we describe a CAF subpopulation with elevated periostin expression (periostin⁺CAFs), located in the primary tumor sites and metastatic lymph nodes, that positively correlated with LNM and poor survival in CSCC patients. Mechanistically, periostin⁺CAFs impaired lymphatic endothelial barriers by activating the integrin‐FAK/Src‐VE‐cadherin signaling pathway in lymphatic endothelial cells and consequently enhanced metastatic dissemination. In contrast, inhibition of the FAK/Src signaling pathway alleviated periostin‐induced lymphatic endothelial barrier dysfunction and its related effects. Notably, periostin^‐CAFs were incapable of impairing endothelial barrier integrity, which may explain the occurrence of CAF‐enriched cases without LNM. In conclusion, we identified a specific periostin⁺CAF subset that promotes LNM in CSCC, mainly by impairing the lymphatic endothelial barriers, thus providing the basis for potential stromal fibroblast‐targeted interventions that block CAF‐dependent metastasis.

Abbreviations

CAFs

cancer‐associated fibroblasts

CM

conditioned medium

CSCC

cervical squamous cell carcinoma

HDLECs

human dermal lymphatic endothelial cells

LN

lymph node

LNM

lymph node metastasis

LVs

lymphatic vessels

MFI

mean fluorescence intensity

NC

negative control

NOFs

normal fibroblasts

SFM

serum‐free media

TAMs

tumor‐associated macrophages

TEM

transmission electron microscopy

TME

tumor microenvironment

     

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

     

A cancer‐associated CDKN1B mutation induces p27 phosphorylation on a novel residue: a new mechanism for tumor suppressor loss‐of‐function

CDKN1B haploinsufficiency promotes the development of several human cancers. The gene encodes p27^Kip1, a protein playing pivotal roles in the control of growth, differentiation, cytoskeleton dynamics, and cytokinesis. CDKN1B haploinsufficiency has been associated with chromosomal or gene aberrations. However, very few data exist on the mechanisms by which CDKN1B missense mutations facilitate carcinogenesis. Here, we report a functional study on a cancer‐associated germinal p27^Kip1 variant, namely glycine9‐>arginine‐p27^Kip1 (G9R‐p27^Kip1) identified in a parathyroid adenoma. We unexpectedly found that G9R‐p27^Kip1 lacks the major tumor suppressor activities of p27^Kip1 including its antiproliferative and pro‐apoptotic functions. In addition, G9R‐p27^Kip1 transfection in cell lines induces the formation of more numerous and larger spheres when compared to wild‐type p27^Kip1‐transfected cells. We demonstrated that the mutation creates a consensus sequence for basophilic kinases causing a massive phosphorylation of G9R‐p27^Kip1 on S12, a residue normally never found modified in p27^Kip1. The novel S12 phosphorylation appears responsible for the loss of function of G9R‐p27^Kip1 since S12AG9R‐p27^Kip1 recovers most of the p27^Kip1 tumor suppressor activities. In addition, the expression of the phosphomimetic S12D‐p27^Kip1 recapitulates G9R‐p27^Kip1 properties. Mechanistically, S12 phosphorylation enhances the nuclear localization of the mutant protein and also reduces its cyclin‐dependent kinase (CDK)2/CDK1 inhibition activity. To our knowledge, this is the first reported case of quantitative phosphorylation of a p27^Kip1 variant on a physiologically unmodified residue associated with the loss of several tumor suppressor activities. In addition, our findings demonstrate that haploinsufficiency might be due to unpredictable post‐translational modifications due to generation of novel consensus sequences by cancer‐associated missense mutations.

Abbreviations

1D/WB

monodimensional western blotting

2D/WB

two‐dimensional western blotting

CDK

cyclin‐dependent kinase

CHX

cycloheximide

G9R‐p27

glycine9‐>arginine‐p27

IUPs

intrinsically unstructured proteins

mAbs

monoclonal antibodies

MEN

multiple endocrine neoplasia

MENX

multiple endocrine neoplasia X

PTMs

post‐translational modifications

rAbs

rabbit antibodies

TSG

tumor suppressor gene

wt‐p27

wild‐type p27

     

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