The LINK-A lncRNA activates normoxic HIF1α signalling in triple-negative breast cancer

The LINK-A lncRNA activates normoxic HIF1α

signalling in triple-negative breast cancer

Aifu Lin

1,13

, Chunlai Li

1,13

, Zhen Xing

1,13

, Qingsong Hu

1

, Ke Liang

1

, Leng Han

2

, Cheng Wang

3

,

David H. Hawke

4

_{, Shouyu Wang}

1

_{, Yanyan Zhang}

1

_{, Yongkun Wei}

1

_{, Guolin Ma}

5

_{, Peter K. Park}

1

_,

Jianwei Zhou

6

,

Yan Zhou

7

_{, Zhibin Hu}

3

_{, Yubin Zhou}

5

_{, Jefery R. Marks}

8

_{, Han Liang}

4,9

_{, Mien-Chie Hung}

1,10,11

_,

Chunru Lin

1,10,14

and Liuqing Yang

1,10,12,14

Although long non-coding RNAs (lncRNAs) predominately reside in the nucleus and exert their functions in many biological

processes, their potential involvement in cytoplasmic signal transduction remains unexplored. Here, we identify a cytoplasmic

lncRNA, LINK-A (long intergenic non-coding RNA for kinase activation), which mediates HB-EGF-triggered, EGFR:GPNMB

heterodimer-dependent HIF1α phosphorylation at Tyr 565 and Ser 797 by BRK and LRRK2, respectively. These events cause

HIF1α stabilization, HIF1α–p300 interaction, and activation of HIF1α transcriptional programs under normoxic conditions.

Mechanistically, LINK-A facilitates the recruitment of BRK to the EGFR:GPNMB complex and BRK kinase activation. The

BRK-dependent HIF1α Tyr 565 phosphorylation interferes with Pro 564 hydroxylation, leading to normoxic HIF1α stabilization.

Both LINK-A expression and LINK-A-dependent signalling pathway activation correlate with triple-negative breast cancer (TNBC),

promoting breast cancer glycolysis reprogramming and tumorigenesis. Our findings illustrate the magnitude and diversity of

cytoplasmic lncRNAs in signal transduction and highlight the important roles of lncRNAs in cancer.

Triple-negative breast cancer (TNBC) continues to

be a severe health problem

1–3

_{, demanding the}

consideration of emerging long non-coding RNAs

(lncRNAs) as biomarkers and therapeutic targets in

combatting this disease

4–6

_{. Accumulating evidence}

demonstrates that lncRNAs have broad functional

roles in the nucleus: regulation of transcriptional

activation,

X

chromosome

inactivation,

heterochromatin formation, and maintenance of

telomeres

7–14

_{. Alterations of these functions promote}

tumour formation, progression and metastasis of many

cancer types

15–20

_{. However, many known lncRNAs}

reside either within the cytosol or shuttle between the

nucleus and cytoplasm

21

_{, playing important roles in}

modulating messenger RNA translation, decay and

cytoplasmic protein trafcking

22–24

_{. Intriguingly, many}

protein kinases and metabolic enzymes bind RNA

through their non-canonical RNA- binding domains

25– 27

_{, raising an important question of whether}

cytoplasmic lncRNAs are relevant in the regulation of

fundamental cellular processes.

The

hypoxia-inducible

factor

(HIF)

transcriptional program is involved in TNBC

progression,

recurrence

and

metabolic

reprogramming

28–30

_{. Although it is well known that the}

hydroxylation

of

HIF1α

mediated

by

proline

hydroxylase domain (PHD) proteins triggers

VHL-dependent HIF1α ubiquitylation and degradation under

normoxic conditions

31,32

_{, under certain circumstances}

in tumour, HIF1α can accumulate under normoxic

conditions, promoting angiogenesis and cancer

progression

33,34

_{. However, the mechanism underlying}

normoxic HIF1α stabilization in TNBC remains elusive.

Here, we identified a highly prognostic lncRNA

in TNBC, long intergenic non-coding RNA for kinase

activation (LINK-A) (also known as LOC339535 and

NR_015407), which is critical for the growth

factor-induced normoxic HIF1α signalling pathway.

1_{Department of Molecular and Cellular Oncology, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA.} 2_{Department of Biochemistry} and Molecular Biology, The University of Texas Health Science Center at Houston McGroven Medical School, Houston, Texas 77030, USA. 3_{Department of} Epidemiology and Biostatistics and Ministry of Education (MOE), School of Public Health, Nanjing Medical University, 210029, China. 4_{Department of System} Biology, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA. 5_{Center for Translational Cancer Research, Institute of Biosciences and} Technology, Texas A&M University Health Science Center, Houston, Texas 77030, USA. 6_{Department of Molecular Cell Biology and Toxicology, School of Public} Health, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China. 7_{Department of Oncology, Yixing People’s Hospital, 75 Zhenguan Road, Yixing} 214200, China.

8_{Department of Surgery, Division of Surgical Science, Duke University, School of Medicine, Durham, North Carolina 27710, USA.} 9_{Department of Bioinformatics and} Computational Biology, Division of Quantitative Sciences, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA. 10_{The Graduate School} of Biomedical Sciences, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA. 11_{Center for Molecular Medicine and Graduate Institute of}

Cancer Biology, China Medical University, Taichung 404, Taiwan. 12_{Center for RNA Interference and Non-Coding RNAs, The University of Texas M D Anderson Cancer} Center, Houston, Texas 77030, USA. 13_{These authors contributed equally to this work.}

a P = 1.38 × 10–3 P = 5.43 × 10–5 P = 2.48 × 10–3 P = 0.06 P = 6.04 × 10–6 P = 2.57 × 10

–6

_{P =} 0.95 P = 0.86 P = 0.82 P = 0.57 P = 1 Training set P = 0.50 P = 0.92 5 4 4 P = 0.88 P = 1 LINK-A P = 2.08 × 10–4 80 _{P = 3.05 × 10}–4 3 3 2 2 40 P = 0.34 P = 0.76 1 1 20 P = 0.35 0 0 d Validation set P = 6.31 × 10–13 P = 6.88 × 10–6 P = 3.07 × 10–5 e 100 Low LINK-A f LINK-A pulldown Peptide number 60 P = 0.034 P = 0.27 P = 0.68 High LINK-A 40 P = 0.69 _{P = 0.11} P = 0.98 P = 0.10 50 20 0 0 P = 0.0189 g MDA-MB-231 LINK-A 0 100 200 Month h Streptavidin pulldown Biotinylated 300 i Streptavidin pulldown Biotinylated Input Beads LINK-A 0.30 0.25 0.20 0.15 0.10 0.05 0 RIP ∗ GAPDH FLAG–BRK IB: FLAG Streptavidin– HRP

Input Beads LINK-A

Myc–LRRK2 IB: Myc-tag Streptavidin– HRP BRK-WT BRK-ΔSH3 SH3 SH2 Kinase domain CT SH2 Kinase LRRK2 WT WD LRRK2 /KD2 1 11 72 78 170 191 321 451 ΔWD40 1 983 1291 1879 2164 2515 Biotinylated j LINK-A (as.)

Biotinylated LINK-A (sen.) _k Streptavidin pulldown

Bio-LINK-A 1 2 3 4 5 6 A B C D E 1 2 3 4 5 6 1 2 3 4 5 6 A A A B B B C C C D D D E E E 1 2 3 4 5 6 1 2 3 4 5 6 A B C D E IB: BRK GST GST–BRK Streptavidin–HRP GST–LRRK2 IB: LRRK2

A1 to E2, individual LINK-A probes (ant.) E3, blank E4, mixed LINK-A probes (ant.) IB: eIF4B

E5, mixed LacZ probes E6, synthesized biotin-scramble probe

1 1540 _{LINK-A (5}

′–3′)

A1 A2 A3 E1 E2 Probes (3′–5′)

Streptavidin–

HRP _MDA-MB-231

Figure 1 LINK-A is a TNBC-upregulated cytoplasmic lncRNA with prognostic value. (a,b) Scatter plots comparing LINK-A expression in breast tumour samples with different ER, PR and HER2 status including ER− _/PR− /HER2−

(n =119), ER− _/PR− _/HER2+ _{(n = 30), ER}+ _/PR+ _/HER2− _{(n =482),} and ER+ _/PR+ _/HER2+ _{(n = 80) (a), or in breast tumour tissue samples} with different subtypes including basal (n = 139), HER2 (n = 67), LumA (n = 417), LumB (n = 191) and normal-like (n = 23) (b). Statistical significance

was determined by two-way ANOVA. The boxes show the median and the interquartile range. The whiskers show the minimum and maximum. (c,d) RNAScope detection of LINK-A expression in human breast cancer and adjacent normal tissues (training (c) and validation (d) set, respectively). Left panel in c: representative images (scale bars, 100 µm); d and right

panel in c: statistical analysis. Training set: TNBC (n = 10), ER− _/PR− /HER2+

(n = 7), ER+ _/PR+ _/HER2− _{(n = 18), and ER}+ _/PR+ _/HER2+ _{(n = 2);} validation

set: ER− _/PR− _/HER2− _{(n = 38), ER}− _/PR− _/HER2+ _{(n = 2), ER}+ _/PR+ /HER2−

(n = 6), ER+ _/PR+ _/HER2+ _{(n =9) and normal tissue (n = 20) (median,}

two-way ANOVA). Horizontal black lines represent median. Coloured error bars represent 95% quantile. (e) Recurrence-free survival analysis of LINK-A status in breast cancer patients detected by qRT–PCR (n = 123 patients,

Bead only

Transcription intermediary factor 1β Prelamin A/C Matrin-3 2 2 4 LINK-A (as.)

Heterogeneous nuclear ribonucleoprotein A1 Tubulin β3 Matrin-3 3 2 3 LINK-A (sen.) Hypoxia-induced factor 1α

Leucine-rich repeat serine/threonine kinase 2

Protein tyrosine kinase 6

Transmembrane glycoprotein NMB

Epidermal growth factor receptor

12

4

5

6

4

ARM ANK LRR ROC/COR Kinase ARM ANK LRR ROC/COR Kinase

E xp re ss io n le ve l (l o g2 ) L IN K -A s ta in in g r e la tiv e i n te ns ity (× 1 0 4) P e rc e n ta g e o f in p u t E R –/P R –/ H E R 2 – T N B C E R –/P R –/ H E R 2 + N o rm a l E xp re ss io n le ve l (l o g2 ) R e cu rr e n ce -f re e s u rv iv a l B a sa l B la n k H E R 2 L u m A L u m B N o rm a li ke P o si tiv e N e g a tiv e L IN K -A s ta in in g r e la tiv e i n te ns ity (× 1 0 4) B la n k W T Δ W D 4 0 In p u t B e a d s T N B C E R –/P R –/ H E R 2 + b c

Gehan–Breslow test). (f) A list of the top LINK-A-associated proteins identified by RNA pulldown and MS analysis in MDA-MB-231 cells. (g) RIP–qPCR detection of indicated RNAs retrieved by BRK-, LRRK2-or eIF4B-specific antibodies in MDA-MB-231 cells. ErrLRRK2-or bars, s.e.m.,

n = 3 independent experiments (∗ _{P < 0.05, two-tailed paired} Student’s

t -test). (h,i) In vitro RNA–protein binding assay showing the interaction

of biotinylated LINK-A with wild-type (WT) FLAG-tagged BRK and a deletion mutant (h), or WT Myc-tagged LRRK2 and a deletion mutant (i). Dot-blot of RNA–protein binding samples indicates equal RNA transcript present in the assay. Bottom panel: graphic illustration of BRK or LRRK2 domain deletion mutants. IB, immunoblot. (j) Upper panel: In vitro RNA– protein binding followed by dot-blot assays using biotinylated LINK-A sense (sen.) or antisense (as.) transcripts and GST-tagged, bacterially expressed BRK or LRRK2 proteins. The hybridized RNA fragments were detected by streptavidin–HRP. Bottom panel: graphic illustration of LINK-A probes. (k) Immunoblot detection of proteins retrieved by in-vitro-transcribed biotinylated LINK-A full-length (FL) or deletion mutants expressed in MDA-MB-231 cells. Unprocessed original scans of blots are shown in Supplementary Fig. 7.

Mechanistically, LINK-A is required for

HB-EGF-triggered, EGFR:GPNMB

heterodimer-mediated

recruitment of BRK to GPNMB, and subsequent

enzymatic activation of BRK. The activated BRK,

together with LRRK2 that is also recruited by

LINK-A, phosphorylates HIF1α at Tyr 565 and Ser 797,

respectively. Whereas the phosphorylation at Tyr 565

inhibits hydroxylation at the adjacent Pro 564, which

prevents

HIF1α

degradation

under

normoxic

conditions, Ser 797 phosphorylation facilitates HIF1α–

p300 interaction, leading to activation of HIF1α

target genes on HB-EGF stimulation. Furthermore, we

demonstrated that LRRK2, a constitutively active

kinase in Parkinson’s disease, is a RNA-binding kinase

that phosphorylates HIF1α in human cancers.

Importantly, both LINK-A expression and activation of

the LINK-A-mediated normoxic HIF1α signalling

pathway correlated with TNBC. Therefore, targeting

LINK-A may serve as a favourable strategy to block a

normoxic HIF1α signalling pathway in TNBC with

promising therapeutic potential.

RESULTS

LINK-A is a cytoplasmic lncRNA with prognostic value

for TNBC

To identify TNBC-relevant lncRNAs, we examined the

lncRNA expression profile in two stage III TNBC tissues

and their paired adjacent non-cancerous tissues,

finding 21 diferentially expressed lncRNAs (ref. 16).

We further searched the expression pattern of these

21 lncRNAs in the TCGA database. Interestingly,

statistical analysis of a combined 711 RNA-seq

transcriptome profiles indicated that the expression of

LINK-A is frequently elevated in TNBC patient cohorts

in comparison with cohorts of ER

−

_/PR

−

_/HER2

+

_,

ER

+

_/PR

+

_/HER2

−

and ER

+

_/PR

+

_/HER2

+

_{patients. Diferential LINK-A}

expression

between

ER

−

_/PR

−

_/HER2

+

_,

_ER

+

_/PR

+

_/HER2

−

_and

ER

+

_/PR

+

_/HER2

+

cohorts was not statistically significant (Fig. 1a).

Consistently,

basal-like breast cancer, which lacks or shows low

levels of ER, PR

and HER2 proteins

35,36

_{, exhibited significantly}

increased LINK-A

expression in comparison with HER2

+

_{, LumA, LumB}

and normal-like

subtypes (Fig.

1b).

LINK-A is a ∼1.5-kb-long intergenic

non-protein-coding RNA

(ref. 37), which was confirmed by our northern

blot and RACE

analyses in MDA-MB-231 cells (Supplementary Fig.

1a,b). Given that LINK-A has a predicted open reading

frame (ORF) of 139 amino acids, we performed in vitro

translation assays, showing that neither the sense nor

the antisense transcript of LINK-A encodes protein

(Supplementary Fig. 1c). We next examined LINK-A

expression in breast cancer tissue microarrays

(clinicopathological

parameters

listed

in

Supplementary Table 1) using the RNAScope 2.0 HD

assay. In both the training and validation sets of tissue

samples, the expression of LINK-A was significantly

increased in TNBC tissues compared

with normal breast tissues, and ER

−

_/PR

−

_/HER2

+

_,

ER

+

_/PR

+

_/HER2

−

_,

and ER

+

_/PR

+

_/HER2

+

_{subtypes (Fig. 1c,d),}

demonstrating the

strong correlation of LINK-A expression with TNBC.

Additionally,

we examined the LINK-A expression level in a

Duke breast

cancer cohort, finding that high levels of LINK-A

correlated with

unfavourable recurrence-free survival for breast

cancer patients (Fig. 1e). Consistently, LINK-A was

highly expressed in TNBC cell lines compared with

oestrogen receptor (ER)- or HER2-positive breast

cancer cell lines (Supplementary

Next, we examined the subcellular localization of

LINK-A, finding that LINK-A predominately resides in

the cytoplasm or close to the cellular membrane,

which was distinct from typical nuclear lncRNAs

including BCAR4 (ref. 16) and HOTAIR (ref. 20)

(Supplementary Fig. 1e–g). Cell fractionation analysis

showed that >90% of LINK-A is localized within the

cytosolic fraction compared with the nuclear

enrichment of BCAR4 (Supplementary Fig. 1h,i). We

reasoned that LINK-A has important roles in the

cytosol.

Identification and characterization of

LINK-A–protein interaction

We performed an RNA pulldown assay followed by

mass spectrometry

15,16

_{(MS) to identify}

LINK-A-associated proteins that might be involved in

cytoplasmic processes. Interestingly, the sense LINK-A,

but not the antisense or beads control, specifically

associated

with

two

transmembrane

proteins,

epidermal growth factor receptor (EGFR) and

transmembrane glycoprotein NMB (GPNMB), tyrosine

protein kinase 6 (also known as breast tumour kinase,

BRK; refs 38,39), leucine-rich repeat kinase 2 (LRRK2;

refs 40,41), and HIF1α in the breast cancer cell (Fig.

1f, Supplementary Fig. 1j and Supplementary Table

2). An RNA pulldown assay in cell lysate and an RNA–

protein binding assay using recombinant EGFR, BRK,

LRRK2, HIF1α and GPNMB confirmed that LINK-A

associated with all of the proteins mentioned above

in vivo, but only BRK and LRRK2 directly

interacted with LINK-A (Supplementary Fig. 1k–n).

The specific interaction between LINK-A and BRK or

LRRK2

was

also

confirmed

by

an

RNA

immunoprecipitation (RIP) assay (Fig. 1g).

To map the BRK domains required for LINK-A

binding, we generated BRK SH3 (amino acids 11–72),

SH2 (amino acids 78–170), kinase domains (amino

acids 191–445), and regulatory carboxy- terminal

(amino acids 446–451) deletion mutants (Fig. 1h,

bottom panel). Deletion of either the SH3 domain or

the C-terminal region of the kinase domain of BRK

alone impaired the interaction between LINK-A and

BRK, suggesting that LINK-A interacts with two

separate domains of BRK (Supplementary Fig. 1o).

Double deletion of these two domains abolished the

LINK-A–BRK interaction in vitro and in vivo (Fig. 1h

and Supplementary Fig. 1p). A similar strategy was

used to map the domain required for LRRK2–LINK-A

interaction, showing that deletion of the WD40

domain, an atypical RNA- binding domain

16,25

_,

abolished the direct interaction (Fig. 1i and

Supplementary Fig. 1q).

To map RNA motifs essential for the LINK-A–protein

interactions, we conducted an in vitro RNA–

protein binding coupled with dot-blot assay

15,16

_,

finding that BRK interacted with LINK-A at two

regions, nucleotides 481–540 (dot B3) and

nucleotides

781–840 (dot C2) (corresponding to the two

domains of BRK at the SH3 domain and the

C-terminal tail) (Fig. 1j). LINK-A nucleotides 1261–1320

(dot D4) interacted with LRRK2 (Fig. 1j). Consistently,

double deletion of LINK-A (nucleotides 471–550

and nucleotides 771–850) abolished the BRK–LINK-A

interaction without afecting the LRRK2–LINK-A

interaction, whereas deletion of LINK-A (nucleotides

1251–1330) specifically abolished LRRK2– LINK-A

association (Fig. 1k). The predicted secondary

structure of LINK-A indicates that the RNA motifs

required for BRK and LRRK2 interactions form

individual branching stem loops,

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IB GPNMB 1.. 7 . .. .-1 MDA-MB-231 Figu陀 2 LfNK-A is involved in an HB-EGF-triggered， EG FR:G

PNMB

mediated signalling pathway，(a) Summary 01 the phosphorylation sites

01 the indicated proteins identilied Irom RNA pulldown lollowed by MS analysis， (b) Immunoprecipitation (IP) lollowed by immunoblot

(IB) detection 01 the indicated proteins in MDA-MB-231 cells treated with the indicated growth lactors lor 30 min， (c) Immunoblot detection using the indicated antibodies in MDA-MB-231 cells

stimulated with vehicle， EGF or HB-EGFlollowed by DTSSP chemical

crosslinking ( 1mM，

30min)， (d，e) His tag (d) or FLAG tag (e) pulldown lollowed

by

immunoblot detectlon using the indicated antibodies 川 MDA-MB-231 cells

translected with the indicated expression vecto內的Ilowed by HB

-EGF

stimulation ，ECD， extracellul司r domain; TM， transmembrane domain;

ICD， intracellular domain ， (f) Immunoprecipitation followed by

immunoblot detection 01 GPNMB， B RK and HIFla phosphorylation in MDA-MB-231

cells treated with the indicated growth lactors，(g) fn vitro kinase assay

using the indicated recombinant proteins， lollowed by Coomassie blue

staining (CBB)， and immunoblot detection using the indicated antibodies ，(h，il Immunoprecipitationlollowed by immunblot

detection using the

indicated antibodies in cells trans ected with the indicated expression vecto

內的Ilowed by HB-EGF stimulation，Lelt panel (自 ): graphic illustration 01

BRK domain delellon mutants，Unprocessed original scans 01 blots are

shown in Supplementary Fi日

7

，

丘 I

suggesting that they contribute to specific RNA–

protein interactions

(Supplementary

Fig. 1r).

Characterization of a HB-EGF-triggered, EGFR:GPNMB-

dependent and LINK-A-mediated signalling pathway in TNBC

Our MS data revealed a series of phosphorylation

sites of GPNMB

(Tyr 525), BRK (Tyr 351), and HIF1α (Tyr 565 and Ser

797) (Fig. 2a

and Supplementary Fig. 2a–d and Supplementary

Table 2), leading us

to generate phosphorylation site-specific antibodies

(Supplementary Fig. 2e–i) to investigate whether

LINK-A modulates a previously unknown signalling pathway.

Given that LINK-A associated with the orphan

receptor GPNMB

and EGFR, which are involved in metastatic TNBC

(refs 42–44),

we reasoned that EGFR and GPNMB may interact

with each other

in TNBC cells on EGF family ligands. Although all

EGFR ligands

efectively activated EGFR (Supplementary Fig. 2j),

HB-EGF robustly

induced the specific interaction between EGFR and

GPNMB (Fig. 2b),

indicating that EGF ligands could diferentially trigger

the formation

of the EGFR homodimer or the heterodimer between

EGFR and other receptors

45

_{. To test this, we}

performed a crosslinking assay, finding that EGF

predominately triggered EGFR homodimerization with

a

lesser

degree

of

EGFR:GPNMB

heterodimerization but HB-EGF stimulated

EGFR:GPNMB heterodimerization with less EGFR

homodimerization (Fig. 2c). Knockdown of LINK-A

exhibited minimal efects on the HB-EGF-induced

EGFR:GPNMB interaction

as

well

as

GPNMB

phosphorylation on ligand stimulation (Supplementary

Fig. 2k,l), suggesting that HB-EGF preferentially

triggered EGFR:GPNMB heterodimer formation. We

further mapped the domains mediating EGFR–GPNMB

binding, and found that the kinase domain (KD) in

EGFR intracellular domains (ICD) interacts

with GPNMB ICD (Fig. 2d,e and Supplementary Fig.

2m).

HB-EGF

robustly

induced

site-specific

phosphorylation of EGFR, GPNMB, BRK and HIF1α (Fig.

2f ) and pretreatment of TNBC cell lines with

cetuximab impaired EGFR–GPNMB interaction

(Supplementary Fig. 2n,o). These observations led

us to fully characterize this HB-EGF-triggered,

EGFR:GPNMB-dependent signalling pathway in TNBC.

First, an in vitro kinase assay indicated that EGFR,

but not BRK,

phosphorylated GPNMB at Tyr 525 (Fig. 2g) and the

exogenously

expressed wild-type GPNMB but not the Y525F mutant

was phosphorylated in vivo on HB-EGF stimulation

(Fig. 2h). Next, we observed the interaction between

GPNMB and BRK following

ligand stimulation, which was abolished in the

presence of the GPNMB Y525F mutant (Fig. 2h).

Furthermore, the ligand-triggered BRK Tyr 351

phosphorylation was abolished in GPNMB

Y525F-overexpressing cells

(Fig.

2h).

Biochemical

experiments showed that BRK SH2 domain deletion

(amino acids 78–170) eliminated the ligand-dependent

interaction with Tyr-525-phosphorylated GPNMB (Fig.

2i). These data suggest that the EGFR-dependent

GPNMB Tyr 525 phosphorylation is required for further

recruitment of BRK through its SH2 domain and

subsequent phosphorylation at Tyr 351.

LINK-A facilitates the recruitment and activation of BRK

We then conducted an immuno-RNA fluorescence in

situ hybridization (FISH) assay to examine the

proximity of LINK-A to the ligand-bound receptors

on ligand treatment, finding the

overlap between LINK-A and EGFR on HB-EGF

stimulation (Supplementary Fig. 3a), which was further

validated by in vivo RIP assay (Supplementary Fig.

3b). We examined the co-localization of BRK and

the EGFR:GPNMB receptor complex in the presence

or absence of LINK-A. Our data indicate that

both BRK and phospho-BRK (Tyr 351) faithfully

co-localized with EGFR on HB- EGF stimulation (Fig. 3a

and Supplementary Fig. 3c). In contrast, depletion of

LINK-A abolished the recruitment of BRK to EGFR

and subsequent phosphorylation of BRK without

afecting the internalization of EGFR (Fig. 3a and

Supplementary Fig. 3c). We then performed rescue

experiments in which LINK-A was knocked down by

locked

nucleic

acids

(LNAs)

followed

by

reintroduction of LNA-resistant full-length LINK-A

or one of the following deletion mutants: ßBRK

(ß471–550 and ß771–850) or ßLRRK2 (ß1251–1330)

(Fig. 3b, lower panel, and Supplementary Fig. 3d,e),

finding that knockdown of LINK-A abolished the

HB-EGF-induced BRK–GPNMB interaction, as well as BRK

Tyr 351 phosphorylation (Fig. 3c,d); reintroduction of

full-length LINK-A or ßLRRK2 but not the ßBRK mutant

rescued these phenotypes (Fig. 3c,d). These data

suggest that LINK-A–BRK interaction facilitates the

recruitment of BRK to the tyrosine-phosphorylated

membrane receptor GPNMB, as well as subsequent

autophosphorylation of BRK.

LINK-A elicits the conformational change of BRK for

kinase activation

It has been reported that the activity of BRK is

auto-inhibited by interaction between the SH2 domain and

the Tyr-447-phosphorylated C-terminal domain

46–48

_.

Our data indicate that LINK-A interacts with BRK at two

regions, the SH3 domain and the C-terminal domain

(see Fig. 1h and Supplementary Fig. 1p), raising a

possible role for LINK-A in eliciting a BRK

conformational change that mitigates the

confor-mation required for self-inhibition. Indeed, we found

that full-length LINK-A and ßLRRK2 LINK-A markedly

enhanced the autophospho- rylation and kinase

activity of BRK, whereas both the control lncRNA and

ßBRK LINK-A showed minimal efects (Fig. 3e,f ).

We next conducted a protease digestion assay by

incubating BRK with caspase-1 in the presence of

full-length LINK-A or ßBRK LINK-A, finding that

caspase-1 barely cleaved BRK at amino acid

397 in the presence of an unrelated lncRNA

RP11-383G10.5, but robustly cleaved BRK only in the

presence of full-length LINK-A (Fig. 3g), suggesting

that a potential conformational change occurred in

BRK to expose the digestion site on LINK-A binding.

Notably, deletion of either of the two regions of LINK-A

involved in BRK interaction failed to promote the

caspase-1-dependent

BRK

cleavage (Fig.

3g),

suggesting that simultaneous binding of LINK-A to

the two BRK domains is required to elicit the

conformational change in BRK. Our data suggest that

the binding of LINK-A to BRK promotes a

conformational

change,

leading

to

increased

accessibility

of

the

SH2

domain

and

the

autophosphorylation sites in the kinase domain. On

ligand stimulation, these events lead to the

recruitment of BRK to Tyr-525-phosphorylated GPNMB

and activation of BRK on Tyr 351 phosphorylation.

LINK-A-interacting BRK and LRRK2 phosphorylate HIF1α

We next performed in vitro phosphorylation assays,

finding that activated BRK phosphorylated HIF1α at

Tyr 565 (Fig. 4a) and

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in vitro-transcribed RNA transcripts as indicated in the presence or absence

01 [且 PlATP. The dot-blot indicates equal RNA transcript present in the assay. (f) Quantilication 01 BRK kinase activity in the presence 01 the indicated in vtiro-transcribed RNA transcripts using HIFl 0:

peptide (amino acids 557-566) as the substrate. Upper panel: release 01 Iree phosphate

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r immunoblot (1 B) detection (d) was perlormed using the indicated

antibodies 川 MDA-MB-231 cells translected with LNA against LfNK-A lollowed by overexpr皂白 ion 01 the indicated rescue plasmids with

HB EGF stimulation. The dotted line on the blots 01 d indicates the position where the images 01 single blots were vertlcally cropped to luxtapose non-adjacent lanes，(e) In vitro kinase assay using recombinant BRK and

experiments ('P<O.05， two-tailed paired Student's t-test ). (g) Immunblot detection 01 BRK using the indicated antibodi的 in the presence 01 the indicated IncRNA transcripts with or without caspase-ldigestion. Lelt panel 皂raphic illustration 01 caspase-l-mediated BRK cleavage in the absence or presence 01 IncR NA. Unprocessed original scans 01 blots are shown In Suppl自mentary F皂i .7.

a Kinase GST BRK LRRK2 GST BRK LRRK2 GST BRK LRRK2 GST BRK LRRK2 b _His–HIF1α His–HIF1α 225 115 80 65 50 35 30 25 15 M_r (K)

CBB 32_{P autoradiography IB: Phospho-HIF1α}

(Tyr 565) IB: Phospho-HIF1α(Ser 797)

GST–BRK ATP – + – + – + IB: GST-tag IB: p-BRK (Tyr 351) IB: His-tag IB: p-HIF1α (Tyr 565) In vitro kinase assay d e f c HB-EGF 0 5 15 30 60 120 (min) EGFR p-EGFR (Tyr 1068) GPNMB p-GPNMB (Tyr 525) BRK p-BRK (Tyr 351) LRRK2 HIF1α PHD2 VHL GAPDH siRNA HB-EGF – + – + – + – + – + EGFR p-EGFR (Tyr 1068) GPNMB p-GPNMB (Tyr 525) BRK p-BRK (Tyr 351) LRRK2 HIF1α PHD2 GAPDH siRNA HB-EGF EGFR p-EGFR (Tyr 1068) GPNMB p-GPNMB (Tyr 525) BRK p-BRK (Tyr 351) LRRK2 HIF1α PHD2 VHL GAPDH – + – + siRNA HB-EGF EGFR p-EGFR (Tyr 1068) GPNMB p-GPNMB (Tyr 525) BRK p-BRK (Tyr 351) LRRK2 HIF1α PHD2 GAPDH – + – + MDA-MB-231 h g HB-EGF 0 5 15 30 60120 (min) MDA-MB-231 siRNA MDA-MB-231 i siRNA MDA-MB-468 HIF1α p-HIF1α (Tyr 565) p-HIF1α (Ser 797) Hydroxy-HIF1α (Pro 564) _MDA-MB-231 HB-EGF HIF1α p-HIF1α (Tyr 565) p-HIF1α (Ser 797) Hydroxy-HIF1α (Pro 564) – + – + HB-EGF – + – + – + – + – + HIF1α p-HIF1α (Tyr 565) p-HIF1α (Ser 797) Hydroxy-HIF1α (Pro 564) j Peptide number detected by LC–MS

k MDA-MB-231 BSA l His–HIF1α WT Y565F MDA-MB-231 Myc–HIF1α MG-132 2° His–PHD1 + – + + – + + – + 1° GST–BRK – + + – + + – + + IB: HIF1α IB: p-HIF1α (Tyr 565) IB: Hydroxy-HIF1α (Pro 564) IB: GST-tag IB: His-tag HB-EGF – + – + – + IB: HIF1α IB: p-HIF1α (Tyr 565) IB: Hydroxy-HIF1α (Pro 564) Input IB: PHD2 MDA-MB-231

Figure 4 LINK-A-dependent BRK phosphorylation of HIF1α at Tyr 565 antagonizes HIF1α Pro 564 hydroxylation. (a) In vitro phosphorylation assay using recombinant proteins (WT or mutants as indicated). IB, immunblot. (b) In vitro kinase assay using bacterially expressed GST-tagged BRK WT or mutant and His-GST-tagged HIF1α. (c–f) Immunoblot detection using the indicated antibodies in MB-231 (c–e) or MDA-MB-468 (f) cells treated with HB-EGF at the indicated time point (c) or transfected with the indicated siRNAs followed by HB-EGF treatment (d–f). (g–i) Immunoblot detection using the indicated antibodies in MDA-MB-231 cells treated with MG-132 followed by HB-EGF treatment at

the indicated time (g) or in cells transfected with the indicated siRNAs followed by MG-132 and HB-EGF treatment (h,i). (j) LC–MS sequencing Sample WT _(P-OH)WT p-Y565 p-Y565 _(P-OH)

WT 20 0 0 0 WT + PHD 8 9 0 0 WT + PHD +DMOG 18 0 0 0 pY565 1 0 33 0 pY565 + PHD 1 0 24 0 pY565 +PHD + DMOG 2 0 32 0 M G-13 2 M G-13 2 M G-13 2 IB IB W T Y 5 6 5 F S 7 9 7 A W T Y 5 6 5 F S 7 9 7 A W T Y 5 6 5 F S 7 9 7 A IB IB C tr l H is -t a g D yn a b e a d E G F R G P N M B B R K C tr l L R R K 2 L IN K -A IB IB C tr l L IN K -A IP : M yc -t a g C tr l IB E G F R B la n k G P N M B W T Y 3 5 1 F B R K B la n k C tr l W T L R R K 2 L IN K -A Y 5 6 5F

of the HIF1α peptide (557–566) in an in vitro hydroxylation assay. The total peptide numbers of HIF1α proline non-hydroxylated versus proline hydroxylated (P-OH) under the indicated conditions are shown. The peptide number of hydroxylated WT peptide is indicated in red. (k) His-tag pulldown followed by immunoblot detection of HIF1α phosphorylation and hydroxylation (WT versus Y565F) in an in vitro kinase assay (1◦ ₎ followed by in vitro hydroxylation assay (2◦ _{). (l) Immunoprecipitation} (IP) followed by immunoblot detection of HIF1α phosphorylation and hydroxylation (WT versus Y565F) in MDA-MB-231 cells transfected with the indicated plasmids and treated with MG-132 followed by HB-EGF treatment. Unprocessed original scans of blots are shown in Supplementary Fig. 7.

LRRK2, another LINK-A-interacting protein kinase,

phosphorylated HIF1α at Ser 797, which was further

demonstrated by the marked inhibition of HIF1α

phosphorylation in the presence of a S797A point

mutant (Fig. 4a). The BRK kinase activity-deficient

mutant, Y351F, diminished the phosphorylation of

HIF1α in vivo (Fig. 4b). Both Tyr 565 and Ser 797 of

HIF1α are conserved (Supplementary Fig. 4a).

HB-EGF induced phosphorylation of GPNMB (Tyr

525) and BRK (Tyr 351), as well as HIF1α

protein stabilization under normoxic conditions (Fig.

4c and Supplementary Fig. 4b). Interestingly,

knockdown of EGFR abolished the ligand-dependent

phosphorylation of GPNMB (Tyr 525) and BRK (Tyr

351), as well as the stabilization of HIF1α; knockdown

of

GPNMB

abolished

HB- EGF-induced

BRK

phosphorylation and HIF1α protein stabilization, but

did not afect EGFR phosphorylation (Tyr 1068) (Fig.

4d). Knockdown of LINK-A in both MDA-MB-231 and

MDA-MB-468 cells eliminated HB-EGF-induced BRK

phosphorylation and HIF1α stabilization, but not

phosphorylation of EGFR or GPNMB (Fig. 4e,f ). In

contrast, LINK-A knockdown exhibited minimal efects

on hypoxia-dependent HIF1α stabilization, and hypoxia

failed to trigger phosphorylation of GPNMB and BRK

(Fig. 4c–f and Supplementary Fig. 4c). Finally,

depletion of BRK decreased ligand-triggered HIF1α

protein accumulation but did not afect the

phosphorylation status of EGFR or GPNMB (Fig. 4d).

Taken together, these data suggest a

linear

EGFR:GPNMB→LINK-A→BRK/LRRK2→HIF1α

signalling

cascade on HB-EGF stimulation under normoxic

conditions.

On HB-EGF stimulation, HIF1α underwent Tyr 565

and Ser 797

phosphorylation but the hydroxylation at Pro 564 was

inhibited, which led to HIF1α stabilization (Fig. 4g).

Knockdown of LINK- A abolished HB-EGF-induced

HIF1α Tyr 565 phosphorylation and enhanced the Pro

564 hydroxylation (Fig. 4h and Supplementary Fig.

4d). A similar pattern was observed with EGFR,

GPNMB and

BRK knockdown (Fig. 4i). These data suggest that

HB-EGF triggers an lncRNA-dependent signalling pathway

to stabilize HIF1α at the protein level.

Tyr 565 phosphorylation antagonizes Pro 564 hydroxylation to

stabilize HIF1α under normoxia

An in vitro hydroxylation assay demonstrated that the

HIF1α peptides (amino acids 557–566) but not

Tyr-565-phosphorylated peptides can be hydroxylated by PHD1

(Fig.

4j

and

Supplementary Fig.

4e–j

and

Supplementary Table 3). An in vitro kinase assay

followed by an in vitro hydroxylation assay further

showed that phosphorylation of wild- type HIF1α but

not the Y565F mutant by BRK prevented subsequent

hydroxylation at Pro 564 (Fig. 4k). Consistently,

HB-EGF-triggered Tyr 565 phosphorylation of HIF1α and

inhibition of hydroxylation at Pro 564, which was

abolished by overexpression of the Y565F mutant of

HIF1α (Fig. 4l).

A cycloheximide treatment experiment revealed that

on HB-EGF

stimulation, the HIF1α protein exhibited ≥4 h half-life

but knocking

down LINK-A reduced it to 1.5 h (Supplementary

Fig. 4k,l). In

TNBC cells exogenously expressing wild-type HIF1α or

the Y565D

mutant, the Y565D mutant exhibited a constitutively

prolonged half- life (Supplementary Fig. 4m–o). These

data

indicate

that

LINK-A- associated

BRK

phosphorylated HIF1α at Tyr 565, which prevents

HIF1α hydroxylation at adjacent Pro 564 and

stabilizes HIF1α

under

normoxia.

LINK-A-recruited LRRK2 phosphorylates Ser 797 of HIF1α to

potentiate its transcriptional activity

Knockdown of LINK-A or LRRK2, or overexpression of

the HIF1α

S797A mutant abolished Ser 797 phosphorylation of

HIF1α as well

as its association with p300, which was concurrent

with the release

of FIH (ref. 49), a protein that binds to HIF1α and

inhibits its

trans-activation function (Fig. 5a,b). We also examined the

kinase activity of LRRK2 in the presence of LINK-A,

finding that full-length LINK- A, ßBRK LINK-A or

ßLRRK2 LINK-A exhibited minimal efect on the kinase

activity of LRRK2 (Supplementary Fig. 5a). The rescue

experiments indicated that full-length LINK-A fully

rescued

HIF1α

phosphorylation and

protein

stabilization; ßBRK LINK-A rescued only HIF1α Ser 797

phosphorylation and ßLRRK2 LINK-A restored HIF1α Tyr

565 phosphorylation and protein stabilization, but

failed to rescue the phosphorylation of HIF1α at Ser

797 (Fig. 5c). Recent studies have shown that certain

lncRNAs encode small protein pep- tides

50–52

_{. Whereas}

our data have demonstrated that a predicted ORF of

LINK-A has no protein-coding products in vitro (see

Supplemen- tary Fig. 1a–c), we further mutated the

predicted translational start codon ATG (nucleotides

318–321), or the potential stop codon TGA

(nucleotides 732–735), of this ORF in a functional

rescue experi- ment, finding that the phosphorylation

of BRK (Tyr 351) and HIF1α (Tyr 565), two major

cellular efects mediated by LINK-A, was fully

rescued by wild-type LINK-A as well as ATG→TAG or

TGA→TGT

mutants of LINK-A (Supplementary Fig. 5b–d). These

observations

suggested that the cellular efect of LINK-A is mainly

dependent on its

RNA function instead of the potential translational

products. Taken together, we demonstrated that

LINK-A, in coordination with two protein kinases BRK and

LRRK2, mediated a growth factor-triggered

signalling cascade to synergistically regulate the

phosphorylation and protein stabilization of HIF1α

under normoxia.

LINK-A-dependent normoxic HIF1α signalling promotes tumour

growth and correlates with TNBC

Next, we examined the transcriptional activity of

HIF1α on HB- EGF stimulation by ChIP-seq, finding that

under normoxia, HB-EGF triggered the recruitment of

HIF1α to the promoters of HIF1α target genes and

regulated

the

HIF1α-dependent

transcriptional

program (Fig. 5d,e and Supplementary Table 4).

Knockdown of LINK-A in TNBC cells impaired

HIF1α-target gene expression on HB-EGF stimulation (Fig.

5f,g and Supplementary Fig. 5e). Consistently, in vitro

glucose uptake and lactate production assays

confirmed that LINK-A deficiency impaired glycolysis

(Supplementary Fig. 5f–l). Consistent with the in vitro

colony formation assays (Fig. 5h), mice with

xenografts of LINK-A-depleted tumour cells rarely

developed tumour mass in vivo (Fig. 5i,j and

Supplementary Fig. 5m).

The LINK-A-mediated signalling pathway was also

activated in TNBC tissues, as evidenced by a

significantly higher staining density of

GPNMB (Tyr 525), BRK (Tyr 351),

phospho-HIF1α (Tyr 565) and phospho-phospho-HIF1α (Ser 797) in TNBC

samples compared with non-TNBC samples (Fig. 6a–c

and Supplementary Fig. 6a). Furthermore, within the

TNBC category, breast cancer with advanced

lymph-node metastasis showed increased phospho-BRK (Tyr

351), phospho-HIF1α (Tyr 565) and phospho-GPNMB

(Tyr 525) levels compared with tissue samples with no

lymph-node metastasis (Fig. 6a–c,d–f, upper panel).

Importantly, there is a strong correlation

a siRNA HB-EGF MG-132 – + – + – + b c MG-132 Myc–HIF1α Blank WT S797A

HB-EGF – + – + – + LNA Scr LINK-A LINK-A no. 5 d P = 0.000001 2 IB: HIF1α IB: p-HIF1α (Ser 797) IB: p300 IB: FIH IB: p300 IB: FIH MDA-MB-231 IB: HIF1α IB: p-HIF1α (Ser 797) IB: p300 IB: FIH IB: FIH IB: p300 MDA-MB-231 HB-EGF IB: HIF1α IB: p-HIF1α (Tyr 565) IB: p-HIF1α (Ser 797) IB: GAPDH – + – + – + – + – + MDA-MB-231 1 0 1 2 3 4 5 6 2 1 0 1 2 3 4 5 6

e Signalling pathway of DE gene

HIF1α signalling pathway Fructose and mannose metabolism Glycolysis/gluconeogenesis Biosynthesis of amino acids Carbon metabolism Oocyte meiosis Progesterone-mediated oocyte maturation

f 0.40 ∗ 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0 ChIP: Anti-HIF1α ∗∗ ∗ Anti-HIF1α IgG ∗ Long-term depression Protein expor HB-EGF – + – + – + – + – + – + – + – + g 25 ∗ 20 15

Enrichment score (–log10(P value))

∗ MDA-MB-231 –HB-EGF +HB-EGF 10 ∗ 5 ∗ 0 siRNA ∗ ∗ ∗ ∗ ∗ _{∗ NS}

ANGPTL4 ALDOA ANKRD37 BHLHE40 EGR1 IGFBP3 LDHA MAPK1 SLC16A3 _{PKM2 RPLP0}

h 200 ∗ 150 i _shRNA Ctrl j _0.5 0.4 0.3 ∗∗ ∗∗ 100 50 LINK-A no. 2 Ctrl LINK-A no. 3 0.2 0.1 0 –0.1 0 shRNA

Figure 5 LINK-A-recruited LRRK2 phosphorylates HIF1α at Ser 797, enhances HIF1α transcriptional activity and promotes tumour growth. (a,b) Immunoprecipitation (IP) followed by immunoblot (IB) detection using the indicated antibodies in MDA-MB-231 cells transfected with the indicated siRNAs (a) or plasmids (b), and treated with MG-132 followed by HB-EGF treatment. (c) Immunoblot detection using the indicated antibodies in MDA-MB-231 cells transfected with LNA against LINK-A followed by overexpression of the indicated rescue plasmids and HB-EGF stimulation. The dotted line indicates the position where the images of single blots were vertically cropped to juxtapose non-adjacent lanes. (d) HIF1α ChIP-seq analysis showing the top enriched HIF-binding consensus motifs. (e) HIF1α ChIP-seq analysis showing signalling pathways in MDA-MB-231 cells treated

with HB-EGF. (f,g) ChIP–qPCR detection of HIF1α occupancy on indicated target gene promoters (f) and qRT–PCR analysis of HIF1α target genes expression (g) in MDA-MB-231 cells transfected with control or

LINK-A siRNLINK-A followed by HB-EGF treatment. (h) Colony formation assay in

MDA-MB-231 cells transduced with control and LINK-A shRNAs. Scale bars, 200 µm. For f–h, error bars, s.e.m.; n = 3 independent experiments

(∗ _{P < 0.05 and} ∗∗ _{P < 0.01, two-tailed paired Student’s t -test). (i,j) In}

vivo

analyses of tumour growth (i) or weight (j) in mice that were subcutaneously injected with MDA-MB-231 cells harbouring control or LINK-A shRNA. Data are mean ± s.e.m., n = 5 mice per group (∗∗ _{P < 0.01,} two-tailed

t siRNA Ctrl LINK-A Ctrl LINK-A Ctrl LINK-A Ctrl LINK-A

0 1 2 3 4 5 6 Promoter ANKRD37 ARRDC3 EGLN3 ERRFI1

In p u t IP : H IF 1α sh R N A 3 sh R N A 2 C tr l R el at iv e ex pr es si on l ev el N um be r of c o lo n ie s In p u t IP : M yc -t a g P er ce nt ag e o f in p u t V ec to r V ec to r F L ΔB R K Δ LR R K 2 T um ou r w ei gh t (m g ) B its B its

paired Student’s t -test). Unprocessed original scans of blots are shown in

a Positive Negative b _{Positive Negative c Positive Negative} 60 P = 6.18 × 10–4 40 20 0 P = 3.67 × 10–5 50 P = 0.0375 40 30 20 10 0 P = 1.08 × 10–5 50 P = 0.0384 P = 4.68 × 10–5 40 30 20 10 0 d _{P = 0.0191} P = 0.0071 60 TNBC e P = 0.018 P = 0.040 60 TNBC f P = 0.0076 P = 0.039 60 TNBC P = 0.24 P = 0.38 P = 0.47 40 40 40 20 20 20 0 0 0 50 40 30 20 10 r2 _{P = 0.0007}= 0.784 0 0 20 40 60 80 LINK-A intensity in TNBC (×104₎ 50 40 30 20 10 r_{P = 0.0289}2 = 0.4689 0 0 20 40 60 80 LINK-A intensity in TNBC (×104₎ 40 30 20 10 r2 _{= 0.5307} P = 0.0169 0 0 20 40 60 80 LINK-A intensity in TNBC (×104₎ g 110 100 90 80 70 60 Low p-BRK (Y351) High p-BRK (Y351) h 150 100 50

Low p-HIF1-α (Y565)

High p-HIF1-α (Y565) i 150

100 50 Low p-GPNMB (Y525) High p-GPNMB (Y525) P = 0.040 P = 0.011 P = 0.035 50 0 50 100 Month 0 150 200 0 50 100 Month 0 150 200 0 50 100 Month 150 200

Figure 6 The LINK-A-dependent normoxic HIF1α signalling pathway correlates with TNBC. (a–c) Immunohistochemical staining using antibodies against phospho-BRK (Tyr 351) (a), phospho-HIF1α (Tyr 565) (b) or phospho-GPNMB (Tyr 525) (c) in human breast cancer tissues. Upper panel: representative images (scale bars, 100 µm; lower panel: statistics analysis based on non-TNBC tissues (n = 5) versus TNBC tissues (n = 40) and non-metastasis (TnN0M0) TNBC (n = 27) versus metastasis

(TnN > 0 M ≥ 0) breast tissues (n = 13) (median, two-way ANOVA). (d–f) Upper panel: statistical analysis of immunohistochemical staining using antibodies against phospho-BRK (Tyr 351) (d), phospho-HIF1α

(Tyr 565) (e) or phospho-GPNMB (Tyr 525) (f) in human breast cancer tissues including TNBC (n = 10), ER− _/PR− _/HER2+ _{(n = 7), ER}+ _/PR+ /HER2− _{(n = 18), and ER}+ _/PR+ _/HER2+ _{(n = 2) (median, two-way} ANOVA). Lower

panel: Pearson’s correlation analysis comparing staining density between

LINK-A expression and phospho-BRK (Tyr 351) (d), phospho-HIF1α (Tyr 565)

(e) or phospho-GPNMB (Tyr 525) (f) within the TNBC group (n = 10 tissue samples, Fisher’s exact test). (g–i) Kaplan–Meier survival analysis of phosphor-BRK (Tyr 351) (g), phosphor-HIF1α (Tyr 565) (h) and phospho-GPNMB (Tyr 525) (i) status in breast cancer patients (n = 160, Gehan–Breslow test). p-B R K ( Y 35 1) in te ns ity in T N B C (× 10 4) p-B R K ( Y 35 1) in te ns ity (× 10 4) p-B R K ( Y 35 1) s ta in in g re la tiv e in te ns ity (× 10 4) P er ce nt ag e o f su rv iv al ( % ) p-B R K ( Y 35 1) N on -T N B C T N B C N on - m et as ta si s (T nN 0M 0) M et as ta si s (T nN > 0 M ≥ 0 ) p-H IF 1α ( Y 56 5) in te ns ity in T N B C (× 10 4) p-H IF 1α ( Y 56 5) p-H IF 1α ( Y 56 5) s ta in in g re la tiv e in te ns ity (× 10 4) P er ce nt ag e of s ur vi va l ( % ) p-H IF 1α (Y 56 5) N on -T N B C T N B C N o m et as ta si s (T nN 0M 0) M et as ta si s (T nN > 0 M ≥ 0 ) p-G P N M B ( Y 52 5) in te ns ity in T N B C (× 10 4) p-G P N M B ( Y 52 5) p-G P N M B ( Y 52 5) s ta in in g re la tiv e in te ns ity (× 10 4) P er ce nt ag e of s ur vi va l ( % ) p-G P N M B ( Y 52 5) N on -T N B C T N B C N on - m et as ta si s (T nN 0M 0) M et as ta si s (T nN > 0 M ≥ 0 )

between LINK-A expression and the phosphorylation

status of BRK, HIF1α and GPNMB in these TNBC tissues

(Fig. 6d–f, lower panel), and breast cancer patients

with higher levels of these phosphoproteins

exhibited a shorter survival time (Fig. 6g–i).

Furthermore, the TCGA database revealed that both

BRK and LRRK2 are highly expressed in invasive breast

carcinoma (Supplementary Fig. 6b). Our data implicate

LINK-A and its associated signalling pathway as

potential biomarkers and therapeutic targets for TNBC.

DISCUSSION

Our study reveals that lncRNA directly interacts with

non-receptor tyrosine kinase and facilitates its

recruitment to the membrane-bound receptor complex

and subsequent activation on ligand stimulation,

broadening the known mechanisms of lncRNA action

(Fig. 6j). The regulatory mechanism of non-receptor

tyrosine kinase activation is largely unknown. We

propose a model in which LINK-A interacts with

non-receptor tyrosine kinases to facilitate their activation.

At the basal level, BRK, a prototype RNA-binding

non-receptor tyrosine kinase, is in a ‘closed’ conformation

and its kinase activity is auto-inhibited, mediated by

the self-inhibitory interaction between the SH2 domain

and the phospho-C-terminus (Tyr 447; ref. 46). The

binding of LINK- A to both the SH3 domain and the

C-terminal region of BRK leads to a more accessible

structure of BRK, which may contribute to higher

accessibility by other regulatory proteins and kinases

for its activation.

Most common cancer types show increased HIF1α

protein levels although hypoxic areas are missing

53,54

_.

Our study delineates an lncRNA–protein kinase module

that regulates normoxic HIF1α stabilization with

respect to functional implications in glycolytic

reprogramming and tumorigenesis. The

LINK-A-dependent

HIF1α signalling cascade and the

consequent efects on cancer cell glycolysis implicate

LINK-A and LINK-A-interacting kinases/receptors as

promising therapeutic targets for TNBC. Analyses of

the LINK-A expression status in the TCGA database and

breast cancer tissues both indicated that LINK-A

significantly correlates with TNBC, revealing an

lncRNA that can serve as a biomarker for further

classification of TNBC.

Our study identifies four previously unknown

phosphorylation sites of GPNMB, BRK and HIF1α in

a LINK-A-regulated signalling pathway for glycolysis

reprogramming in TNBC. These phosphorylation

events predict a worse outcome in TNBC patients,

suggesting that the LINK-A-dependent signalling

pathway plays a critical role in TNBC and may

provide wide-ranging therapeutic

targets

for

treating

TNBC.

口

METHODS

Methods and any associated references are available

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ACKNOWLEDGEMENT S

We thank S. Kopetz for providing cetuximab and J. Chen for providing SFB-tagged

expression vector. We thank D. Aten for assistance with figure presentation. This

work was supported by the NIH R00 award (R00DK094981), UT Startup and UT

STARS grants to C.Lin, and the NIH R00 award (R00CA166527), CPRIT award

(R1218), UT Startup and UT STARS grants to L.Y.

AUTHOR CONTRIBUTIONS

C.Lin, L.Y. and A.L. designed the research, and A.L., C.Li and Z.X. performed

most of the experiments, with participation of K.L., S.W., Q.H., Y.Zhang, G.M.

and Yubin Z. D.H.H. executed mass spectrometry analysis. Clinical specimens were

ascertained and processed by S.W., J.Z., Yan Z. and J.R.M. The histological staining

and corresponding analysis were performed by K.L. and Y.W. P.K.P. helped with

manuscript preparation. TCGA data and microarray data analysis was performed

by C.W., Z.H., L.H. and H.L. M.-C.H. provided reagents and conceptual advice L.Y.,

C.Lin and A.L. wrote the manuscript.