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Oncogenic Ras induces inflammatory cytokine production by up-regulating the squamous cell carcinoma antigens SerpinB3/B4

April 2014
Nature Communications 5(1):3729

DOI:10.1038/ncomms4729

Source
PubMed

Authors:

Namratha Sheshadri

Rutgers, The State University of New Jersey

Yu Sun

Stony Brook University

Show all 9 authorsHide

Mounting evidence indicates that oncogenic Ras can modulate cell autonomous inflammatory cytokine production, although the underlying mechanism remains unclear. Here we show that squamous cell carcinoma antigens 1 and 2 (SCCA1/2), members of the Serpin family of serine/cysteine protease inhibitors, are transcriptionally upregulated by oncogenic Ras via MAPK and the ETS family transcription factor PEA3. Increased SCCA expression leads to inhibition of protein turnover, unfolded protein response, activation of NF-κB and is essential for Ras-mediated cytokine production and tumour growth. Analysis of human colorectal and pancreatic tumour samples reveals a positive correlation between Ras mutation, enhanced SCCA expression and IL-6 expression. These results indicate that SCCA is a Ras-responsive factor that plays an important role in Ras-associated cytokine production and tumorigenesis.

SCCA promotes cytokine production by inducing UPR. (a,b) Vector-control or HRasV12-expressing IMR90 cells were stably infected with lentiviral shRNA control (shNTC) or two independent hairpins targeting SCCA. Whole-cell lysates were analysed by western blot with indicated antibodies. Quantification of the ubiquitin blot in a was performed by Image J using the full-length of each lane. (c–g) Vector-control or HRasV12 IMR90 cells were stably infected with lentiviral shRNA control (shNTC) or shRNA hairpins targeting ATF6 or XBP1. (c,g) Whole-cell lysates were analysed by western blot with indicated antibodies. (d) Culture media were collected and subjected to a cytokine antibody array. The blots of indicated cytokines are shown. (e) The relative amount of cytokines was quantified and normalized to that of RasV12-shNTC cells. (f) Total RNA was extracted and cytokine transcript levels were analysed via qRT–PCR and normalized against that in RasV12-shNTC cells. Data shown are mean+s.e.m. of three independent experiments performed in triplicate. (h,i) Vector-control or HRasV12 IMR90 cells were stably infected with lentiviral shRNA control (shNTC) or shSCCA, together with vector-control or XBP1s-expressing construct. (h) Whole-cell lysates were analysed by western blot with indicated antibodies. (i) Total RNA was extracted and cytokine transcript levels were analysed via qRT–PCR and normalized against that in RasV12-shNTC cells. Data shown are mean+s.e.m. of three independent experiments performed in triplicate. **P<0.01; ***P<0.001; NS, non-significant by t-test.

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ARTICLE

Received 29 Oct 2013 |Accepted 26 Mar 2014 |Published 23 Apr 2014

Oncogenic Ras induces inﬂammatory cytokine

production by upregulating the squamous cell

carcinoma antigens SerpinB3/B4

Joseph M. Catanzaro1,*, Namratha Sheshadri1,*, Ji-An Pan1, Yu Sun1, Chanjuan Shi2, Jinyu Li3, R. Scott Powers3,

Howard C. Crawford4& Wei-Xing Zong1

Mounting evidence indicates that oncogenic Ras can modulate cell autonomous inﬂammatory

cytokine production, although the underlying mechanism remains unclear. Here we show that

squamous cell carcinoma antigens 1 and 2 (SCCA1/2), members of the Serpin family of

serine/cysteine protease inhibitors, are transcriptionally upregulated by oncogenic Ras via

MAPK and the ETS family transcription factor PEA3. Increased SCCA expression leads to

inhibition of protein turnover, unfolded protein response, activation of NF-kB and is essential

for Ras-mediated cytokine production and tumour growth. Analysis of human colorectal and

pancreatic tumour samples reveals a positive correlation between Ras mutation, enhanced

SCCA expression and IL-6 expression. These results indicate that SCCA is a Ras-responsive

factor that plays an important role in Ras-associated cytokine production and tumorigenesis.

DOI: 10.1038/ncomms4729

1Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794, USA. 2Department of Pathology, Microbiology

and Immunology, Vanderbilt University, Nashville, Tennessee 37232, USA. 3Cancer Genome Center, Cold Spring Harbor Laboratory, Woodbury, New York

11797, USA. 4Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida 32224, USA. *These authors contributed equally to this work. Correspondence

and requests for materials should be addressed to W.-X.Z. (email: weixing.zong@stonybrook.edu).

NATURE COMMUNICATIONS | 5:3729 | DOI: 10.1038/ncomms4729| www.nature.com/naturecommunications 1

Constitutively active mutants of Ras are found in a large

number of human cancers1,2. Among the oncogenic

activities of mutant Ras, its ability to alter the tumour

microenvironment has been well appreciated with numerous

studies implicating Ras in various non-cell autonomous processes

including basement membrane degradation, immune cell

inﬁltration and angiogenesis3,4. This tumour microenvironment

altering function of Ras is at least partially caused by the ability of

Ras to induce the production and secretion of proinﬂammatory

and pro-tumorigenic cytokines. As cellular senescence has been

largely attributed to the cytokine secretory response termed

senescence-associated secretion phenotype (SASP)5,6, the

possibility remains that oncogene-induced secretory proﬁle is a

senescence-independent process on Ras activation. Several

reports have implicated Ras in the ability to modulate the

tumour microenvironment at least due in part to Ras’ ability to

promote the production of cytokines and chemokines such as

Interleukin (IL)-6, IL-8, granulocyte–macrophage-colony-

stimulating factor (GM-CSF) in a cell autonomous

manner3,4,7,8. Therefore, it remains to be determined whether

premature senescence is a prerequisite of oncogene-induced

secretory phenotype, and how Ras mechanistically regulates the

expression of the proinﬂammatory cytokines.

Squamous cell carcinoma antigens (SCCAs) are members

of the serpin family of endogenous protease inhibitors.

Approximately 98 and 92% homologous at their nucleotide and

amino-acid levels, respectively, SCCA1 (SerpinB3) and SCCA2

(SerpinB4) are ‘suicide-substrate’ protease inhibitors with differ-

ing substrate speciﬁcities because of amino-acid differences

within their reactive site loop domain9. Upregulated in

numerous cancers (the cervical, lung, head and neck, liver and

breast)10–13, both SCCA1 and SCCA2 are thought to promote cell

survival through the inhibition of cell death14,15. Moreover, the

level of SCCA has been shown to predict pathological grade,

disease stage, recurrence and response to both radiotherapy and

chemotherapy16–18. Despite SCCA’s well-reported involvement in

cancer, how SCCA is transcriptionally upregulated during

transformation and contributes to tumour development remains

largely unknown. Here, we study the oncogenic regulation of

SCCA and uncover a novel proinﬂammatory role for SCCA

downstream of mutant Ras. We demonstrate that through MAPK

signalling and the ETS transcription factor PEA3, oncogenic Ras

upregulates the expression of SCCA1/2 (SerpinB3/B4) to promote

inﬂammatory cytokine production and xenograft tumour growth.

Moreover, SCCA upregulation is observed in human colorectal

and pancreatic cancer. Our ﬁndings suggest an important role of

serpins in Ras-driven tumorigenesis.

Results

Oncogenic Ras upregulates SCCA expression. Elevated

expression of SCCA has been found in numerous human

cancers. However, the underlying molecular mechanism of its

upregulation remains unclear. We began to study this by

expressing several oncoproteins (HRasV12, myristolated-Akt

(myr-Akt), and c-Myc) in IMR90 primary human lung ﬁbro-

blasts. While myr-Akt and c-Myc failed to induce SCCA

expression, HRasV12 led to a marked increase in SCCA protein

levels (Fig. 1a). The SCCA antibody utilized was unable to

distinguish between the SCCA isoforms10, although quantitative

reverse-transcription PCR (qRT–PCR) analysis revealed an

increase in the transcript levels of both SCCA1 and SCCA2 in

response to RasV12 expression (Fig. 1b). This is not surprising

as the two SCCA isoforms are tandemly arranged on

human chromosome 18q and their promoters are highly

homologous19.

64 kDa Akt

Myc

Ras

SCCA

Vector

HRas

Vector

HRas

Vector

KRas

Vector

KRas

Vector

KRas

β-Tubulin

Vector

myr-Akt

c-Myc

HRas

Vector

NRas

HRas

KRas

Ras

SCCA

β-Tubulin

Ras

64 kDa

22 kDa

22 kDa 22 kDa

IMR90 BJ HT-29 Caco-2 HeLa

ERK1/2

P-ERK1/2

:P-ERK/T-ERK

β-Tubulin

50 kDa

64 kDa

***

Vector

Ras

10,000

1,000

100

0.1

Relative expression

4-OHT: – –++

1.0

1.0 3.7 4.2 2.9

1.9 0.1

SCCA

ER:Ras

Day 12

Day 8

Day 0

:SCCA/tubulin

50 kDa

50 kDa 50 kDa

50 kDa

50 kDa 50 kDa

ER:Ras Day 0

ER:Ras+OHT Day 8

ER:Ras+OHT Day 12

ER:Ras-withdraw

***

1.2

1.0

0.8

Relative expression

0.6

0.4

0.2

0SCCA1 SCCA2

SCCA1 SCCA2

***

Figure 1 | Oncogenic Ras upregulates SCCA expression. (a) Indicated oncogenes were stably expressed in IMR90 cells. Whole-cell lysates were obtained

and analysed by western blot with indicated antibodies. (b) Total RNA was extracted from vector-control or RasV12-expressing IMR90 cells. SCCA1

and SCCA2 transcript levels were analysed via qRT–PCR and normalized to that in vector-control cells. Data shown are mean þs.e.m. of three independent

experiments performed in triplicate. (c,d) IMR90 cells expressing the ER:RasV12 fusion protein were treated with 4-OHT for 8 days, split and either cultured

in media containing 4-OHT or withdrew 4-OHT for additional 4 days. (c) Whole-cell lysates were analysed by western blot with indicated antibodies.

(d) Total RNA was extracted, and SCCA1 and SCCA2 transcript levels were analysed via qRT–PCR, and normalized to that of Day 12 ER:Ras with

4-OHT cells. Data shown are mean þs.e.m. of three independent experiments performed in triplicate. (e) Indicated oncogenic Ras proteins were

stably expressed in IMR90 cells. Whole-cell lysates were obtained and analysed by western blot with indicated antibodies. (f) HRasV12 was stably

expressed in IMR90 and BJ cell lines. KRasV12 was stably expressed in HT-29, Caco-2 and HeLa cells. Whole-cell lysates were obtained and analysed

by western blot with indicated antibodies. *Po0.05; ***Po0.001 by t-test.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4729

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To determine whether persistent Ras signalling is required for

SCCA expression, we used the oestrogen receptor (ER):RasV12

fusion protein that allows for RasV12 stabilization on the addition

of 4-hydroxytamoxifen (4-OHT) to the culture medium20. While

4-OHT induced the expression of Ras and SCCA (Fig. 1c),

removal of 4-OHT led to a drastic reduction in RasV12 protein

levels and diminished downstream signalling indicated by

decreased phospho-ERK, accompanied by a reduction of SCCA

at both protein and transcript levels (Fig. 1c,d), indicating that

sustained Ras signalling is required for maintaining SCCA

expression.

In addition to HRasV12, expression of KRasV12 or NRasQ61 also

resulted in elevated SCCA expression in IMR90 cells (Fig. 1e).

Similar to IMR90 cells, increased SCCA expression was observed

by expressing HRasV12 in the BJ primary human foreskin

ﬁbroblast cells, and by expression of KRasV12 in a number of

cancer cell lines with wild-type Ras, including the colon cancer

cell lines HT-29 and Caco-2, and HeLa cells (Fig. 1f). Taken

together, these results indicate that upregulation of SCCA

expression is a general feature of oncogenic Ras proteins in

various human cell lines.

It is important to note that in IMR90 and BJ ﬁbroblasts

expression of RasV12 elicits oncogene-induced senescence21. The

upregulation of SCCA in response to RasV12 in Caco-2, HT-29

and HeLa cells, which do not undergo senescence3,22, suggests

that upregulation of SCCA by RasV12 is independent of cellular

senescence. To further elucidate that the increased SCCA

expression is not a consequence of cellular senescence, we

induced premature senescence by treating IMR90 cells with

etoposide or H

and replicative senescence by continually

passaging the cells. Indeed, unlike RasV12, these senescence-

inducing conditions failed to upregulate SCCA expression

(Fig. 2). These results indicate that expression of oncogenic Ras

stimulates expression of SCCA in a manner that is independent of

oncogene-induced senescence.

Ras-induced SCCA is dependent on the MAPK/PEA3 pathway.

To elucidate the mechanism by which RasV12 stimulates SCCA

expression, we used pharmacological inhibitors to selectively

inhibit MAPK and Akt pathways that are well characterized to be

activated by Ras2,23. While inhibition of Akt signalling had

little to no effect on SCCA expression (Fig. 3a,b), the MEK

inhibitor U0126 caused a marked inhibition of both SCCA1 and

SCCA2 transcription in both IMR90 (Fig. 3c,d) and HeLa

cells (Supplementary Fig. 1a,b). Similarly, expression of the

dominant-negative ERK2 blocked Ras-induced SCCA expression

(Supplementary Fig. 1c,d). Along the Ras/Raf/MAPK/ERK

pathway, expression of oncogenic BRafV600E also induced

SCCA expression (Supplementary Fig. 1e). We next sought to

identify which transcription factor may be mediating Ras-induced

expression of SCCA. The ETS transcription factor family member

PEA3 has been shown to be modulated by MAPK via

sumolyation24 and can activate SCCA transcription25. Indeed,

silencing of PEA3 in RasV12-expressing cells (Fig. 3e) resulted in a

drastic decrease of both SCCA1 and SCCA2 at protein and

transcript levels (Fig. 3f,g), without affecting MAPK signalling

indicated by similar levels of phospho-ERK in RasV12-expressing

cells (Fig. 3f). This PEA3-dependent SCCA expression was also

observed in RasV12-expressing HeLa cells (Supplementary

Vehicle Etoposide

H2O2Ras

Early passage Late passage

Veh

Eto

H2O2

Ras

22 kDa

50 kDa

22 kDa

50 kDa β-Tubulin

p21

SCCA

Ras

Vector

Ras

22 kDa

50 kDa

22 kDa

50 kDa β-Tubulin

p21

SCCA

Ras

Figure 2 | DNA damage-induced and replicative senescence fails to upregulate SCCA. (a,b) IMR90 cells were treated with vehicle-control, etoposide

(10 mM) for 48 h, H

(10 mM) for 1 h, or stably transduced with HRasV12, then analysed 7 days post-treatment. (a) Cells were stained for b-Gal activity.

Representative images are shown. (b) Whole-cell lysates were analysed by western blot with indicated antibodies. (c,d) IMR90 cells were continuously

passaged and harvested at passage 15 as early passage (EP) or at passage 30 as late passage. (c) Cells were stained for b-galactosidase activity.

Representative images are shown. (d) Whole-cell lysates were analysed by western blot with indicated antibodies. Note that while all the conditions induce

cellular senescence, only RasV12 led to SCCA expression. Scale bar ¼100 mm.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4729 ARTICLE

NATURE COMMUNICATIONS | 5:3729 | DOI: 10.1038/ncomms4729 | www.nature.com/naturecommunications 3

Fig. 2a–c) and was rescued by expression of wild-type murine

PEA3, but not by a PEA3 sumolyation-defective mutant24

(Supplementary Fig. 2d–f). These results indicate that mutant

Ras leads to SCCA upregulation via the MAPK/PEA3 pathway.

SCCA is essential for Ras-induced cytokine production. Next

we examined the biological role of elevated SCCA expression. We

chose to focus on using the IMR90 cell line that is widely used for

studying the effect of Ras activation during the early phase of

tumorigenesis. We began by examining the downstream signal-

ling pathways activated by oncogenic Ras (MAPK, Akt, NF-kB).

To this end, SCCA expression was silenced in RasV12-expressing

cells using two independent shRNA constructs that effectively

silenced both SCCA1 and SCCA2 (Fig. 4a), and the signalling

pathways examined by respective phosphorylation antibodies.

While loss of SCCA had little to no effect on the ability of mutant

Ras to activate Akt and MAPK phosphorylation (Fig. 4b), loss of

SCCA signiﬁcantly diminished RasV12-induced RelA/p65 phos-

phorylation that is indicative of NF-kB activation (Fig. 4b). This

effect was further conﬁrmed by utilizing a NF-kB luciferase

reporter construct (Fig. 4c).

Activation of NF-kB signalling has been attributed to cytokine

production downstream of Ras activation26. Indeed, inhibition

of NF-kB signalling using the pharmacological inhibitor BAY11-

7082 abrogated Ras-induced cytokine production in IMR90 and

HeLa cells (Supplementary Fig. 3). Therefore, we examined

whether SCCA plays a role in Ras-induced cytokine production

and performed a quantitative cytokine array analysis using cell

culture media. In agreement with literature5,6, RasV12-expressing

cells displayed a marked increase in a spectrum of cytokines

including IL-6, IL-8, CXCL1, G-CSF and GM-CSF (Fig. 4d).

Vector Ras

1.6

1.4

1.2

1.0

0.2

0.4

0.6

0.8

SCCA1 SCCA2

Vector Ras

Ras

SCCA

+–+–

AKTi:

22 kDa

50 kDa

1.0 0.9

Ras

SCCA

:SCCA/tubulin

P-Akt (Ser473)

:P-Akt/T-Akt

β-Tubulin

0.72.10.3

50 kDa

22 kDa

MEKi:

64 kDa

1.0

Akt

+–+–

P-ERK1/2

:SCCA/tubulin

ERK1/2

β-Tubulin

shNTC

shPEA3 ***

3.0

2.5

2.0

1.5

1.0

0.5

0Vector Ras

Relative expression

:P-ERK/T-ERK

1.4 0.60.3

0.3

1.0

50 kDa

Vector Ras

Ras

SCCA

50 kDa

22 kDa

shPEA3

shNTC

shREA3

shNTC

P-ERK1/2

:SCCA/tubulin

ERK1/2

β-Tubulin

:P-ERK/T-ERK1.9 2.11.0

0.5

1.0

50 kDa

Vector + vehicle

Vector + AKTi

Ras + vehicle

Ras + AKTi

Relative expression

1.2

1.0

0.2

0.4

0.6

0.8

SCCA1 SCCA2

Vector + vehicle

Vector + MEKi

Ras + vehicle

Ras + MEKi

Relative expression

1.2

1.0

0.2

0.4

0.6

0.8

SCCA1 SCCA2

***

Vector-shNTC

Vector-shPEA3

Ras-shNTC

Ras-shPEA3

Relative expression

Figure 3 | SCCA upregulation is mediated by MAPK/PEA3. (a,b) Vector-control or HRasV12-expressing IMR90 cells were treated with either

vehicle-control or AKTi (10 mM) for 24 h. (a) Whole-cell lysates were analysed by western blot with indicated antibodies. (b) Total RNA was extracted

and SCCA1 and SCCA2 transcript levels were analysed via qRT–PCR, and normalized to RasV12 cells treated with vehicle-control. Data shown are

mean þs.e.m. of two independent experiments performed in triplicate. (c,d) Vector-control or HRasV12-expressing IMR90 cells were treated with

either vehicle-control (DMSO) or U1026 (MEKi, 10mM) for 24 h. (c) Whole-cell lysates were analysed by western blot with indicated antibodies.

(d) Total RNA was extracted, and SCCA1 and SCCA2 transcript levels were analysed via qRT–PCR and normalized to that of Ras-expressing cells

treated with vehicle. Data shown are mean þs.e.m. of three independent experiments performed in triplicate. (e–g) Vector-control or HRasV12-expressing

IMR90 cells were stably infected with lentiviral shNTC (non-target control) or shPEA3. (e) Successful silencing of PEA3 was conﬁrmed via qRT–PCR

and normalized to that in vector-control cells with shNTC. Data shown are mean þs.e.m. of three independent experiments performed in triplicate.

(f) Whole-cell lysates were analysed through western blot with indicated antibodies. (g) Total RNA was analysed for the transcript levels of SCCA1

and SCCA2 via qRT–PCR and normalized to that in Ras-expressing cells with shNTC. Data shown are meanþs.e.m. of three independent experiments

performed in triplicate. **Po0.01; ***Po0.001; NS, non-signiﬁcant by t-test.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4729

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Consistent with the observed decrease in NF-kB signalling

(Fig. 4b,c), this increased cytokine expression was signiﬁcantly

abrogated on SCCA silencing (Fig. 4d,e). Quantitative RT-PCR

analysis revealed that the suppression of cytokine production was

due to transcriptional downregulation (Fig. 4f) and was rescued

by ectopically activating NF-kB signalling with TNFatreatment

(Fig. 4g). The SCCA-dependent NF-kB activation and cytokine

production downstream of oncogenic Ras were also observed in

HeLa and Caco-2 cells (Supplementary Fig. 4). The introduction

of oncogenic Ras in IMR90 elicits a DNA damage response that

has been reported to mediate cytokine production6,27. However,

silencing of SCCA had virtually no effect on Ras-induced DNA

damage indicated by the phosphorylation of H2A.X (Fig. 5a,b)

or the senescence-associated cell cycle arrest (Fig. 5c).

Taken together, these results indicate that SCCA plays an

essential role in Ras-mediated NF-kB activation and

inﬂammatory cytokine production that is independent of the

DNA-damage response.

GM-CSF

G-CSF

CXCL1

IL-8

IL-6

shSCCA#2

shSCCA#1

shNTC

Vector

***

1.2

1.0

0.8

Relative expression

0.6

0.4

0.2

IL-6 IL-8 CXCL1 G-CSF GM-CSF

1.2

1.0

0.8

Relative amount of

secreted protein

0.6

0.4

0.2

0IL-6 IL-8 CXCL1 G-CSF GM-CSF

***

*** ***

***

Vector

***

Relative luminescence

(NF-kB activity)

Ras-shNTC

Ras-shSCCA#1

Ras-shSCCA#2

***

4.0

3.0

2.0

Relative expression

1.0

IL-6 IL-8 CXCL1 G-CSF GM-CSF

Ras

64 kDa

50 kDa

64 kDa

1.0 3.7 3.0 4.2

P-Akt (Ser473)

Akt

:P-Akt/T-Akt

:P-ERK/T-ERK

P-RelA/p65

RelA/p65

:P-RelA/T-RelA2.42.13.81.0

1.81.92.01.0

β-Tubulin

P-ERK1/2

ERK1/2

22 kDa

50 kDa

Ras

Vector

shNTC

shSCCA#1

shSCCA#2

Vector

shNTC

shSCCA#1

shSCCA#2

SCCA

β-Tubulin

Ras

Vector

Ras-shNTC

Ras-shSCCA

Ras-shSCCA+TNFa

Vector

Ras-shNTC

Ras-shSCCA#1

Ras-shSCCA#2

Vector

Ras-shNTC

Ras-shSCCA#1

Ras-shSCCA#2

Figure 4 | SCCA modulates Ras-induced cytokine production. (a–f) Vector-control or HRasV12-expressing IMR90 cells were stably infected with lentiviral

shRNA control (shNTC) or two independent hairpins targeting SCCA. (a,b) Whole-cell lysates were analysed by western blot with indicated antibodies.

(c) Cells were transfected with an NF-kB luciferase reporter and a renilla luciferase construct. Twenty-four hours post transfection, cells were lysed

and luminescence was quantiﬁed. NF-kB luciferase activity was standardized based on renilla luciferase activity and normalized to that of vector-control

cells. Data shown are mean þs.e.m. of three independent experiments performed in triplicate. (d) Culture media were collected and subjected to a cytokine

antibody array. The representative of two independent blots of indicated cytokines are shown. (e) The relative amount of cytokines was quantiﬁed and

normalized to that of RasV12-shNTC cells. (f) Total RNA was extracted and cytokine transcript levels were analysed via qRT–PCR, and normalized to

that of RasV12-shNTC cells. Data shown are mean þs.e.m. of three independent experiments performed in triplicate. (g) Ras-shSCCA cells were

treated with TNFa(50 ng ml1) for 16 h. Total RNA was extracted and cytokine transcript levels were analysed via qRT–PCR and normalized to that of

RasV12-shNTC cells. Data shown are mean þs.e.m. of two independent experiments performed in triplicate. *Po0.05; **Po0.01; ***Po0.001 by t-test.

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SCCA promotes cytokine production by inducing the unfolded

protein response. We next sought to understand how SCCA

promotes Ras-mediated NF-kB activation and cytokine produc-

tion. A possible mechanism is through upregulation of the

unfolded protein response (UPR), which has been implicated in

mediating NF-kB activation and inﬂammatory transcriptional

programmes28. Our previous work showed that SCCA leads to

decreased lysosomal and proteasomal protein turnover, two

major protein degradation pathways, and hence increases the

steady-state level of ER stress29. Thus, we speculated that mutant

Ras induces ER stress through upregulation of SCCA. Indeed,

the ability of RasV12 to promote an ER stress response has

been reported30. Silencing of SCCA relieved the Ras-induced

inhibition of protein turnover as measured through total

ubiquitinated proteins (Fig. 6a). Furthermore, while RasV12 cells

showed a robust increase in UPR indicated by the appearance of

proteolytically cleaved ATF6 as well as both unspliced and spliced

XBP1, this response was signiﬁcantly reduced in SCCA-silenced

cells (Fig. 6b), indicating that SCCA mediates the UPR induced

by RasV12. Silencing of ATF6 or XBP1 in RasV12-expressing

cells (Fig. 6c) drastically diminished RasV12-induced cytokine

production (Fig. 6d–f; Supplementary Fig. 5) that was

accompanied by decreased RelA/p65 phosphorylation (Fig. 6g;

Supplementary Fig. 5). Conversely, ectopic expression of the

transcriptionally active spliced variant of XBP1 (XBP1s) restored

cytokine production in RasV12 cells when SCCA was silenced

(Fig. 6h,i). These results indicate that SCCA modulates cytokine

production downstream of mutant Ras by stimulating UPR.

SCCA is upregulated in colorectal and pancreatic cancers. Our

results thus far show that SCCA is a Ras-responsive gene that

plays an essential role in Ras-induced cytokine production. We

next sought to identify an in vivo connection between Ras

and SCCA expression. Using TCGA (The Cancer Genome Atlas)

data of human colorectal cancer, which were available with

sizeable samples with the KRas mutation, we identiﬁed a positive

correlation between the presence of mutant Ras and SCCA

upregulation (Fig. 7a). We also chose to examine pancreatic

ductal adenocarcinoma (PDAC), where the mutant KRas func-

tions as the primary driver31. It also offers a unique model where

the precursor lesions to PDAC have a deﬁned pathological

progression31. To this end, we analysed the pancreatic cancer

gene expression data sets for SCCA expression through

Oncomine. In the seven available patient data sets, SCCA1

mRNA was upregulated in six and SCCA2 mRNA in three

independent data sets32–37 (Fig. 7b–d). Of note, the Logsdon data

set37 identiﬁed both SCCA1 and SCCA2 to be upregulated in

pancreatic cancer when compared with chronic pancreatitis

(Fig. 7d). To further examine the association of SCCA expression

in human pancreatic cancer, we performed immunohisto-

chemistry (IHC) for SCCA using a pancreatic cancer tissue

microarray. While all non-neoplastic/normal pancreatic samples

were negative for SCCA expression, positive staining for SCCA

was seen throughout all stages of pancreatic cancer progression.

The incidence of SCCA positivity increased in relation to the

progression of pancreatic cancer: 2 out of 17 (11.8%) PanIN-1

lesions, 5 out of 19 (26.3%) PanIN-2 lesions, 8 out of 15 (53.3%)

PanIN-3 lesions and 20 out of 30 (66.7%) of PDAC (Fig. 7e;

Po0.001). Moreover, consistent with the result that SCCA

promotes inﬂammatory cytokine production in cultured cells,

IHC of IL-6 on the same panel of pancreatic tissues showed a

positive correlation between IL-6 and SCCA in both PDAC

(Fig. 7f,g) and PanIN (Supplementary Fig. 6) samples.

Endogenous SCCA promotes IL-6 production and tumor-

igenesis. To further explore the physiological relevance of SCCA,

we tested the expression and function of SCCA in a setting when

mutant Ras is expressed at endogenous levels. RasV12 failed to

induce the expression of the SCCA paralogues Serpinb3a or Ser-

pinb3b in immortalized MEF NIH3T3 (Supplementary Fig. 7a) or

in primary MEFs (Supplementary Fig. 7b). The failure to detect

upregulation of SCCA paralogues in the murine system prevented

us from using the well-established LSL-KRas mouse model38.

Hence, we instead examined a panel of human pancreatic cancer

cell lines that harbour the mutant KRas. We found ﬁve out of nine

cell lines to be positive for SCCA expression (Fig. 8a). In the cell

lines that displayed detectable levels of SCCA and can tolerate loss

of KRas (CFPAC-1, L3.6 and Capan-1), silencing of mutant KRas

signiﬁcantly reduced SCCA expression (Fig. 8b). Consistent with

the essential role of SCCA in Ras-mediated cytokine production,

silencing of SCCA in three SCCA-positive cell lines resulted in

decreased expression of IL-6, whereas the SCCA shRNAs did not

affect IL-6 expression in the SCCA-low PANC-1 cells (Fig. 8c).

Moreover, while silencing of SCCA in CFPAC-1, L3.6 and HPAF-II

cells did not compromise cell proliferation in cell culture

(Supplementary Fig. 8), it led to impaired xenograft tumour

growth in all three cell lines (Fig. 8d). These results, taken together

with the above ﬁnding that SCCA positivity correlates with

progression of pancreatic cancer development (Fig. 7e), indicate

that SCCA expression is under the control of mutant Ras expressed

at endogenous level, and that the SCCA-mediated inﬂammatory

response plays a pro-tumorigenic role in Ras-driven cancer.

DAPI

Vector

Ras-shNTC

Ras-shSCCA#1

Ras-shSCCA#2

γH2AX Merge Vector

Ras-shNTC

Ras-shSCCA#1

% γH2AX-positive cells

BrdU

SAHF

Vector

Ras

shNTC

Ras

shSCCA#1

Ras

shSCCA#2

% Positive cells

–5

Ras-shSCCA#2

Figure 5 | Silencing of SCCA does not interfere with the DNA-damage response. (a–c) Vector-control or HRasV12-expressing IMR90 cells were stably

infected with lentiviral shRNA control (shNTC) or two independent hairpins targeting SCCA (same cells as shown in Fig. 4). (a) Immunoﬂuorescence

against gH2A.X was performed. Representative images are shown; scale bar, 20 mm. (b) Quantiﬁcation of percent gH2A.X-positive cells is shown. Note that

silencing of SCCA does not compromise Ras-induced DNA damage. Data shown are mean þs.e.m. of three independent experiments. (c) Cells were

cultured with BrdU (10 mM) for 6 h and immunoﬂuorescence against BrdU was performed. Quantiﬁcation of BrdU-positive and senescence-associated

heterochromatic foci (SAHF)-positive cells are shown. Data shown are mean þs.e.m. of three independent experiments. NS, non-signiﬁcant by t-test.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4729

6NATURE COMMUNICATIONS | 5:3729 | DOI: 10.1038/ncomms4729 | www.nature.com/naturecommunications

Discussion

In this study, we show that the endogenous serine/cysteine

proteinase inhibitor SCCA is upregulated on activation of

oncogenic Ras, in a manner that is dependent on hyper-active

MAPK signalling and the ETS transcription factor PEA3.

Downstream of oncogenic Ras, SCCA promotes activation of

NF-kB signalling and cytokine production by blocking protein

turnover and inducing the UPR.

Our study implicates SCCA as a direct link between oncogenic

Ras mutation, NF-kB activation and cytokine production.

Vector

shNTC

shSCCA#1

shSCCA#2

Ras

Vector

shNTC

shSCCA#1

shSCCA#2

Ras

Vector

shNTC

shATF6

shXBP1

Ras

β-Tubulin

RT-PCR

GAPDH

ATF6

XBP1

Ras

22 kDa

50 kDa

96 kDa

36 kDa

50 kDa

Ras

Vector

shNTC

shATF6

shXBP1

β-Tubulin

:XBP1u/tubulin

:XBP1s/tubulin

:ATF6/tubulin1.0

1.0 2.2 1.2

5.4 3.6 4.0

1.0

1.0 2.6 1.0 0.3

ATF6 p50

ATF6 p90

Ras

XBP1u

XBP1s

96 kDa

64 kDa

50 kDa

1.0 4.0 1.6 1.7

Ubiquitin

:Ubiquitin/tubulin

β-Tubulin

IL-6

IL-8

CXCL1

G-CSF

GM-CSF 0

IL-6 IL-8 CXCL1 G-CSF GM-CSF

0.2

0.4

0.6

Relative amount of

secreted protein

Relative expression

0.8

1.0

Ras – +

Ras

SCCA

1.0

1.0 8.7

53 2.0 6.2

4.3 10

:SCCA/tubulin

XBP1s

:XBP1s/tubulin

β-Tubulin

–

–––

–

––

–

shNTC

shSCCA

Vector

XBP1s

22 kDa

50 kDa

0.2

0.4

0.6

0.8

1.0

1.2

Vector

Ras-shNTC

Ras-shATF6

Ras-shXBP1

0IL-6 IL-8 CXCL1 G-CSF GM-CSF

0.2

0.4

0.6

Relative expression

0.8

1.0

1.2

Vector

Ras-shNTC

Ras-shSCCA

Ras-shSCCA-XBP1s

***

Vector

Ras-shNTC

Ras-shATF6

Ras-shXBP1

***

*** ***

64 kDa

50 kDa

1.0 2.0 1.0 1.4

Ras

Vector

shNTC

shATF6

shXBP1

β-Tubulin

P-RelA/p65

:P-RelA/T-RelA

RelA/p65

***

1.2

IL-6 IL-8 CXCL1 G-CSF GM-CSF

Figure 6 | SCCA promotes cytokine production by inducing UPR. (a,b) Vector-control or HRasV12-expressing IMR90 cells were stably infected with

lentiviral shRNA control (shNTC) or two independent hairpins targeting SCCA. Whole-cell lysates were analysed by western blot with indicated antibodies.

Quantiﬁcation of the ubiquitin blot in awas performed by Image J using the full-length of each lane. (c–g) Vector-control or HRasV12 IMR90 cells

were stably infected with lentiviral shRNA control (shNTC) or shRNA hairpins targeting ATF6 or XBP1. (c,g) Whole-cell lysates were analysed by western

blot with indicated antibodies. (d) Culture media were collected and subjected to a cytokine antibody array. The blots of indicated cytokines are shown.

(e) The relative amount of cytokines was quantiﬁed and normalized to that of RasV12-shNTC cells. (f) Total RNA was extracted and cytokine transcript

levels were analysed via qRT–PCR and normalized against that in RasV12-shNTC cells. Data shown are mean þs.e.m. of three independent experiments

performed in triplicate. (h,i) Vector-control or HRasV12 IMR90 cells were stably infected with lentiviral shRNA control (shNTC) or shSCCA, together

with vector-control or XBP1s-expressing construct. (h) Whole-cell lysates were analysed by western blot with indicated antibodies. (i) Total RNA was

extracted and cytokine transcript levels were analysed via qRT–PCR and normalized against that in RasV12-shNTC cells. Data shown are mean þs.e.m.

of three independent experiments performed in triplicate. **Po0.01; ***Po0.001; NS, non-signiﬁcant by t-test.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4729 ARTICLE

NATURE COMMUNICATIONS | 5:3729 | DOI: 10.1038/ncomms4729 | www.nature.com/naturecommunications 7

While it has been clearly demonstrated that the mutant Ras can

activate NF-kB and induce inﬂammatory cytokine production in

various settings, the underlying molecular mechanism has

remained elusive. In numerous settings, inﬂammatory cytokine

production has been attributed to oncogene-induced senescence

that is associated with excess production of reactive oxygen

species and subsequent DNA damage or loss of p53 (refs 6,27).

We found that oncogenic Ras upregulates SCCA to promote

TCGA colorectal

Wild-type

KRas

Mutant

KRas

P= 0.012

Pei pancreas

–2

–1.5

–3.0

–1.5

1.5

3.0

1.5

3.0

–2

P= 3.95E-7

P= 8.85E-6 P= 7.51E-4 P= 0.002

–1.5

1.5

3.0

P= 0.050

–4

–2

Pei pancreas Badea pancreas SCCA1

Logsdon pancreas

SCCA2

Logsdon pancreas

Normal

Cancer

Normal

SCCA

SCCA negative SCCA positive

100

% Cases

SCCA

negative

SCCA

positive

P= 0.0385

High

Moderate

Low

Negative

IL-6

PanIN-1 PanIN-2 PanIN-3 PDAC

P= 1.03E-5

1.5

3.0

–1.5

–3

–2

–1

–6

–2

–4

P= 7.37E-4 P= 0.006 P=0.043

Normal

Cancer

Pancreatitis

Badea pancreas Iacoduzio-Donahue

pancreas

Segara

pancreas

Grutzmann

pancreas

mRNA expression

(log2 intensity)

mRNA expression

(log2 intensity)

mRNA expression

(log2 intensity)

mRNA expression

(log2 intensity)

–6

–4

–2

Figure 7 | SCCA is upregulated in human colorectal and pancreatic cancer. (a) TCGA human colorectal cancer data of somatic mutation and RNA

expression from Broad Institute’s Genome Data Analysis Center were analysed. There were 207 human colorectal tumours that have both somatic

mutation and mRNA expression data available. KRas was mutated in 87 out of the 207 samples. By comparing SCCA mRNA expression level of the groups

with wild-type and mutated KRas, SCCA expression was found to be signiﬁcantly higher in the group with KRas mutation. Boxplots with whisker from 10 to

90 percentile are shown. SCCA expression log2 intensity values for wild-type (n¼120) and mutant (n¼87) KRas samples are shown. P¼0.012 by

Wilcoxon Rank Sum test. (b–d) Oncomine (www.oncomine.org) data sets were analysed for SCCA1 (b) or SCCA2 (c) mRNA expression levels in normal

pancreatic tissue and pancreatic cancer, or for SCCA1 and SCCA2 mRNA expression levels in chronic pancreatitis and pancreatic cancer (d). The boxes

represent the interquartile range. Whiskers represent the 10th–90th percentile range. Bars represent the median. P-values were calculated by two-sample

t-test. (e–g) IHC against SCCA and IL-6 was performed on the corresponding serial pancreatic tissue microarrays. (e) Representative images of

normal pancreatic tissue and SCCA-positive PanIN 1, PanIN-2, PanIN-3 and PDAC samples are shown; scale bar, 100 mm. (f) Representative images of

SCCA/IL-6-negative and SCCA/IL-6-positive grade III PDAC samples; scale bar, 50 mm. (g) Quantiﬁcation of IL-6 staining in SCCA-negative and

SCCA-positive samples. w2-test for trend was used to determine signiﬁcance, P¼0.0385.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4729

8NATURE COMMUNICATIONS | 5:3729 | DOI: 10.1038/ncomms4729 | www.nature.com/naturecommunications

cytokine production, whereas SCCA is not upregulated in the

setting of drug-induced and replicative senescence. These results

suggest that the secretory response seen in cells with oncogenic

Ras mutations may be distinct from SASP induced by DNA

damage or loss of p53. Although the physiological signiﬁcance of

Ras-induced cellular senescence remains an open topic, our

results show that Ras can induce SCCA expression in cells where

Ras does not induce senescence (Fig. 1f). Furthermore, we found

that SCCA-dependent cytokine production occurs in human cells

expressing endogenous level of mutant Ras (Fig. 8) and this

provides a mechanism that uncouples cellular senescence

from inﬂammatory cytokine production on Ras activation.

Importantly, our study shows that the incidence of SCCA

positivity within PanIN lesions and PDAC increases with disease

progression (Fig. 7e). Therefore, monitoring SCCA levels may

become an attractive diagnostic tool in pancreatic cancer patients,

as the levels of SCCA have been used in numerous studies to

successfully predict disease stage and response to therapy16–18.

Our work uncovers SCCA as a critical mediator of Ras-induced

UPR. Until now, SCCA’s involvement in cancer was largely

attributed to its protective role against lysosomal permeability

that leads to unscheduled activation of lysosomal proteases29,39.

We have previously reported that, through its protease inhibitory

activity, SCCA leads to a decrease in both lysosomal and

proteasomal protein turnover and elicits a non-lethal level of ER

stress response29. It remains to be determined how SCCA

mechanistically suppresses protein turnover, and whether other

functions of SCCA such as its possible role in the nucleus

contribute to increased UPR. Nevertheless, here we show that

SCCA-mediated UPR plays a critical role in the context of

oncogenic Ras mutation. Enhanced UPR has been well implicated

in cancer, largely owing to the highly proliferative status of cancer

50 kDa

AsPC-1

Capan-1

Capan-2

CFPAC-1

HPAF-II

L3.6

PANC-1

PL45

S2-013

SCCA

22 kDa

shNTC

shKRas

shNTC

shKRas

shNTC

shKRas

50 kDa

1.2

1.0

0.8

Relative expression

0.6

0.4

0.2

CFPAC-1

50 kDa

***

*** *

IL-6

***

IL-6

1.5

1.0

0.5

0IL-6IL-6

1.2

1.0

0.8

0.6

0.4

Relative

expression

0.2

L3.6

shNTC

shSCCA#1

shSCCA#2

shNTC

shSCCA#1

shSCCA#2

shNTC

shSCCA#1

shSCCA#2

shNTC

CFPAC-1

60 5 10 15 2050403020

Time post injection (days) Time post injection (days)

10 20 30

Time post injection (days)

100

150

200

250

300

350

200

300

400

100

400

600

800

1,000

1,200

Tumour volume (mm3)

shSCCA#1

shSCCA#2

shSCCA#2 shNTC

L3.6 HPAF-II **

shSCCA#1 *

shNTC

shSCCA#1

shSCCA#2

HPAF-II PANC-1

1.0 0.6

1.0 0.3

SCCA

KRas

CFPAC-1 L3.6 Capan-1

β-Tubulin

SCCA2SCCA1 SCCA2SCCA1 SCCA2SCCA1

1.0 0.3

1.0 0.5

SCCA

KRas

β-Tubulin

SCCA

:SCCA/tubulin

shNTC

shKRas

*********

*** *

:KRas/tubulin

KRas

β-Tubulin

SCCA

β-Tubulin

Figure 8 | Ras-dependent SCCA expression promotes IL-6 production and tumorigenesis in human pancreatic cancer cell lines. (a) Whole-cell lysates

from a panel of pancreatic cancer cells were obtained and analysed through western blot with indicated antibodies. (b) Indicated cell lines were stably

infected with lentiviral shNTC or shKRas. Whole-cell lysates were analysed through western blot with indicated antibodies. Note that the Ras antibody

utilized is a pan-Ras antibody and KRas is indicated by arrowhead. Total RNA was extracted, and SCCA1 and SCCA2 transcript levels were analysed via

qRT–PCR and normalized to that of shNTC cells. Data shown are mean þs.e.m. of three independent experiments performed in triplicate. (c) Indicated cell

lines were stably infected with lentiviral shNTC or shSCCA. Whole-cells lysates were analysed through western blot with indicated antibodies. Total RNA

was extracted and IL-6 transcript levels were analysed via qRT–PCR. Data shown are meanþs.e.m. of three independent experiments performed in

triplicate. Relative level of transcript was normalized to that of shNTC cells. Note that silencing of SCCA in PANC-1 cells, which have undetectable SCCA

expression, had virtually no effect on IL-6 production. (d) CFPAC-1, L3.6 and HPAF-II cells were injected into the ﬂanks of athymic nude mice and monitored

for tumour growth; n¼5. Representative images of tumour and the tumour growth curve±s.e.m. are shown; scale bar, 1 cm. *Po0.05, **Po0.01,

***Po0.001 by t-test.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4729 ARTICLE

NATURE COMMUNICATIONS | 5:3729 | DOI: 10.1038/ncomms4729 | www.nature.com/naturecommunications 9

cells and their encounter of growth limiting conditions such as

nutrient deprivation and hypoxia40,41. Targeting the enhanced

UPR of cancer cells remains an attractive therapeutic option,

although the precise prediction of sensitivity has been difﬁcult.

Consistent with this notion, tumours driven by the mutant Ras

have been shown to be susceptible to proteotoxic therapies42. Our

current work showing that SCCA expression is stimulated by

oncogenic Ras and plays an essential role in Ras-induced ER

stress response, together with our previous report demonstrating

SCCA sensitizes cells to proteotoxic stress29, suggest that SCCA

may be a valuable predictor of response to proteotoxic agents in

tumours harbouring oncogenic Ras.

The contribution of the tumour stroma and inﬂammation to

tumour development and progression, especially in pancreatic

and colorectal cancer, is well accepted43,44. While NF-kB

signalling has been shown to mediate the SASP response26,

it is critical for the development of Ras-driven lung

adenocarcinoma45. The pro-tumorigenic role of SCCA

mediating NF-kB activation and inﬂammatory cytokine

production is consistent with these reports. This adds a new

pro-tumour mechanism of SCCA in addition to its well-

documented anti-cell death function15,46–49. In a parallel study,

we ﬁnd that ectopic expression of SCCA can independently

promote the transformation of the non-tumorigenic mammary

epithelial cell line MCF10A (Sheshadri et al., in preparation).

As numerous studies utilizing anti-inﬂammatories to target the

tumour microenvironment have shown great promise in the

treatment of malignancies50,51, the ability of SCCA to promote

cytokine production and tumorigenesis suggests a therapeutic

value of targeting its protease inhibitory activity.

Methods

Cell lines and culture.293T, IMR90, BJ, HT-29, Caco-2, HeLa, AsPC-1, Capan-1,

Capan-2, CFPAC-1, HPAF-II, L3.6, PANC-1, PL45 and S2-013 cells were cultured

according to ATCC recommendations. 293T, HeLa, PANC-1, PL45 and S2-013

cells were cultured in DMEM supplemented with 10% FBS (HyClone). IMR90,

BJ and HPAF-II cells were cultured in MEM supplemented with 10% FBS. HT-29

and Capan-2 cells were cultured in McCoy’s 5a supplemented with 10% FBS.

Caco-2 cells were cultured in MEM supplemented with 20% FBS. AsPC-1 and L3.6

cells were cultured in RPMI supplemented with 10% FBS. Capan-1 cells were

cultured in Iscove’s supplemented with 20% FBS. CFPAC-1 cells were cultured in

Iscove’s supplemented with 10% FBS. All media were supplemented with 100 units

per ml penicillin and 100 mgml1streptomycin (Invitrogen).

Plasmids and reagents.Retroviral expression vectors for WZL-hygro and

WZL-HRasV12 were a kind gift of Dr Alea Mills at Cold Spring Harbor

Laboratory21. pBABE-puro and pBABE-KRasV12 were a kind gift of Dr Scott Lowe

at Memorial Sloan-Kettering Cancer Institute. pBABE-NRasQ61 was purchased

from Addgene (Dr Channing Der; Plasmid 12543)52. pLNCX-ER:HRasV12 was a

kind gift of Dr Masashi Narita at University of Cambridge20. pcDNA3-FLAG-

ERK2-WT and pcDNA3-FLAG-ERK2-T138A (DN) were a kind gift of Dr Scott

Eblen at the Medical University of South Carolina53. pcDNA3-PEA3-WT and

pcDNA3-PEA3-K123R (MUT) were a kind gift of Dr Andrew Sharrocks at the

University of Manchester24. Human XBP1s was cloned by RT-PCR from total

RNA from IMR90 cells. Primers used were as follows, forward with HindIII

restriction and FLAG tag: 50-AAGCTTATGGATTACAAGGATGACGATGAC

AAGTGGTGGTGGCAGCCGCGCCGAACCC-30and reverse primer with HindIII

restriction site: 50-AAGCTTTTAGACACTAATCAGCTGGGGAAAG-30. The RT-

PCR product was digested with HindIII and ligated into the pLPC retroviral

expression vector. All shRNA lentiviral constructs were in the pLKO (Sigma)

backbone. shRNA targeting sequences used are shGFP: 50-TACAACAG

CCACAACGTCTAT-30; shScramble: 50-CAACAAGATGAAGAGCACCAA-30;

shSCCA#1: 50-GCACAACAGATTAAGAAGGTT-30; shSCCA#2: 50-CCGCTGTA

GTAGGGATTCGGAT-30; shPEA3: 50-GCTCCGATACTATTATGAGAA-30;

shATF6: 50-GCAGCAACCAATTATCAGTTT-30; shXBP1: 50-GCCTGTCTGTA

CTTCCATTCAA-30; shKRas: 50-CAGTTGAGACCTTCTAATTGG-30. Bay-11-

7082 (B5556), 4-hydroxytamoxifen (H7904) and crystal violet (C0775) were

purchased from Sigma. U0126 (V1121) was purchased from Promega. AKT

inhibitor VIII (124018) was purchased from Calbiochem. TNFa(210-TA) was

purchased from R&D Systems. X-gal (15520-018) was purchased from Invitrogen.

Etoposide (8154-1) was purchased from Clontech.

DNA transfection and viral infection.Both retrovirus and shRNA lentivirus were

generated in 293T cells. In brief, 293T were transfected by Lipofectamine 2000

(Invitrogen) with the plasmid of interest, packaging plasmid, and a plasmid

encoding for the VSV-G envelope protein. Forty-eight and seventy-two hours after

initial transfection, viral supernatant was collected, ﬁltered, supplemented with

polybrene (10 mgml1) and used to infect target cells. Forty-eight hours after last

infection, cells were selected with appropriate antibiotics. IMR90 cells were selected

for 2 days with 100 mgml1of hygromycin, 2 mgml1of puromycin or

1.75 mg ml 1of G418. HeLa and L3.6 cells were selected with 0.5 mgml1of

puromycin for 2 days. Caco-2, Capan-1, CFPAC-1 and PANC-1 cells were selected

with 1.0 mgml1of puromycin for 3 days.

Immunoblot analysis.Cell lysates were prepared in RIPA buffer (1% sodium

deoxycholate, 0.1% SDS, 1% Triton X-100, 0.01 M Tris pH 8.0, 0.14 M NaCl).

Protein expression was examined by western blotting using antibodies against

SCCA1/2 (FL-390; Santa Cruz; 1:1,000), Ras (Clone Ras10; Millipore; 1:10,000),

p21 (C-19; Santa Cruz; 1:500), ERK1/2 (4,695; Cell Signaling; 1:10,000), P-ERK1/2

(4,370; Cell Signaling; 1:2;000), Akt (9,272; Cell Signaling; 1;2,000), P-Akt (4,058;

Cell Signaling; 1:2,000), P-RelA/p65 (3,031; Cell Signaling; 1;1,000), RelA/p65 (F-6;

Santa Cruz; 1:2,000), ATF6 (F-7; Santa Cruz; 1:500), XBP1 (M-186; Santa Cruz;

1:500), Ubiquitin (P4D1; Covance; 1:1,000) and b-tubulin (Sigma; 1:10,000). All

primary antibodies were incubated overnight at 4 °C. Horseradish peroxidase or

Alexaﬂuor-conjugated goat anti-rabbit (Rockland) or goat anti-mouse (Rockland)

antibodies were used as secondary antibodies (1:2,000). Western blots were

developed using an ECL detection kit (Thermo Scientiﬁc) or an Odyssey Imager

(LI-COR). Full scans of uncropped blots can be found in Supplementary Figs 9,10.

Gene expression analysis and quantitative PCR.Total RNA was isolated and

puriﬁed using the RNeasy kit (Qiagen). cDNA was obtained by reverse transcribing

1–2 mg of total RNA using SuperscriptIII Reverse Transcriptase (Invitrogen) and

used for qPCR. qPCR reactions were performed in triplicate using SYBR Green

reagents (Quanta Biosciences) on a StepOnePlus (Life Technologies). GAPDH was

used as an endogenous control. All results were normalized to GAPDH. Primer sets

used are: GAPDH: 50-AAGGTCGGAGTCAACGGATTT G-30and 50-CCATGGG

TGGAATCATATTGGAA-30; SCCA1: 50-AGCCGCGGTCTCGTGC-30and

50-GGCAGCTGCAGCTTCTG-30; SCCA2: 50-AGCCACGGTCTCTCAG-30and

5-GCAGCTGCAGCTTCCA-30; Serpinb3a: 50-CATTTGTTTGCTGAAGCCAC

TAC-30and 50-CATGTTCGAAATCCAGTGATTCC-30; Serpinb3b: 50-ATTCGT

TTTCATGCAGCTGATGT-30and 50-GAAAGCTGAAGTTAAATTTGTTCG-30;

PEA3: 50-GGACTTCGCCTACGACTCAG-30and 50-CGCAGAGGTTTCTCA

TAGCC-30; IL-6: 50-TCCACAAGCGCCTTCGGTCCA30and 50-AGGGCTGA

GATGCCGTCGAGGA-30; IL-8: 50-AAGGAAAACTGGGTGCAGAG-30and

50-ATTGCATCTGGCAACCCTAC-30; CXCL1: 50-CACCCCAAGAACATCCA

AAG-30and 50-TAACTATGGGGGATGCAGGA-30; G-CSF: 50-ACTACAAGCA

GCACTGCCCT-30and 50-AGCAGTCAAAGGGGATGACA-30; GM-CSF: 50-CAA

GTGAGGAAGATCCAGGG-30; and 50-AGAGAGTGTCCGAGCAGCAC-30.

Cytokine array.Cells were washed once with PBS and incubated for 8 h with fresh

media. Supernatant was collected, cleared by centrifugation and used immediately.

The amount of supernatant used was normalized to cell number and used with the

human cytokine array kit (R&D Systems). IRDye 800CW Streptavidin (Rockland)

was used as secondary (1:2,000) and arrays were imaged and quantiﬁed on an

Odyssey Imager.

Senescence assays.Senescence was induced by oncogenic Ras, etoposide

(100 mM, 48 h), H

(100 mM, 1 h) or long-term passaging (replicative senes-

cence). All cells were analysed 7 days post-selection or post-treatment. For

SA-b-gal staining, cells were ﬁxed in 2% formaldehyde, 0.2% glutaraldehyde in PBS

for 15 min and stained (150 mM NaCl, 2 mM MgCl

,5mMK

Fe(CN)

,5mM

Fe(CN)

, 40 mM NaPi, pH 6.0, 1 mgml1X-Gal) overnight at 37 °C. For BrdU

staining, cells were cultured with BrdU (10 nM) for 6 h, ﬁxed with acid ethanol

(90% ethanol, 5% acetic acid, 5% H

O) for 30 min at room temperature. Cells were

then washed once with PBS, incubated with 2 M HCl for 20 min, 0.1 M sodium

borate, pH 8.5 for 2 min and washed once with PBS. Cells were blocked in 10%

bovine serum albumin (BSA) in PBS for 1 h at room temperature and incubated

with anti-BrdU (BD Pharmingen, 1:500 in 5% BSA in 0.1% PBS-tween) overnight

at 4 °C. Cells were washed, incubated with anti-mouse Alexa-594 (1:500) for 1 h at

room temperature, washed, co-stained with 40,6-diamidino-2-phenylindole (DAPI)

and mounted.

NF-jB luciferase activity assay.NF-kB activity was determined by using an

NF-kB luciferase reporter construct where the luciferase gene is under control of

the IL-6 promoter and internal control plasmid pCMV-RL using a dual-luciferase

reporter system (Promega). Cells were plated 24 h before transfection at 5 104

cells per well of 24-well plate. NF-kB-luciferase vector (250 ng) and 100 ng

pCMV-RL were used for transfection. Twenty-four hours post transfection, cells

were washed with PBS and lysed in 100 ml passive lysis buffer for 10 min. Luciferase

activity was determined following the manufacturer’s recommended protocol with

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4729

10 NATURE COMMUNICATIONS | 5:3729 | DOI: 10.1038/ncomms4729 | www.nature.com/naturecommunications

a SpectraMax M5 Microplate Reader. The ratios of ﬁreﬂy luciferase versus renilla

luciferase are used as relative luciferase activities.

TCGA analysis.TCGA human colorectal cancer (study abbreviation:

COADREAD) data were downloaded from Broad Institute’s Genome Data

Analysis Center (GDAC). Standard data of somatic mutations (Mutation_

Packager_Calls_Level_3) and RNA expression (Merge_transcriptome__

agilentg4502a_07_3__unc_edu__Level_3__unc_lowess_normalization_gene_

level__data.Level_3) were used. There were 207 human colorectal tumours that

have both somatic mutation and mRNA expression data available. KRas was

mutated in 87 out of the 207 samples. SCCA mRNA expression level was compared

between the groups with wild-type and mutant KRas.

Immunohistochemistry.Tissue microarrays came from the Vanderbilt GI SPORE

Tissue Core. Distribution and the use of all human samples were approved by the

Institutional Review Boards of Vanderbilt University Medical Center and Stony

Brook University, and samples were obtained with informed consent. IHC was

performed on a Ventana XT (Tucson, AZ, USA) autostainer, according to the

manufacturer’s directions.

Tissue microarray analysis.Damaged core spots and those that did not contain

cancerous tissue were eliminated from scoring. The sections were scored inde-

pendently by two evaluators blinded to the clinical status of the patients. Samples

were scored as previously described10. In brief, samples were scored as percent of

tumour cells with SCCA or IL-6 expression: 0, no expression; 1, o10%; 2, 10–50%;

3, 450%. For SCCA, a score Z1 was considered positive. For IL-6, 0, negative; 1,

weak; 2, moderate; 3, strong.

Xenograft tumour experiments.Male athymic nude mice, 6- to 8 weeks old, were

obtained from Taconic Farms. Mice were housed and monitored at the Division of

Laboratory Animal Resources at Stony Brook University. All experimental proce-

dures and protocols were approved by the Stony Brook University institutional

animal care and use committee (IACUC). Tumours were established by

resuspending 1 106tumour cells in 100 ml PBS and injecting the cells into the

mid-ﬂanks of mice using a 26-gauge needle. For each tumour, the tumour length (l)

and width (w) was measured every 4–5 days with an electronic caliper. Tumour

volume (v) was calculated using the formula v¼(lw2)/2 and plotted in mm3.

Statistical analysis.Statistical analyses were performed with GraphPad Prism

(Graphpad Software Inc). For gene expression, luciferase activity and xenograft

tumour growth signiﬁcance was calculated with t-tests. w2-tests were used to

assess statistical signiﬁcance of various categorical clinical features between

SCCA-negative and SCCA-positive samples. For the TCGA colorectal cancer data

analysis, Wilcoxon Rank Sum test was performed. For all tests, P-values were

considered statistically signiﬁcant when o0.05.

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Acknowledgements

We thank Drs Alea Mills, Scott Lowe, Masashi Narita, Scott Eblen, Andrew Sharrocks

and Erich Mackow for reagents and Drs Richard Lin and Dafna Bar-Sagi for critical

reading. J.M.C. was supported by the NCI T32 training grant (T32CA009176). This work

was supported by grants from NIH (R01CA129536 and R01GM97355 to W.-X.Z.,

U01CA168409 to R.S.P., and R01CA159222 and R01CA100126 to H.C.C.), and the Carol

Baldwin Breast Cancer Research Foundation (to W.-X.Z.). P50CA095103 for the

Vanderbilt University Medical Center GI Spore Tissue Core.

Author contributions

J.M.C., N.S., J.-A.P., Y.S. and C.S. performed experiments. J.L. R.S.P., and H.C.C. aided

with data analysis. J.M.C. and W.-X.Z. wrote the paper.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/

naturecommunications

Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests.

Reprints and permission information is available online at http://npg.nature.com/

reprintsandpermissions/

How to cite this article: Catanzaro, J. M. et al. Oncogenic Ras induces inﬂammatory

cytokine production by upregulating the squamous cell carcinoma antigens SerpinB3/B4.

Nat. Commun. 5:3729 doi: 10.1038/ncomms4729 (2014).

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4729

12 NATURE COMMUNICATIONS | 5:3729 | DOI: 10.1038/ncomms4729 | www.nature.com/naturecommunications

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Article

Apr 2006

Katagiri et al. 2006. J. Cell Biol. doi:10.1083/jcb.200508064 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200508064%26rft_id%253Dinfo%253Apmid%252F16549498%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%

Targeting RAS signaling pathways in cancer therapy

Article

Jan 2003
NAT REV CANCER

Julian Downward

Senescence-associated phenotypes reveal cell-autonomous of oncogenic RAS and the p53 tumor suppressor

Article

Jan 2008

J.P. Coppe

SCCA2 inhibits TNF‐mediated apoptosis in transfected HeLa cells.

Article

Nov 2001
Eur J Biochem

The squamous cell carcinoma antigens, SCCA1 and SCCA2, are members of the serine protease inhibitors (serpin) superfamily and are transcribed by two tandomly arrayed genes. A number of serpins are known to inhibit apoptosis in mammalian cells. In this study we demonstrate the ability of SCCA2 to inhibit tumor necrosis factor-alpha (TNFα)-induced apoptosis. HeLa cells stably transfected with SCCA2 cDNA had increased percentage cell survival and reduced DNA fragmentation. We investigated if the reactive centre loop (RCL) was necessary to allow SCCA2 to inhibit TNFα-mediated apoptosis. The RCL amino acids (E353Q, L354G, S355A), flanking the predicted cleavage site, were mutated and the resulting SCCA2 lost both the ability to inhibit cathepsin G and to protect stably transfected cells from TNFα-induced apoptosis. The presence of SCCA2 caused a decrease in the activation of caspase-3 upon induction with TNFα but no direct inhibition of caspases by SCCA2 has been found. Expression of cathepsin G was found to be induced in HeLa cells following treatment with TNFα. This protease has recently been shown to have a role in apoptosis through cleavage of substrates, so maybe the relevant target for SCCA2 in this system.

Pancreatic Cancer

Article

Jan 2007

The past two decades have witnessed an explosion in Our understanding of pancreatic cancer, and it is now clear that pancreatic cancer is a disease of inherited (germ-line) and somatic gene mutations. The genes mutated in pancreatic cancer include KRAS2, p16/CDKN2A, TP53, and SMAD4/DPC4, and these are accompanied by a substantial compendium of genomic and transcriptomic alterations that facilitate cell cycle deregulation, cell survival, invasion, and metastases. Pancreatic cancers do not arise de novo, and three distinct precursor lesions have been identified. Experimental models of pancreatic cancer have been developed in genetically engineered mice, which recapitulate the multistep progression of the cognate human disease. Although the putative cell of origin for pancreatic cancer remains elusive, minor populations of cells with stem-like properties have been identified that appear responsible for tumor initiation, metastases, and resistance of Pancreatic cancer to conventional therapies.

Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW.. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4A. Cell 88: 593-602

Article

Mar 1997
CELL

Oncogenic ras can transform most immortal rodent cells to a tumorigenic state. However, transformation of primary cells by ras requires either a cooperating oncogene or the inactivation of tumor suppressors such as p53 or p16. Here we show that expression of oncogenic ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by ras is accompanied by accumulation of p53 and p16, and is phenotypically indistinguishable from cellular senescence. Inactivation of either p53 or p16 prevents ras-induced arrest in rodent cells, and E1A achieves a similar effect in human cells. These observations suggest that the onset of cellular senescence does not simply reflect the accumulation of cell divisions, but can be prematurely activated in response to an oncogenic stimulus. Negation of ras-induced senescence may be relevant during multistep tumorigenesis.

A randomized trial of calcium supplementation to prevent colorectal adenomas

Article

Apr 1998

John A Baron

ER stress-induced inflammation: Does it aid or impede disease progression?

Article

Aug 2012

Different lines of research have revealed that pathways activated by the endoplasmic reticulum (ER) stress response induce sterile inflammation. When activated, all three sensors of the unfolded protein response (UPR), PERK, IRE1, and ATF6, participate in upregulating inflammatory processes. ER stress in various cells plays an important role in the pathogenesis of several diseases, including obesity, type 2 diabetes, cancer, and intestinal bowel and airway diseases. Moreover, it has been suggested that ER stress-induced inflammation contributes substantially to disease progression. However, this generalization can be challenged at least in the case of cancer. In this review, we emphasize that ER stress can either aid or impede disease progression via inflammatory pathways depending on the cell type, disease stage, and type of ER stressor.

Oncogenic Ras induces inflammatory cytokine production by up-regulating the squamous cell carcinoma antigens SerpinB3/B4

Abstract and Figures

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