American Association for Cancer Research - MCT4 ......2018/02/24 · Total cellular RNA was...

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MCT4 Expression is a Potential Therapeutic Target in Colorectal Cancer

with Peritoneal Carcinomatosis

Hee Kyung Kim1*

, InKyoung Lee2*

, Heejin Bang3, Hee Cheol Kim

4, Woo Yong Lee

4, Seong

Hyeon Yun4, Jeeyun Lee

1, Su Jin Lee

1, Young Suk Park

1, Kyoung-Mee Kim

3, and Won Ki

Kang1

1Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center,

Sungkyunkwan University School of Medicine, Seoul, Korea

2Biological Research Institute, Samsung Medical Center, Sungkyunkwan University School

of Medicine, Seoul, Korea

3Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of

Medicine, Seoul, Korea

4Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of

Medicine, Seoul, Korea

*These authors contributed equally to this work.

Running Title: Monocarboxylate transporter4 in colorectal cancer

Keywords: MCT4, Colorectal Cancer, Peritoneal carcinomatosis

Corresponding author: Won Ki Kang, MD

Research. on September 18, 2020. © 2018 American Association for Cancermct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 26, 2018; DOI: 10.1158/1535-7163.MCT-17-0535

http://mct.aacrjournals.org/

2

Division of Hematology-Oncology, Department of Internal Medicine, Samsung Medical

Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul

06351, Korea

Tel: +82-2-3410-3451, Fax: +82-2-3410-1754

E-mail: [email protected]

Acknowledgments

This research was supported by a grant of the Korea Health Technology R&D Project through

the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health

& Welfare, Republic of Korea (grant number; HI14C3418). WK Kang had been awarded a

grant.

Conflict of Interest Statement: The authors declare no potential conflicts of interest.



mailto:[email protected]


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Abstract

Monocarboxylate transporters (MCTs) are transmembrane proteins which control the lactate

metabolism and associated with poor prognosis in solid tumors including colorectal cancer

(CRC). Here we aimed to investigate the biological and clinical role of MCTs in CRC and to

assess the potential of therapeutic application.

A total of 16 human CRC cell lines, 11 patient-derived cells from malignant ascites (PDC),

and 39 matched pairs of primary CRC and normal colorectal tissues were used to assess the

role of MCT in vitro and in vivo. siRNA methodology was used to determine the effect of

MCT inhibition and molecular mechanism of hypoxia- and angiogenesis-related factors in

addition to MCT4. The effect of MCT inhibition was confirmed in mouse xenograft models.

MCT4 expression in surgical tissue was evaluated by immunohistochemistry (IHC) and used

for survival analysis.

Expression of MCTs was demonstrated in CRC cell lines. siRNA-mediated MCT silencing

caused significant decline of cell proliferation both in vitro and in vivo. An additive effect of

MCT inhibition was induced by combined treatment with chemotherapy or radiotherapy. In

particular, the expression of MTC4 was markedly increased in PDCs and MCT4 inhibition

significantly decreased PDC proliferation. Hypoxia inducible factor 1-α (HIF1α) was also

highly expressed in PDCs, whereas HIF1α knockdown reduced MCT4 expression and of

other angiogenesis-related mediators. The patients with high MCT4 expression by IHC

showed shorter relapse-free survival compared with low expression.

These findings suggest that MCT4 may represent a new therapeutic target for CRC with

peritoneal carcinomatosis and serve as a prognostic indicator.




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Introduction

Colorectal cancer (CRC) represents the third most common cancer in males and the

second in females worldwide, with 1.2 million new diagnoses and 608,700 deaths estimated

in 2008 (1). The survival of metastatic CRC has been markedly improved in the past decades

resulting from the development of targeted therapies such as cetuximab, panitumumab,

bevacizumab, and regorafenib (2-6). However, tumor heterogeneity and selective pressure in

patients with CRC leads to the development of resistance to anti-cancer treatments and

several studies have attempted to seek new therapeutic targets by addressing the CRC

molecular landscape (7-10). In particular, the targeting of metabolic enzymes or transporters

represents a different approach for cancer therapy and is expected to overcome the resistance

to current therapies (11).

Monocarboxylate transporters (MCTs) are proteins that are expressed in the cell

membrane and control the lactate metabolic pathway. The level of lactate is increased in

rapidly growing cancer cells (12, 13) as it is generated by glycolytic metabolism, on which

the survival of cancer cell is largely dependent (14, 15). MCTs prevent intracellular

acidification consequent to the increased intracellular lactate production in cancer cells by

balancing its influx and efflux to maintain a steady intracellular pH and protect against loss of

cancer cell viability (16). Particularly MCT1 and MCT4 require the formation of complex

with chaperone, CD147. CD147 is a transmembrane protein of the immunoglobulin

superfamily and is also known as Basigin (17-19).

The MCT family comprises 14 members in total, among which MCTs 1-4 play various

roles in mammals (20). MCT1 and MCT2 take part in lactate uptake, thus initiating the

oxidative pathway of lactate, whereas, MCT4 functions in lactate excretion (21-23).




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Furthermore, the pattern of MCT expression differs according to cell type. MCT1 but not

MCT2 expression has been shown in almost all tissues (23). Similarly, MCT expression in

tumor cells depends upon tumor type; for example, MCT4 expression in lung cancer may be

down-regulated whereas both MCT1 and 4 are generally up-regulated in breast cancer (24).

In addition, tumor microenvironment such as high oxidative stress, lactate metabolism is

main determinant controlling MCT expression (25, 26).

MCTs also correlate with cancer progression, infiltration, and angiogenesis, with cells

exhibiting high MCT expression demonstrating invasiveness and poor prognosis in several

solid tumors including CRC (27). Accordingly, several studies have evaluated MCT inhibitors

as anti-cancer treatments. For example, the MCT1/2 inhibitor AR-C155858 and MCT1

inhibitor AZD3965 showed preclinical anti-cancer activity (28, 29) and a Phase 1 trial of

AZD3965 for refractory and advanced solid tumors or lymphoma is ongoing (NCT07197595).

We previously studied the clinical implication of MCT4 in metastatic gastric cancer with

peritoneal carcinomatosis, which revealed that the inhibition of MCT4 reduced tumor cell

proliferation and the export of lactate (30). However, little information is available regarding

the relationship between MCT expression and clinical outcome in CRC. In the current study,

we investigated the expression patterns of MCTs in CRC cell lines, and their therapeutic

potential as target proteins in the treatment of CRC. We also analyzed survival outcomes

according to MCT4 expression in primary CRC tissues derived from patients to determine its

prognostic impact on relapse-free and overall survival.




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Materials and Methods

Cell culture

We utilized fibroblasts from normal colon (CCD-18Co) and 16 known human CRC cell

lines: CoLo320, LS513, SNU175, and SW620 cells were purchased from the Korean Cell

Line Bank in 2010. SW48, HT29, RKO, WiDr, DLD1, HCT8, HCT116, LoVo, LS174T,

NCI-H716, and SW480 cells were purchased from American Type Culture Collection

(Manassas, VA, USA) in 2010. CCD-18Co was purchased from the Korean Cell Line Bank

in 2017. DiFi was kindly provided by Dr. JO Park (Samsung Medical Center, Seoul, Korea)

in 2009.

To establish patient-derived cells (PDCs) from patients with metastatic CRC and

malignant effusion, the individuals who were enrolled onto the SMC Oncology Biomarker

study (NCT#01831609) were screened for MCT expression by western blot. All patients

provided an informed consent form according to the SMC Institutional Review Board

guidelines. Briefly, collected effusions (1–5 L) were divided into 50 mL tubes, centrifuged at

1500 rpm for 10 min, and washed twice with PBS.

All cells and PDCs were grown in RPMI-1640 medium supplemented with 10% FBS, an

antibiotic, and an antimycotic (Invitrogen Corporation, Carlsbad, CA, USA). Cells were

cultured at 37°C in a humidified 5% CO2 environment.

All cell lines were tested for mycoplasma contamination and authenticated by STR DNA

profiling (Samsung Medical Center Basic Research Support Center, Seoul, Korea) every 6

months. And all cells were cultured according to the manufacturer's manual and had been

passaged for fewer than two months after thawing.




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Western blot analyses

Total cell extracts were obtained using lysis buffer (20 mM HEPES [pH 7.4], 1% Triton

X-100, 1 mM EDTA, 1 mM MgCl2, 150 mM NaCl, 10% glycerol, and protease inhibitor

cocktail [Invitrogen]) and protein concentration was determined using the micro-BCA protein

reagent (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of proteins (30 μg per

well) from the clarified lysates were separated by SDS-PAGE and transferred onto

nitrocellulose membranes having a 0.45-μm pore size (Whatman, Maidstone, UK). The

membranes were sequentially incubated in 5% dry milk and antibodies against MCT1 (H-1,

1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), MCT2 (HPA005911, 1:400; Atlas

Antibodies, Stockholm, Sweden), MCT4 (H-90, 1:500; Santa Cruz), β-actin (AC-15, 1:5,000;

Sigma, St Louis, MO, USA), PCNA PC10, 1:1,000; (Santa Cruz), HIF1α (1:1,000; Cell

Signaling Technologies [CST], Danvers, MA, USA), phospho-STAT3 (Tyr705) (3E2,

1:1,000; CST), STAT3 (124H6, 1:1,000; CST), angiopoietin 2 (1:500; AbFrontier, Seoul,

Korea), BiP (1:1,000; BD Biosciences, San Jose, CA, USA), and VEGF (A-20, 1:500; Santa

Cruz). The ECL system was used for protein detection (Invitrogen).

Reverse transcription-PCR (RT-PCR)

Total cellular RNA was extracted using an RNeasy MiniKit (Qiagen, Valencia, CA, USA)

and treated with DNase I (Qiagen). Then, 1 μg RNA was converted to cDNA using an

Omniscript RT Kit (Qiagen). The primer sequences designed from the coding region of

human MCT cDNA were as follows: MCT1 cDNA forward, 5'-GGA AGG TGG ACC AGA

ATG AA-3' and reverse, 5'-CAA TTT AGC AAG GCC CAA AA-3' (GenBank accession no.

AK312500); MCT2 cDNA forward, 5'-AGG ATT AAT TGC AAA CTC CA-3', and reverse,




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5'- CCG AAT GTT TAG ATT TGC TC-3' (GenBank accession no. AF058056); and MCT4

cDNA forward, 5'-AGG AGC TCA TAC AGG AGT TT-3' and reverse, 5'-GTG AGG TAG

ACC TGG ATG AT-3' (GenBank accession no. AK223040). The PCR conditions were as

follows: 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s.

Real-time PCR was performed using a Prism 7900HT Sequence Detection System (PE

Applied Biosystems, Foster City, CA, USA). MCT1, MCT2, MCT4 mRNA and 18S rRNA

were detected using TaqMan Gene Expression Master Mix Reagent and TaqMan probes

(Applied Biosystems). Data were normalized using 18S rRNA as an endogenous control and

calculated using the comparative Ct method (2-delta delta Ct).

RNA interference and transfection

The 21-nucleotide-long siRNAs targeting MCT1, MCT2, MCT4, and the negative control

siRNA (siC) were purchased from Dharmacon (Lafayette, CO, USA). Cells (3 × 105 cells per

60-mm dish) were transfected with 20 nM siRNAs using HiPerfect transfection reagents

(Qiagen) according to the manufacturer’s instructions and were used for western blot analysis

48 h after transfection. Sequences of the siRNAs used were: MCT1-targeted siRNA (siMCT1)

(5-CCA AGG CAG GGA AAG AUA AGU CUA A-3), MCT2-targeted siRNA (siMCT2)

(5-GGA UUU AAC UGG AGA AUA U-3), MCT4-targeted siRNA (siMCT4) (5-CGA CCC

ACG UCU ACA UGU ACG UGU UUU-3), HIF1α-targeted siRNA (siHIF1α) (5- ACA

CGC AAA UAG CUG AUG GUA AGC CUC-3), and control nontargeting siRNA (5-UAG

CGA CUA AAC ACA UCA A-3).

Cell growth assessment and colony formation assay




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To assess cell numbers, cells (1 × 105 cells per 6-well plate, Corning Costar Corp, NY,

USA) were transfected with siRNAs and incubated for 3 days. Adhered cells were trypsinized,

stained with 0.2% trypan blue (Sigma), and counted using a hemocytometer. Cell

proliferation following each treatment was compared that of with untreated cells.

For the clonogenic assay, cells were transfected with siRNAs for 24 h, irradiated with a

137Cs source (2.01 Gy/min, IBL-437C, CIS-US Inc.), trypsinized, and counted. Then, 400

cells were replated in 6-well plate. After incubation at 37°C for 10–14 days, colonies were

stained with 0.005% (w/v) crystal violet (Sigma). Colonies (> 50 cells) were counted and

surviving fractions following the given treatments were calculated based on the survival of

nonirradiated cells.

ELISA assay

Secreted protein levels of VEGF and Angiopoietin 2 were assessed using culture media

(200 μL) collected from siHIF1a or siMCT4 transfected cells. Protein levels of VEGF and

Angiopoietin 2 were measured using an ELISA kit for human VEGF and Angiopoietin 2

(R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions and

quantified using a microtiter plate reader at 450 nm wavelength.

Xenograft study

Male BALB/c nude mice, 4–6 weeks old, were obtained from Orient Bio Inc. (Seongnam,

Korea). Mice were implanted subcutaneously with SW602 (5 × 106) cells in a 100 μL volume.

The mice were randomized and the treatment started when the tumor size reached 60 mm3 at

11 days after inoculation. Mice were assigned into five groups: siC, siMCT1, siMCT2,




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siMCT4, or a combination of siMCT1 and siMCT4. siMCTs (1 μg with HiPerfect

transfection reagent/tumor, intratumoral injection) were administered 2 times per week.

Tumor growth was measured using a digital caliper (Proinsa, Vitoria, Spain) every 3–4

days and average tumor volumes were calculated using the following formula: V = (L × W2) /

2, where V = volume (in cubic millimeters), L = length (in millimeters), and W = width (in

millimeters). The mice were sacrificed and the tumors (three tumors per treatment group)

were resected and frozen in liquid nitrogen until later use for western blot analyses. All

mouse experiments were conducted in accordance with the Institute for Laboratory Animal

Research Guide for the Care and Use of Laboratory Animals and the protocols were approved

by the appropriate Institutional Review Boards at Samsung Medical Center (Agreement-

20141211001).

Human CRC and PDCs

We collected 39 matched pairs of primary CRC and normal colorectal tissues and PDCs

(N = 11) at the Samsung Medical Center. All CRC tumors and control tissues were confirmed

by the hospital’s Clinical Pathology department. The normal colorectal tissue was collected at

a distance of at least of 10 cm from the tumor site. To establish PDCs from patients with

metastatic CRC and malignant effusion, individuals who were enrolled in the SMC Oncology

Biomarker study (NCT#01831609) were screened for the expression of MCTs by western

blot and real-time PCR.

All patients provided written informed consent. This study was performed in accordance

with the Declaration of Helsinki and was approved by the Institutional Review Board of

Samsung Medical Center.




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Patients for immunohistochemistry and survival analysis

We collected data from the electronic medical records of patients who were diagnosed

with CRC and who underwent curative surgery at the Samsung Medical Center between June

2008 and May 2009 (N=586). Data including sex, node-metastasis, staging, location of tumor,

histology, lymphatic invasion, perineural invasion, and vascular invasion were collected. This

study was approved by the institutional review board of the Samsung Medical Center, Seoul,

Korea.

MCT4 immunohistochemistry

Immunohistochemistry (IHC) analysis was performed on tissue microarray blocks. Two

tissue cores with a diameter of 2.0 mm from representative tumor areas marked by

pathologists were punched from tumor tissue blocks and included in each array block.

Formalin-fixed, paraffin-embedded blocks were cut into 4-m serial sections. Antigen

retrieval was performed for 20 min with pH 6.0 LOW buffer (EnVision™ FLEX Target

Retrieval Solution Low pH (50x) (Dako, Denmark)) in a 97C water bath. Endogenous

peroxidase blocking was conducted for 5 min. The sections were incubated using a primary

antibody to MCT4. MCT4 expression was evaluated by two independent pathologists (Kim

KM and Bang H). To determine MCT4 expression, the intensity and proportion of staining in

the tumor cell membrane were assessed semi-quantitatively. The intensity was classified into

negative, weak, moderate, and positive and scored as 0, 1, 2, and 3, respectively. The

proportion of stained tumor cells was scored on a scale 0 to 4 (0, < 5%; 1, 5%-25%; 2, 26%-

50%; 3, 51%-75%; 4, > 75%).




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Statistical analysis

Preclinical data were evaluated by a two-tailed t-test or one-way ANOVA using GraphPad

Prism version 4.01 (San Diego, CA, USA). For analysis of the MCT4 IHC cohort, SPSS

statistical software version 23 (Chicago, IL, USA) was used. All comparisons were examined

by the χ2 test or Fisher exact test. Recurrence-free survival (RFS) was measured from the date

of curative surgery to the date of first cancer recurrence or death. Overall survival (OS) was

measured from the date of curative surgery to the date of death or date the patient was last

seen. The Kaplan-Meier method was used to estimate RFS, OS, and survival curves were

compared by the log-rank test. To evaluate the prognostic value of MCT4, we utilized a

multivariable Cox proportional hazard regression model. All tests were two-tailed and P

values < 0.05 were considered significant.




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Results

MCTs are expressed in CRC cell lines and MCT knockdown suppresses tumor

proliferation

Fig. 1A illustrates the expression of MCT mRNA and proteins in 16 CRC cell lines as

assessed by RT-PCR and western blot. All CRC cell lines expressed MCT1, whereas MCT2

and MCT4 demonstrated different patterns of expression. Specifically, MCT2 was expressed

at a lower level than in KRAS-wild or BRAF-mutant type CRC cell lines in comparison with

KRAS-mutant cell lines. Fig. 1D demonstrated the inhibition of MCT1 and MCT4 decreased

Basigin expression, but not MCT2 expression.

The proliferation of CRC cell lines decreased following MCT inhibition by transfection

with siRNA (Fig. 1B and Supplementary Fig. S1). MCTs were effectively knocked down by

siMCT1, 2, or 4 after 72 h, and cell proliferation was significantly decreased depending on

the type of MCTs expressed in each respective cell line. In particular, MCT1 expression was

confirmed by western blot in 5 cell lines (CoLo320, NCI-H716, SW620, SW480 and DiFi)

which showed decreased proliferation by the addition of siMCT1. MCT2 was expressed in

NCI-H716 and SW480 cell lines, and transfection with siMCT2 hindered the expression of

MCT2 as well as cell proliferation in these lines. Similarly, SW620 and SW480 cell lines

exhibited MCT4 expression and their cell proliferation was inhibited by siMCT4. Fig. 1C

illustrated that normal colorectal cell line (CCD-18Co) did not express MCT4 and was not

affected by siMCT4. These results suggested that knockdown of MCT1, 2 and 4 inhibited

cancer cell proliferation which expressed MCT1, 2, 4, respectively.

Effect of MCT inhibition on CRC growth in vivo




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To determine whether the anti-tumor effect of MCT inhibition could also be observed in

vivo, we implanted SW620 cells, which expressed MCT1 and 4, into mice and assigned them

to the following 5 groups (n = 5 mice per treatment group): untreated control, siMCT1,

siMCT2, siMCT4, and the combination siMCT1 and 4. Mice were subjected to 2

subcutaneous injections in each flank to promote the growth of 2 tumors (10 tumors per

treatment group). The combination siMCT1 and siMCT4 treatment resulted in the most

significant decrease of tumor volume on day 17 (siControl vs siMCT4, mean tumor volume

on day 17, 857.5 vs 381.2 cm3; mean difference, 476.3 cm

3; 95% CI, 320.4–632.3; P <

0.0001, Fig. 2A). A single treatment with siMCT1 or siMCT4 but not siMCT2 also showed a

significant reduction of tumor volume.

The xenograft tumors were resected and protein expression was evaluated using IHC.

IHC was performed by applying a monoclonal anti-PCNA antibody for the validation of

tumor growth inhibition with SW620 in xenografts (Fig. 2B). The expression level of PCNA

in IHC was lowest in the combination siMCT1 and MCT4 group; siMCT1 or siMCT4 alone

also lessened the level of protein expression in the xenograft tumors. Similar results of

protein expression were observed by western blot (Fig. 2C).

MCT inhibition in conjunction with 5-fluorouracil or radiotherapy showed additive

anti-tumor effects

We next assessed the additive effects of MCT inhibition and radiotherapy or cytotoxic

chemotherapy. SW620 cells were transfected with MCT siRNA and then irradiated with a

137Cs source or treated with 5-fluorouracil (5-FU) after 24 h. As the dose of radiation

increased, the cell proliferation became further decreased, demonstrating a radiation dose-




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dependent inhibition in cells with concomitant knockdown of MCT1, or 4 (Fig. 3A).

Knockdown of MCT1 or 4 significantly induced cell death upon treatment of 5-FU, whereas

in the case of MCT2 the level of cell proliferation was not significantly different than that of

the control (Fig. 3B). The other tested cell lines (CoLo320, NCI-H716, and SW480) also

demonstrated best additive effects of MCT inhibition with irradiation or 5-FU

(Supplementary Fig. S2)

MCT4 expression is increased in malignant ascites and MCT4 knockdown decreases

PDC cell proliferation

We next investigated the MCT expression levels in 39 matched paired normal colon tissue,

CRC tissue, and 11 PDCs acquired from malignant ascites using qRT-PCR, which

demonstrated markedly increased MCT4 expression in PDCs from malignant ascites (Fig.

4A). IHC performed to evaluate the MCT4 expression in tissue samples and PDCs also

showed strong expression in PDCs from malignant ascites (Fig. 4B). Treatment of 3 PDCs

with siMCT4 indicated that knockdown of MCT4 also induced the reduction of MCT4

expression and cell death (Fig. 4C), similar to the effects in the CRC cell lines.

Hypoxia and angiogenesis related-factors are correlated with MCT4 expression

We analyzed expression of MCT4, hypoxia-inducible factor alpha (HIFα), STAT3

phosphorylation, angiogenesis-related mediators including angiopoietin 2 (ANGPT2),

binding immunoglobulin protein (BiP), and VEGF in PDCs using western blot. All the

factors were distinctly expressed to a greater degree in PDCs compared with normal and

primary tumor tissue (Fig. 5A).




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To determine the order of contribution of these factors to the underlying mechanism, we

induced a hypoxic condition using 1% oxygen and transfected the SW48 cells with siHIFα or

siMCT4. Hypoxia induced the expression of HIFα and MCT4 , ANGPT2, and BiP expression

were decreased after knockdown of HIFα. However, knockdown of MCT4 did not influence

the expression of HIFα and MCT4 expression was up-regulated following overexpression of

HIF1α (Fig. 5B).

We also measured the expression of the angiogenesis-related mediators, ANGPT2 and

VEGF by ELISA in PDCs. Transfection with siHIFα or siMCT4 in the hypoxic condition in

PDCs induced a decrease of VEGF and ANGPT2 expression (Fig. 5C). A similar

phenomenon was also observed in other cell lines (HT29, HCT8) treated with siHIFα or

siMCT4 (Supplementary Fig. S3 and S4).

Strong MCT4 expression by IHC is associated with significantly shorter RFS in patients

with CRC

A total of 586 patients who had been diagnosed with CRC received curative surgery

between June 2008 and May 2009. We compared baseline characteristics and survival

outcome by dividing the patients into two groups, MCT4 positive and negative (Table 1). The

MCT4 positive group was defined as exhibiting strong positive MCT4 expression by IHC

(intensity score 3) and the MCT4 negative group had an intensity score of 0 to 2 by IHC (Fig.

4B). During the 83.5 months of median follow-up duration, the patients with positive MCT4

expression by IHC showed significantly shorter RFS than the MCT4 negative group (5-year

RFS rate, 83.5% vs. 73.1%, P = 0.013, Fig. 6). Notably, in the multivariate analysis, the

MCT4 positive state was identified as an independent risk factor for RFS (hazard ratio 1.29,




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95% CI, 1.11–1.49, P = 0.001).




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Discussion

MCT expression has been known to correlate with tumor invasion or metastasis in several

cancers in addition to CRC (31) including gastric (32), lung (33), breast (34), and prostate (35)

cancers. In the current study, we showed that MCT proteins are expressed in CRC cell lines

and tumor tissue and present the first demonstration that MCT4 expression was significantly

elevated in malignant cells in CRC ascites. In our previous report regarding MCT4 in gastric

cancer, MCT4 also was found to be overexpressed in malignant cells in ascites. However,

MCT4 expression in gastric cancer was not revealed as a factor for poor prognosis in survival

analysis (30). An association between MCT4 expression and poor survival has been reported

in pancreas cancer (36) and clear renal cell carcinoma (37); in comparison, here we provide

the first report that in patients with CRC, MCT4 expression instead represents a risk factor

for early relapse and shorter RFS.

MCT4 is up-regulated by hypoxia as a transporter and is known to be under the control of

HIF-1α (37, 38), which performs essential roles in pH regulation, metabolism, and cell

invasion in addition to controlling VEGF and ANGPT2 in the hypoxia-signaling pathway

(39). We identified significantly higher expression of HIF-1α and and MCT1, 2, 4, VEGF,

and ANGPT2 in PDCs from malignant ascites in comparison with normal and tumor tissue.

These results implied that hypoxic conditions in the peritoneal space induced metabolic

adaptation including up-regulation of MCT4 in CRC with peritoneal seeding. We also

verified HIF-1α as an additional key upstream regulatory factor of MCT4 because HIF-1α

knockdown or up-regulation led to reduced or increased expression of MCT4, respectively.

MCT4 also has been reported to be associated with VEGF in CRC (40). VEGF is usually

up-regulated and induced by angiogenesis and hypoxia in cancer cells (41) and is known as a




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therapeutic target of metastatic CRC for the anti-VEGF antibody, bevacizumab. Bevacizumab

function depends on the presence of a specific genomic alteration and does not induce

marked survival benefits. Recent studies reported the mechanisms between adaptive

resistance to anti-angiogenic treatments and metabolic symbiosis. Metabolic symbiosis based

on the exchange of lactate and depends on MCTs is also responsible for tumor resistance to

anti-angiogenic treatments (42-44). In the current study, MCT4 inhibition was also able to

down-regulate VEGF expression in CRC cell lines. Therefore, an MCT4 inhibitor might have

a potential role in the treatment of CRC along with bevacizumab.

We used RPMI-1640 medium supplemented with 10% FBS (fetal bovine serum) for

culture of all cells and PDCs. In current study, not only MCT4 but also MCT1 and MCT2

inhibition demonstrated the suppression of tumor growth in the absence of exogenous lactate.

However, lactate could be generated from metabolism of cancer cells and transported through

the membrane. In case of MCT2, we previously reported that knockdown of MCT2 could

suppress tumor growth in colorectal cancer in the absence of exogenous lactate (45).

According to the findings following clinical applications of MCT inhibitor (17, 29, 46),

therapeutic approaches for targeting MCTs appear to hold promise. Here, we demonstrated

that MCT knockdown was able to inhibit tumor cell proliferation and enhance the efficacy of

radiotherapy or cytotoxic chemotherapy, which is consistent with the previous findings that

hypoxia and lactate concentration are linked with the treatment failure of radiotherapy or

chemotherapy in many cancers (47-49). Similarly, Sonveaux P, et al. demonstrated that MCT

inhibition increased tumor radiosensitivity (50) and Bola et al. showed that the MCT1

inhibitor AZD3965 augmented radiosensitivity in the treatment of small cell lung cancer (51).

Notably, our finding that elevated MCT4 expression in CRC as measured by IHC could




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serve as a negative prognostic factor reflected the observation that RFS was significantly

worse with high MCT4 expression; however, no difference was noted in terms of OS

(Supplementary Fig. S5). The association between MCT4 expression and shorter OS has

previously been described in several studies involving CRC and other cancers (49, 52). One

potential explanation for this discrepancy may be selection bias. The patients assessed in the

current study were not diagnosed with metastatic disease and all had received curative

resection at the time of tissue archiving. As MCT4 expression is associated with metastatic

and advanced disease, the survival analysis of MCT4 expression might be more conclusive in

patients with metastatic CRC.

Metastatic CRC with peritoneal carcinomatosis carries a grave prognosis with a median

survival of 6 to 9 months following diagnosis (53, 54). Approximately 4 to 7% of patients

with CRC present with peritoneal carcinomatosis despite early detection, with the peritoneum

serving in second place to the liver as a site of metastasis (55). MCT4 was strongly expressed

in cells from malignant ascites of CRC in our study; thus, this result indicates that an MCT4

inhibitor may overcome the treatment failure in patients with peritoneal carcinomatosis.

However, we observed that the expression of basigin could be decreased by MCT1 and

MCT4 inhibition. The possible role of basigin in peritoneal carcinomatosis could not be

excluded.

In conclusion, the result of this study showed that MCT4 was expressed in CRC and most

notably in PDCs from malignant ascites, and that it may serve as a factor indicating poor

prognosis in this disease. MCT4 inhibitor might be a key treatment for hypoxic tumors in

conjunction with radiotherapy or chemotherapy. Further studies such as clinical trials to

elucidate the benefit of MCT4 inhibitor treatment and the role of basigin are therefore




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warranted.




22

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.




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Table 1. Patients’ characteristics

All patients

(n=586)

MCT4 (-)

(n=485)

MCT4 (+)

(n=91)

P-value

Median age (range) 59 (28-90) 59 (28-90) 60 (33-88) 0.207

Sex

Male

Female

365 (62.3%)

221 (37.7%)

305 (62.9%)

180 (37.1%)

53 (58.2%)

38 (41.8%)

0.402

Stage at diagnosis

II

III

308 (52.6%)

278 (47.4%)

251 (51.8%)

234 (48.2%)

57 (62.6%)

34 (37.4%)

0.056

Location

Colon

Rectum

434 (74.3%)

150 (25.7%)

357 (73.6%)

127 (26.2%)

70 (76.9%)

20 (22.0%)

0.423

Histology 0.848

Adenocarcinoma

Mucinous carcinoma

Signet ring cell

carcinoma

559 (95.4%)

25 (4.3%)

2 (0.3%)

463 (95.5%)

20 (4.1%)

2 (0.4%)

88 (96.7%)

3 (3.3%)

0

Lymphatic invasion

no

yes

430 (73.4%)

156 (26.6%)

355 (73.2%)

130 (26.8%)

69 (75.8%)

22 (24.2%)

0.602

Perineural invasion

no

yes

543 (92.7%)

43 (7.3%)

450 (92.8%)

35 (7.2%)

85 (93.4%)

6 (6.6%)

0.832

Vascular invasion

no

yes

504 (86.0%)

82 (14.0%)

412 (84.9%)

73 (15.1%)

84 (92.3%)

7 (7.7%)

0.063




31

Figure legend

Figure 1: The expression pattern of MCTs (MCT1, 2 and 4) in colorectal cancer (CRC)

cell lines.

A. RT-PCR and western blot analysis of MCTs expression in 16 CRC cell lines (KRAS wild

type, N = 3; BRAF mutant, N = 3; KRAS mutant, N = 10)

B. MCTs knockdown by transfection of MCTs-siRNA suppressed CRC cell proliferation and

protein expression by western blot analysis.

C. MCTs knockdown suppressed SW620 cell proliferation but no significant effect was

observed in the normal colon fibroblast cell line (CCD-18Co).

D. Knockdown of MCT1 and MCT4 reduced Basigin expression, but not MCT2.

*P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 2: Effect of MCTs knockdown on tumor growth in vivo. BALB/c nude mice were

injected subcutaneously in the bilateral flank with SW620 cells (5 x 106 cells). One week

after injection, mice were treated 2 times per week with an intratumoral injection of siMCTs

(1 μg with HiPerfect transfection reagents /tumor).

A. Time course of tumor growth following injection of siMCTs (left) and mean tumor volume

and standard deviation (right).

B. Cell proliferation in xenograft of SW620 was analyzed by IHC with anti-PCNA antibody

(Santa Cruz Biotechnology, PC-10, 1:2000). The brown staining in the nucleus is the PCNA

signal, and cell proliferation was decreased by siMCT1 and/or siMCT4.




32

C. The level of MCTs proteins and Basigin were decreased by siMCTs in western blot

analysis.

*P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 3: Additive effect of MCTs inhibition with radiotherapy or chemotherapy.

SW620 cells were transfected with indicating siMCTs, then the cells were irradiated with a

137Cs source or 5-fluorouracil after 24 hours.

A. Downregulation of MCTs is associated with the radiosensitizing effects. Radiosensitivity

assessed by clonogenic cell survival. SW620 cell lines treated with siMCTs and radiation

were plated (300 cells/well in 6-well) and maintained at for 14 days. Colonies were stained

with 0.1% crystal violet and colonies larger than 50 cells were counted. Surviving fractions

following the given treatments were calculated based on the survival of non-irradiated cells.

B. Additive effect of MCTs inhibition with 5-fluorouracil. SW620 cell lines treated with

siMCTs and 5-fluorouracil for 3 days and stained with 0.4% trypan blue. Results of cell

counts are expressed as percentage of cell proliferation using the control as reference.

*P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 4: MCTs expression in patients’ tumor tissues.

A. MCT1, MCT2, and MCT4 mRNA and 18S rRNA were detected using real-time PCR in

patients’ tissues according to tissue (normal tissue, primary tumor tissue, and PDCs collected

from malignant ascites). MCT4 expression in PDCs from malignant ascites was markedly

increased in comparison with increment of MCT1 and MTC2. Data were normalized using

18S rRNA as an endogenous control.




33

B. MCT4 immunostaining of colorectal cancer tissues and PDCs. Intensity MCT4

immunostaining was measured as score 0 (negative), score 1 (weak), score 2 (moderate), and

score 3 (strong) for positive cases of colorectal cancer tissue (left panel). Right panel

represents two cases of PDCs with highly strong MCT4 expression. Original magnification, x

200.

C. MCT4 expression in PDCs collected from malignant ascites and the effects of MCT4

inhibition on cell proliferation

*P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 5: Correlation between MCT4 expression and hypoxia- or angiogenesis-related

factors.

A. Strong expression of MCT4 and hypoxia/angiogenesis-related factors in PDCs compared

to normal and tumor tissue.

B. Left panel showed the protein expression when siRNA of HIF1α and MCT4 were

transfected on SW48 cell line, in hypoxic condition with 1% of oxygen. The MCT4

expression was decreased by knockdown of HIF1α but silencing of MCT4 could not reduce

the HIF1α expression. Right panel showed that the transfection with vector of HIF1α induced

the MCT4 expression.

C. Angiogenesis-related factors including VEGF and Angiopoietin 2 were also decreased by

siHIF1α or siMCT4 in PDCs with ELISA assay. Left two graphs showed that VEGF and

ANGPT2 were significantly decreased by siRNA of HIF1α and MCT4. Right two graphs

showed that the transfection with vector of HIF1α increased VEGF and ANGPT2.




34

*P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 6: Relapse-free survival according to MCT4 expression by IHC




Published OnlineFirst February 26, 2018.Mol Cancer Ther Hee Kyung Kim, InKyoung Lee, Heejin Bang, et al. Colorectal Cancer with Peritoneal CarcinomatosisMCT4 Expression is a Potential Therapeutic Target in

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