Itraconazole-induced inhibition on human esophageal cancer cell … · 2018-03-28 · AMP-activated...

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Itraconazole-induced inhibition on human

esophageal cancer cell growth requires AMPK

activation

Min-Bin Chen 1#, Yuan-Yuan Liu 2#, Zhao-yu Xing 3#, Zhi-qing Zhang 4, Qin Jiang 5*, Pei-Hua

Lu 6* and Cong Cao 4, 5, 7*

1 Department of Radiotherapy & Oncology, Kunshan First People’s Hospital Affiliated to

Jiangsu University, Kunshan, China. 2 Department of Laboratory Center, Kunshan First People’s Hospital Affiliated to Jiangsu

University, Kunshan, China. 3 The Department of Urology, the Third Affiliated Hospital of Soochow University,

Changzhou, China. 4 Institute of Neuroscience, Soochow University, Suzhou, China. 5 The Affiliated Eye Hospital of Nanjing Medical University, Nanjing, China 6 Department of Medical Oncology, Wuxi People’s Hospital Affiliated to Nanjing Medical

University, Wuxi, China. 7 North District, The Municipal Hospital of Suzhou, Suzhou, China

# Co-first authors.

Correspondence to:

* Prof. Qin Jiang M.D. Ph.D., the Affiliated Eye Hospital, Nanjing Medical University. 138

Han-zhong Road, Nanjing, 210029, China. Tel/Fax:+86-025-86677699. E-mail:

[email protected].

* Prof. Pei-Hua Lu, M.D. Ph.D., Department of Medical Oncology, Wuxi People’s Hospital

Affiliated to Nanjing Medical University, No. 299, Qingyang Road, Wuxi, 214023, China

Tel/Fax: +86-510-85350070. Email: [email protected].

* Prof. Cong Cao, M.D. Ph.D., Institute of Neuroscience, Soochow University, 199 Ren’ai

Road, SIP, Suzhou, 215021, China. Tel/Fax: +86-512-65883602. E-mail:

[email protected]

Running head. Itraconazole activates AMPK to inhibit cancer cells.

The authors declare no potential conflicts of interest.

Research. on November 17, 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 March 28, 2018; DOI: 10.1158/1535-7163.MCT-17-1094

http://mct.aacrjournals.org/

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Abstract.

We here evaluated the anti-esophageal cancer cell activity by the antifungal drug

itraconazole. Our results show that μg/mL concentrations of itraconazole potently inhibited

survival and proliferation of established (TE-1 and Eca-109) and primary human esophageal

cancer cells. Itraconazole activated AMP-activated protein kinase (AMPK) signaling, which

was required for subsequent esophageal cancer cell death. Pharmacological AMPK

inhibition, AMPKα1 shRNA or dominant negative mutation (T172A) almost completely

abolished itraconazole-induced cytotoxicity against esophageal cancer cells. Significantly,

itraconazole induced AMPK-dependent autophagic cell death (but not apoptosis) in

esophageal cancer cells. Further, AMPK activation by itraconazole induced multiple receptor

tyrosine kinases (RTKs: EGFR, PDGFRα and PDGFRβ) lysosomal translocation and

degradation to inhibit downstream Akt activation. In vivo, itraconazole oral gavage potently

inhibited Eca-109 tumor growth in severe combined immunodeficient (SCID) mice. It was yet

ineffective against AMPKα1 shRNA-expressing Eca-109 tumors. The in vivo growth of the

primary human esophageal cancer cells was also significantly inhibited by itraconazole

administration. AMPK activation, RTKs degradation and Akt inhibition were observed in

itraconazole-treated tumors. Together, itraconazole inhibits esophageal cancer cell growth

via activating AMPK signaling.

Keywords. Esophageal cancer; Itraconazole; AMPK signaling; Autophagy; Receptor

tyrosine kinase (RTK).

Introduction

Esophageal cancer is one important cause of cancer-related human mortalities (1).

Treatment of this devastating disease in the past decades has achieved significant

achievements (1). The application of conventional cytotoxic drugs and recently-developed

molecular-targeted agents are however discouraging against cancer cells with pre-existing

and/or acquired resistance (1). Therefore, there is an urgent need to explore novel, more

efficient, but less toxic anti-esophageal cancer agents (1).

Itraconazole blocks the synthesis of ergosterol in the fungal cell membrane, and it

functions as a systematic antifungal drug (2). Studies have tested itraconazole as a novel

anti-cancer drug (2-5). Preclinical studies have demonstrated its superior anti-cancer activity

against a number of different cancer cells (3-5). It is currently under phase II clinical trials of

non-small-cell lung cancer, basal cell carcinoma, and prostate cancer (5-7). The potential

effect of this antifungal drug in human esophageal cancer cells has not been studied. The

underlying signaling mechanism of itraconazole-mediated anti-cancer activity is still largely

unclear (5).

AMP-activated protein kinase (AMPK) activation maintains cellular energy homeostasis

under many stress conditions (8, 9). It is also critical in controlling cell survival and death




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(see review (8-10)). Our group and others have been focusing on the anti-cancer ability by

AMPK. AMPK activation could promote cancer cell death via regulating its downstream

signaling effectors, including p53 activation, mammalian target of rapamycin (mTOR)

complex 1 (mTORC1) inhibition as well as autophagy induction and oncogenic protein

degradation (11). Here, we show that itraconazole activates AMPK signaling to promote

esophageal cancer cell death.

Materials and methods

Ethics approval and consent to participate. The animal procedures in this study

were approved by Soochow University Ethics Review Board and IACUC. All studies

requiring human tissue samples were conducted according to the principles of Declaration of

Helsinki, and protocols were approved by the Soochow University Ethics Review Board.

Each of the participant provided written-informed consent.

Chemicals, reagents and antibodies. Itraconazole,

5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, catalog no. A9978) (12),

ammonium chloride (NH4Cl) and 3-methyaldenine (3-MA) were purchased from Sigma

(Shanghai, St. Louis, MO). Cell permeable short-chain C6 ceramide was described

previously (13). The applied caspase inhibitors, Z-DEVD-fmk, Z-LEHD-fmk and Z-VAD-fmk

were purchased from Merck-Sigma (Catalog nos. C0605, C1355, V116). The molecular

structures can be found from the supplier’s website (https://www.sigmaaldrich.com/) and

from https://pubchem.ncbi.nlm.nih.gov (SID: 329774802, 329774878 and 347914303). All

the antibodies were obtained from Cell Signaling Technology (Beverly, MA). Necrostatin-1

and ferrostatin-1 were purchased from Sigma.

Culture of established cell lines. The human esophageal cancer lines, Eca-109 and

TE-1, as well as the human esophageal epithelial cell (HEEC) line (14), were purchased

from the Cell Bank of Shanghai Institute of Biological Science (Shanghai, China, 08-2015).

Cells were maintained in the DMEM plus 10% FBS (Sigma) medium. All cell lines were

routinely subjected to mycoplasma and microbial contamination examination. STR profiling,

population doubling time, colony forming efficiency, and morphology were checked every

three months to confirm the genotype.

Primary culture of human esophageal cancer cells. One patient (47 year old, female)

with primary esophageal cancer (Stage-III) at Wuxi People’s Hospital of Nanjing Medical

University (Wuxi, China) was enrolled in the study. Written-informed consent was obtained

from the patient, who received no therapy before surgery. As described early (15), the

surgery-dissected esophageal cancer tissue specimen was washed and minced. Cancer

tissues were then mechanically dissociated and filtered through a 70 µm strainer. Single-cell

suspensions of primary cancer cells were achieved by incubation cells in collagenase I

(Sigma)-containing DMEM. Afterwards, individual cells were pelleted and rinsed, which were

re-suspended in the cell culture medium as described (15).




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Methyl thiazol tetrazolium (MTT) assay of cell viability. Cell viability was examined

by the routine MTT (Sigma) assay as described (15-18).

BrdU incorporation assay of cell proliferation. Cell proliferation was tested by the

incorporation of 5-bromo-2’-deoxyuridine (BrdU) assay via an ELISA format, the detailed

protocol was described previously (19).

Colony formation assay. Cells with applied treatment were suspended in 1 mL of

DMEM with 0.5% agar (Sigma). The suspension was then added on the pre-solidified cell

culture dish. After 10-day incubation, the remaining colonies were stained and counted.

Trypan blue staining assay of cell death. As described (15), the “dead” cell

percentage was determined via counting cells in an automatic hemocytometer with trypan

blue dye, which stains dead cells.

Apoptosis assays, including Histone-DNA ELISA assay, Annexin V

fluorescence-activated cell sorting (FACS) assay and TUNEL assay were described in detail

in our studies elsewhere (16-18).

Western blot assay. Western blot assay was described in detail in our previous reports

(15-18). Blot intensity was quantified via the ImageJ software (NIH). Note that the exact

same set of lysate samples were run in sister gels to examine different proteins, and the blot

was stripped and re-probed. For each lane, the exact amount (30 μg) of total cell/tissue

lysates were loaded. The intensity of each band was quantified via Image J software (NIH).

LC3B immunochemistry. After applied treatment, Eca-109 cells were fixed, washed

and blocked. The cells were then incubated with the primary antibody (anti-LC3B-Alexa

Fluor 555 Conjugate, Cell Signaling Tech, 1:25). Afterwards, LC3B fluorescence puncta was

visualized under the Leica microscope. The percentage LC3B puncta positive cells (green

fluorescence) was recorded. For each treatment, total 100 cells (DAPI stained) in 5 random

views were counted (20).

Subcellular fractionation. Isolation of lysosomes: Following the treatment, 20 millions

cells per treatment were re-suspended in 250 mM sucrose/10 mM Tris/HCl buffer solution,

which were then disrupted by sonication with two 15s pulses (Sonics and Materials VCX 600

Watt, Danbury, CT) (21). The disrupted cells were then subjected to rotation (1,200 g for 15

min) to remove nuclei and cell debris, and the supernatants were submitted to centrifugation

at 30,000 g for 30 min to achieve the enriched lysosome fraction. Lysosome-enriched

protein LAMP1 (Lysosome-associated membrane protein 1) was tested in the fraction.

Isolation of cell plasma membrane was described exactly in detail in other studies (22). Na,

K-ATPase was tested as a plasma membrane marker protein.

Short hairpin RNA (shRNA) knockdown. As described (13), lentiviral AMPKα1




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shRNA (Santa Cruz, sc-29673-V, 10 μL/mL per well) was added to cultured esophageal

cancer cells for 24 hours. Afterwards, puromycin (2.0 μg/mL, Sigma) was added to the

culture medium for 2 passages. shRNA knockdown of EGFR, PDGFRα and PDGFRβ was

performed similarly.

AMPKα1 mutation. The dominant-negative (dn) mutant of AMPKα1

(dn-AMPKα1,T172A, Flag-tagged) construct, the constrictively-active AMPKα1 (ca-AMPKα1,

T172D, Flag-tagged) and the empty vector were described previously (13). The construct

(0.25 μg/mL per well) was transfected to cultured esophageal cancer cells using the

Lipofectamine 2000 reagent (13), and stable cells were selected for 8 days. Transfection

efficiency was determined again by Western blot assay.

Tumor xenograft assay. Briefly, Eca-109 cells at logarithmic growing phase (5×106

cells per mouse), expressing either scramble control shRNA or AMPKα1 shRNA, were

inoculated (via s.c. injection) to the female severe combined immuno-deficient (SCID) mice

(4-5 week old, 17-18 g weight). Within 3-4 weeks, the tumors reached the average volume of

150 mm3, the mice were treated with/without itraconazole through oral gavage. Tumor

volumes were recorded weekly, calculated via the following formula: π/6 × larger diameter ×

(smaller diameter)2. Estimated daily tumor growth (in mm3 per day) was also calculated as

described (19). The establishment of xenograft tumors using the primary human cancer cells

was performed similarly.

Statistical analysis. Data were presented as mean ± standard deviation (SD).

Statistics were analyzed by one-way ANOVA followed by the Scheffe' and Tukey Test using

SPSS software. Significance was chosen as P < 0.05. To determine significance between

only 2 treatment groups, a 2-tailed unpaired t test was used (Excel 2007 for Windows).

Results

Itraconazole is cytotoxic and anti-proliferative to human esophageal cancer cells

First, Eca-109 esophageal cancer cells were treated with gradually-increasing

concentrations of itraconazole. The MTT viability assay results in Figure 1A demonstrate

that itraconazole dose-dependently inhibited Eca-109 cell survival. Itraconazole also

displayed a time-dependent response in inhibiting Eca-109 cells, it would require at least 24

to 48 hours for itraconazole (0.3-3.0 μg/mL) to exert a significant anti-survival activity (Figure

1A). Clonogenicity assay results in Figure 1B show that treatment with itraconazole (0.3-3.0

μg/mL) dramatically decreased the number of viable Eca-109 colonies. Meanwhile, with the

itraconazole (0.3-3.0 μg/mL) treatment, the number of trypan blue-stained cells (“dead” cells)

was significantly increased (Figure 1C).

The potential effect of itraconazole on other esophageal cancer cells was also studied.

MTT assay results in Figure 1D show that itraconazole (0.3-3.0 μg/mL) was cytotoxic to

TE-1 cells (another established esophageal cancer cell line) and patient-derived primary




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esophageal cancer cells. The same itraconazole treatment failed to inhibit survival of HEEC

esophageal epithelial cells (14) (Figure 1D). These results demonstrate that itraconazole is

only cytotoxic to human esophageal cancer cells.

Next, we tested the potential effect of itraconazole on esophageal cancer cell

proliferation. BrdU incorporation assay results in Figure 1E show that itraconazole (0.3-3.0

μg/mL, 24 hours) significantly inhibited Eca-109 cell proliferation (BrdU ELISA OD reduction).

In TE-1 cells and primary esophageal cancer cells, BrdU incorporation was also inhibited by

itraconazole (Figure 1F). However, proliferation of HEEC esophageal epithelial cells was

unchanged (Figure 1F). These results imply that itraconazole inhibits esophageal cancer cell

proliferation.

Itraconazole fails to induce apoptosis in esophageal cancer cells

The potential effect of itraconazole on cell apoptosis was tested. Several different

apoptosis assays were performed, including the Annexin V FACS assay (Figure 2A),

Histone DNA apoptosis ELISA assay (Figure 2B) and TUNEL staining assay (Figure 2C).

Results from all the assays show that itraconazole (48 hours treatment) failed to induce

significant apoptosis in Eca-109 cells (Figure 2A-C). On the other hand, short-chain C6

ceramide (13), tested as a positive control, induced significant apoptosis activation in

Eca-109 cells (Figure 2A-C).

Next, the caspase-3 specific inhibitor Z-DEVD-fmk, the caspase-8 specific inhibitor

Z-IETD-fmk and the pan caspase inhibitor Z-VAD-fmk were applied. Results show that the

caspase inhibitors failed to inhibit itraconazole (3.0 μg/mL)-induced Eca-109 cell viability

reduction (Figure 2D) and cell death (Figure 2E). Similarly, in TE-1 cells and primary

esophageal cancer cells, treatment with itraconazole (3.0 μg/mL) failed to induce apparent

cell apoptosis (TUNEL staining assay, Figure 2F). No apoptosis was induced in itraconazole

(3.0 μg/mL)-treated HEEC epithelial cells (Figure 2F).

Necrostatin-1 is a specific inhibitor of necroptosis, acting by directly blocking

receptor-interacting serine/threonine-protein kinase 1/3 (RIP1) (23). Ferrostatin-1 is the

known ferroptosis inhibitor (24). We show that pre-treatment with necrostatin-1 or

ferrostatin-1 failed to inhibit itraconazole-induced cytotoxicity against Eca-109 cells (Figure

S1A and B). The results suggest that necroptosis and ferroptosis are unlikely involved in

itraconazole-induced cancer cell death.

AMPK activation mediates itraconazole-induced esophageal cancer autophagic cell

death

The tumor-suppressing function of AMPK has been well-established. AMPK signaling in

itraconazole-treated cells was then tested. Western blot results in Figure 3A demonstrate

that itraconazole (3.0 μg/mL) treatment in Eca-109 cells induced increased phosphorylation

(“p-“) of AMPKα1 (Thr-172) and its major downstream substrate acetyl-CoA carboxylase




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(ACC, Ser-79), indicating AMPK signaling activation. AMPK activation lasted for at least 12

hours (Figure 3A). In order to study the role of AMPK activation in itraconazole-induced

activity, the AMPK inhibitor Compound C (“CC”) was applied. Compound C completely

blocked itraconazole-induced AMPK activation (AMPKα1-ACC phosphorylation) (Figure 3B),

which also largely attenuated itraconazole-induced Eca-109 cell viability reduction (Figure

3C).

Next, the lentiviral shRNA strategy was applied to stably knockdown AMPKα1 in

Eca-109 cells. AMPKα1 protein expression was almost completely silenced in AMPKα1

shRNA-expressing stable cells (Figure 3B). Consequently, itraconazole-induced AMPK

activation was blocked (Figure 3B). Itraconazole-induced cytotoxicity in Eca-109 cells was

also largely ameliorated by AMPKα1-shRNA (Figure 3C). In TE-1 cells, treatment with

itraconazole also induced AMPK activation (AMPKα/ACC phosphorylation, Figure S2A),

which was completely blocked by AMPKα1 shRNA (Figure S2B). Significantly, AMPKα1

shRNA almost nullified itraconazole-induced TE-1 cell viability reduction (Figure S2C) and

cell death (Figure S2D). The results further support the requirement of AMPK activation in

itraconazole-mediated esophageal cancer cell death.

The dominant negative mutant AMPKα1 (“dn-AMPKα1”, T172A, Flag-tagged) (13, 19)

was transfected to Eca-109 cells. As shown in Figure 3D, dn-AMPKα1 almost abolished

itraconazole-induced AMPK activation, and also largely protected cells from itraconazole

(Figure 3E). Conversely, the constitutively-active AMPKα1 (“ca-AMPKα1”, T172D) (25) was

introduced to Eca-109 cells. Results in Figure 3F confirmed expression of the exogenous

ca-AMPKα1 (Flag-tagged) in Eca-109 cells. AMPKα1-ACC phosphorylation was significantly

elevated in the ca-AMPKα1-expressing cells (Figure 3F). As compared to the vector-control

cells, expression of ca-AMPKα1 reduced cell viability in Eca-109 cells (Figure 3G).

AMPK activation was able to trigger autophagic cell death, via phosphorylating and

activating Ulk1 (the direct mechanism) (26) or inhibiting mTORC1 (the indirect mechanism)

(26). Results in Figure 3H show that itraconazole (3.0 μg/mL) induced Ulk1 phosphorylation

(at Ser-317 (26)), light chain 3B-I (LC3B-I) to LC3B-II convention, Beclin-1 and ATG-5

upregulation as well as p62 downregulation in Eca-109 cells. Further, the percentage of

Eca-109 cells with LC3B-GFP puncta was significantly increased following itraconazole

treatment (Figure 3I), indicating autophagy activation. Significantly, AMPKα1 shRNA or

dominant negative mutation almost completely blocked itraconazole-induced LC3B-GFP

puncta formation (Figure 3I). The results suggest that itraconazole-provoked autophagy is

dependent on AMPK. To study the role of autophagy in itraconazole-induced cytotoxicity, two

known autophagy inhibitors were applied: ammonium chloride (NH4Cl) and 3-methyaldenine

(3-MA). Both largely attenuated itraconazole-induced Eca-109 cell death (Figure 3J).

Collectively, the results suggest that itraconazole induces AMPK-dependent autophagic

death of esophageal cancer cells.

Itraconazole in-activates Akt-mTORC1 signaling and induces multiple receptor

tyrosine kinases (RTKs) degradation




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mTORC1 is important for esophageal cancer cell progression (27, 28). As shown in

Figure 4A, treatment with itraconazole (3.0 μg/mL, 12 hours) in Eca-109 cells inhibited

phosphorylation of the mTORC1 substrates 4E-binding protein 1 (4E-BP1, Ser-65) and S6

(Ser-235/236), suggesting mTORC1 inhibition (29, 30). Compound C or AMPKα1 shRNA

(see Figure 3) almost reversed itraconazole-induced mTORC1 inhibition (Figure 4A).

Conversely, AICAR or ca-AMPKα1 inhibited mTORC1 activation (p-4E-BP1/p-S6) in

Eca-109 cells (Figure 4B). Akt activation, tested by p-Akt at both Ser-473 and Thr-308, was

also largely inhibited by itraconazole (Figure 4C), which was almost completely blocked by

Compound C or AMPKα1 shRNA (Figure 4C). Further, AICAR or ca-AMPKα1 similarly

inhibited Akt activation in Eca-109 cells (Figure 4D). The results suggest that AMPK

activation by itraconazole also inhibits Akt-mTORC1 signaling.

Co-current activation of multiple receptor tyrosine kinases (RTKs) is responsible for

downstream Akt-mTORC1 activation (31, 32). Expression of several RTKs in

itraconazole-treated cells were then tested. As demonstrated, treatment with itraconazole in

Eca-109 cells induced downregulation of several RTKs, including epidermal growth factor

receptor (EGFR), platelet-derived growth factor receptor α (PDGFRα) and PDGFRβ (Figure

4E). Notably, such effect by itraconazole was also dependent on AMPK, and was abolished

by Compound C or AMPKα1 shRNA (Figure 4E). AICAR or ca-AMPKα1 similarly induced

downregulation of RTKs (EGFR, PDGFRα and PDGFRβ) (Figure 4F). In the patient-derived

primary esophageal cancer cells, itraconazole treatment also induced AMPK activation

(Figure S2E), RTKs degradation (Figure S2F) and Akt inhibition (Figure S2G). Notably,

RTKs expression and pAkt were quite low in the non-cancerous HEEC cells (Figure S2F-G),

which could explain why the epithelial cells were not killed by itraconazole (Figure 1D), even

though AMPK was activated in the cells (Figure S2E). The results indicate that AMPK

activation by itraconazole induces degradation of several RTKs, causing downstream Akt

inhibition.

AMPK activation by itraconazole dictates RTKs lysosomal enrichment and

degradation

Next, the underlying mechanism of itraconazole-induced RTKs degradation was

assessed. As shown in Figure 5A, itraconazole (3.0 μg/mL, 1 hour) treatment in Eca-109

cells induced RTKs (EGFR, PDGFRα and PDGFRβ) translocation from plasma membrane

to lysosome. The plasma membrane-located RTKs were decreased, and the

lysosome-enriched RTKs were increased after itraconazole treatment (Figure 5A).

Na-K-ATPase was tested as the plasma membrane marker protein, and LAMP1

(Lysosome-associated membrane protein 1) is the lysosomal maker (Figure 5A). Such

activity by itraconazole was almost abolished by AMPKα1 shRNA (Figure 5A). ca-AMPKα1

also promoted RTKs translocation to lysosome (Figure 5B). The results suggest that

itraconazole dictates RTKs (EGFR, PDGFRα and PDGFRβ) lysosomal translocation in a

AMPK-dependent manner.




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NH4Cl increases the intra-lysosomal pH and prevents autophagic protein degradations

(33). Further, 3-MA is known to block conversion of LC3B-I to LC3B-II, and to inhibit

autophagosome formation (33). Remarkably, the autophagy/lysosomal inhibitors almost

completely blocked RTKs degradation in Eca-109 cells (Figure 5C). The results suggest that

itraconazole promotes RTKs translocation to lysosome for possible degradation.

Considering that the two autophagy inhibitors also largely attenuated

itraconazole-induced cytotoxicity (Figure 3), we speculate that RTKs degradation might be

the key mechanism of itraconazole-induced cytotoxicity. Therefore, shRNA strategy was

applied to knockdown the RTKs (EGFR, PDGFRα and PDGFRβ) in Eca-109 cells. Western

blot assay results (Figure 5D) confirmed knockdown of all three RTKs by the designated

shRNAs, and activation of Akt (p-Akt at Thr-308) was also largely inhibited (Figure 5D).

Compared to the control cells, shRNA knockdown of all three RTKs inhibited Eca-109 cell

survival (Figure 5E). Significantly, itraconazole was unable to further exert cytotoxicity to the

cells with depleted-RTKs (Figure 5E). The results imply that degradation of RTKs (EGFR,

PDGFRα and PDGFRβ) by itraconazole could be the main cause of Akt inhibition and

subsequent Eca-109 cell death.

Itraconazole oral administration inhibits esophageal cancer cell growth in mice

The potential in vivo anti-esophageal cancer activity by itraconazole was also tested.

Eca-109 cells were inoculated (s.c. injection) to SCID mice, and xenograft tumors were

established. Tumor growth curve results show that daily administration of itraconazole (50

mg/kg, gavage) efficiently inhibited Eca-109 tumor growth (Figure 6A). Estimated daily

tumor growth (in mm3 per day) was also significantly lower in itraconazole-treated mice

(Figure 6B). The Eca-109 tumors were lighter after itraconazole treatment (Figure 6C).

Significantly, same itraconazole (50 mg/kg, daily, gavage) treatment was almost in-effective

against tumors of AMPKα1-shRNA-expressing Eca-109 cells (Figure 6A-C). Thus,

itraconazole failed to inhibit Eca-109 tumor growth when AMPKα1 was silenced. Notably,

mice body weight was not different between the groups (Figure 6D). We also failed to

observe any apparent toxicities in the experimental mice. Thus, the mice were well-tolerated

to the itraconazole administration, as reported by other studies (3, 4).

The signalings were also tested in itraconazole-treated tumor tissues. Western blot

assay was employed. Results show that itraconazole administration in vivo activated AMPK

signaling in Eca-109 tumor tissues, and p-AMPKα1 and p-ACC were significantly increased

at Day-1 after the itraconazole administration (Figure 6E). Further, RTKs (EGFR, PDGFRα

and PDGFRβ) were downregulated in tumor tissues with itraconazole treatment (Figure 6F).

Remarkably, AMPKα1-shRNA-expressing tumors show depleted AMPKα1 (Figure 6E and F).

Itraconazole-induced AMPK activation (p-AMPKα1/p-ACC) and RTKs degradation were

almost completely reversed in AMPKα1-shRNA-expressing tumors (Figure 6E and F). The in

vivo growth of sh-AMPKα1-expresssing Eca-109 tumors was not significantly different from

the control tumors with shRNA-C (Figure S3A-C). The mice body weight was indifferent

between the two groups as well (Figure S3D). One possibility is that the basal AMPK




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activation (AMPK/ACC phosphorylation) was quite low in the control Eca-109 tumors (Figure

S3E). The results indicate that AMPK activation likely mediates itraconazole-induced

anti-tumor activity in vivo.

In order to further confirm the anti-esophageal cancer cell activity in vivo, we next

inoculated primary human esophageal cancer cells to SCID mice. Within three weeks,

tumors of primary cancer cells (“primary tumors”) were established. As shown in Figure 6G

and H, growth of the primary tumors in SCID mice was largely inhibited with the itraconazole

treatment (50 mg/kg, oral administration, daily). Tumor weights (at the terminal) were also

significantly lower after itraconazole administration (Figure 6I). The mice body weight was

not affected (Figure 6J). Therefore, itraconazole efficiently inhibited in vivo growth of the

primary human cancer cells.

Discussion

The results of this study suggest that AMPK activation is the key signaling mechanism

responsible for itraconazole-mediated anti-esophageal cancer cell activity. Pharmacological

AMPK inhibition, AMPKα1 shRNA or dominant negative mutation (T172A) almost

completely nullified itraconazole-induced cytotoxicity in esophageal cancer cells. Conversely,

AMPK activator AICAR or exogenous expression of ca-AMPKα1 mimicked itraconazole

actions. Further mechanistic studies show that itraconazole provoked AMPK-dependent

autophagic cell death (but not apoptosis) in esophageal cancer cells. In vivo, itraconazole

gavage potently inhibited esophageal cancer cell growth in SCID mice. It was however

ineffective against AMPKα1-shRNA tumors.

There are several mechanisms responsible for AMPK-mediated cancer suppressing

actions. For example, activated AMPK directly (by phosphorylating Raptor (34)) or indirectly

(by phosphorylating TSC2 (35)) inhibits mTORC1 signaling (26). Meanwhile, AMPK

activation could provoke pro-apoptotic p53 (36) and JNK (37) cascades. AMPK could also

induce autophagic cancer cell death, directly (vs. phosphorylating Ulk1 (38)) or indirectly (via

inhibiting mTORC1 (26)). In this study, mTORC1 inhibition and autophagy induction were

observed in itraconazole-treated cancer cells. Remarkably, we here propose another

AMPK-dependent mechanism to inhibit cancer cells: by inducing degradation of multiple

RTKs (EGFR, PDGFRα and PDGFRβ).

Concurrent activation of multiple RTKs in human esophageal cancer cells leads to

sustained activation of oncogenic signaling cascades (i.e. PI3K-Akt-mTOR) to promote

cancer progression (39). Tyrosine kinase inhibitor (TKI) against single RTKs could only

result in part or even no inhibition of the downstream cascades, it is therefore less effective

against human cancer cells. The novel finding of this study is that AMPK activation by

itraconazole induces lysosomal enrichment and subsequent degradation of multiple RTKs

(EGFR, PDGFRα and PDGFRβ), causing downstream Akt inhibition and esophageal cancer

cell autophagic death. Such activity by itraconazole was almost reversed with AMPK

blockage, silence or mutation. Notably, the anti-cancer cell activity by itraconazole was




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compromised in Eca-109 cells with depleted-RTKs (EGFR, PDGFRα and PDGFRβ).

Additionally, expression of RTKs as well as downstream Akt activation were quite low in the

HEEC epithelial cells (Figure S2), this might explain why these cells were not killed by

itraconazole (Figure 1D). Therefore, itraconazole-induced AMPK-dependent lysosomal

degradation of RTKs (EGFR, PDGFRα and PDGFRβ) could be the primary reason of its

superior and specific anti-cancer cell activity.

The clinical studies have demonstrated that patients with prostate cancer, lung cancer,

and basal cell carcinoma shall benefit from the itraconazole treatment (5-7). Further studies

also proposed the superior anti-cancer activity of itraconazole again leukemia, ovarian,

breast, and pancreatic cancers (5-7). Itraconazole has safe pharmacokinetics as well as a

defined toxicity profile (5-7). Given these information, it would be certainly interesting to

further test itraconazole as a promising anti-esophageal cancer agent in clinical settings.

Declarations

Fundings. This work is supported by the National Natural Science Foundation

(81771457/81302195/31371139/81571282 to C. Cao, 81472786/81773192 to M. B. Chen,

81472305 to P. H. Lu, 81502162 to Z.Q. Zhang), the Six Talents Peak Project of Jiangsu

Province (2014-WSN-012 to M. B. Chen, 2014-WSN-061 to P. H. Lu), Kunshan Science and

Technology Program (KS1418 to M. B. Chen), and by grants from Natural Science

Foundation of Jiangsu Province (BK20130301/BK20170060 to C. Cao, and BK20171248 to

M. B. Chen). The funders had no role in study design, data collection and analysis, decision

to publish, or preparation of the manuscript.

Competing interests. The authors declare that they have no competing interests.

Consent for publication. Not applicable

Availability of data and materials. All data generated or analyzed during this study are

included in this published article [and its supplementary information files].

Acknowledgements. Not applicable.

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Figure legends

Figure 1. Itraconazole is cytotoxic and anti-proliferative to human esophageal cancer

cells. Established esophageal cancer cells (Eca-109 and TE-1 lines), primary human

esophageal cancer cells (“Primary cancer cells”) or HEEC esophageal epithelial cells were

treated with/without applied concentrations (0.1-3.0 μg/mL, 0.14-4.25 μM) of itraconazole

(“Itra”), cells were then cultured in conditional medium for designated time, and were

subjected to MTT assay (A and D), clonogenicity assay (B) and trypan blue staining assay

(C). Cell proliferation was evaluated by the BrdU ELISA assay (E and F). n=5 for each assay.

“Ctrl” stands for untreated control group (Same for all figures). * P < 0.05 vs. group “Ctrl”.




14

Experiments in this and all following figures of in vitro experiments were repeated three

times, with similar results obtained.

Figure 2. Itraconazole fails to induce apoptosis in esophageal cancer cells. Listed cells

were treated with/without applied concentrations (0.3-3.0 μg/mL) of itraconazole (“Itra”) or

C6 ceramide (“C6 Cer”), cells were then cultured in conditional medium for designated time,

and were tested by designated apoptosis assays (A-C and F, data were quantified). Eca-109

cells were pre-treated for 1 hour with 50 μM of Z-DEVD-fmk (“DEVD”), Z-IETD-fmk (“IETD”)

or Z-VAD-fmk (“VAD”), followed by itraconazole (“Itra”, 3.0 μg/mL) treatment for 72 hours,

cell viability (MTT assay, D) and cell death (trypan blue staining assay, E) were tested. * P <

0.05 vs. group “Ctrl”.

Figure 3. AMPK activation mediates itraconazole-induced esophageal cancer

autophagic cell death. Eca-109 cells were treated with itraconazole (“Itra”, 3.0 μg/mL) for

applied time, expression of listed proteins were shown (A and H). Puromycin-selected stable

Eca-109 cells, with scramble control shRNA (“shRNA-Ctrl”) or AMPKα1 shRNA

(“shRNA-AMPKα1”), were treated with itraconazole, or plus AMPK inhibitor Compound C

(“CC”, 10 μM); Expression of listed proteins were shown (B); Cell viability (C) was tested.

Eca-109 cells with dominant negative mutant AMPKα1 (T172A, “dn-AMPKα1”, Flag-tagged),

constitutively-active AMPKα1 (T172D, “ca-AMPKα1”, Flag-tagged) or empty vector

(“Vector”), were treated with/without itraconazole (3.0 μg/mL); Expression of listed proteins

were shown (D and F); Cell viability was also tested (E and G). Stable Eca-109 cells, with

shRNA-Ctrl, shRNA-AMPKα or dn-AMPKα1, were treated with itraconazole (24 hours),

LC3B-GFP positive cells were counted (I); Eca-109 cells, pretreated with 3-methyladenine

(“3-MA”, 2.5 mM) or ammonium chloride (“NH4Cl”, 1 mM) for 1 hour, were treated

with/without itraconazole (3.0 μg/mL) for 72 hours, cell viability was tested (J). Note that the

exact same set of lysate samples were run in sister gels to examine different proteins, and

the blot was stripped and re-probed (Same for all Figures). “DMSO” stands for 0.1% of

DMSO. “C” stands for parental Eca-109 cells (D, E and G). * P < 0.05 vs. group “Ctrl”. # P <

0.05 vs. group “shRNA-Ctrl” or “Vector” (E and G). & P < 0.05 (I and J).

Figure 4. Itraconazole in-activates Akt-mTORC1 signaling and induces multiple

receptor tyrosine kinases (RTKs) degradation. Puromycin-selected stable Eca-109 cells,

expressing scramble control shRNA (“shRNA-Ctrl”) or AMPKα1 shRNA (“shRNA-AMPKα1”),

were treated with itraconazole (3.0 μg/mL) or plus Compound C (“CC”, 10 μM) for applied

time; Expression of listed proteins were tested (A, C and E); Eca-109 cells with

constitutively-active AMPKα1 (T172D, “ca-AMPKα1”) or empty vector (“Vector”,

p-Super-puro), were treated with/without AICAR (1 mM, 12 hours), expression of listed

proteins were shown (B, D and F); Expression of indicated proteins was quantified.

Figure 5. AMPK activation by itraconazole dictates RTKs lysosomal enrichment and

degradation. Eca-109 cells, expressing scramble control shRNA (“shRNA-C”), AMPKα1

shRNA (“sh-AMPKα1”) or constitutively-active AMPKα1 (T172D, “ca-AMPKα1”), were

treated with/without itraconazole (“Itra”, 3.0 μg/mL) for 1 hour; Cell plasma fraction and




15

lysosomal fraction were isolated, expression of listed proteins were shown (A and B);

Eca-109 cells, pretreated with 3-methyladenine (“3-MA”, 2.5 mM) or ammonium chloride

(“NH4Cl”, 1 mM) for 1 hour, were treated with/without itraconazole (“Itra”,3.0 μg/mL) for 24

hours, RTKs expression were tested (C). Expression of the listed RTKs in Eca-109 cells

infected with EGFR-shRNA plus PDGFRα/β-shRNA or scramble control shRNA (“sc-sh”)

were shown, p-Akt and regular Akt1 were also tested (D). Above cells were also treated with

itraconazole (“Itra”, 3.0 μg/mL) for 72 hours, cell viability (E) was tested. * P < 0.05.

Figure 6. Itraconazole-induced the anti-esophageal cancer cell activity in vivo. Exact

same amount of stable Eca-109 cells, expressing scramble control shRNA (“shRNA-C”) or

AMPKα1 shRNA (“sh-AMPKα1”) (A-F), as well as the primary human esophageal cancer

cells (G-J), were inoculated to the SCID mice. When the tumor volumes were around 150

mm3, mice were randomly assigned to the listed groups, with 10 mice per group.

Itraconazole (50 mg/kg, gavage, daily) or vehicle (“Veh”) administration was performed, the

tumor volume (A and G) and the mice body weight (D and J) were recorded every 7 days;

Daily tumor growth (in mm3 per day) was also calculated (B and H); At day-42, all animals

were subjected to surgery, tumors were separated and weighted (C and I). At experimental

Day-1, 16 hours after the itraconazole administration, one Eca-109 tumor per group was

isolated, expression of listed proteins in tumor lysates were shown (E and F). * P < 0.05 vs.

group “Veh” (A and G). # P < 0.05 vs. group “Itra+shRNA-C” (A).* P < 0.05 (B-C).




0

20

40

60

80

100

120

Ctrl 0.1 0.3 1.0 3.0

24h48h72h96h

** *

** *

** **

Itra (μg/mL)

MTT

OD

(% v

s. “

Ctr

l”)

0

20

40

60

80

100

120

Ctrl 0.1 0.3 1.0 3.0

TE-1Primary cancer cellsHEEC

Itra (μg/mL), 72 h

MTT

OD

(% v

s. “

Ctr

l”)

* **

*

**

*

Eca-109

0

10

20

30

40

50

Ctrl 0.1 0.3 1.0 3.0

*

**N

umbe

r of c

olon

ies

per d

ish

Itra (μg/mL), 10 days

Eca-109

0

10

20

30

Ctrl 0.1 0.3 1.0 3.0

*

* *

Tryp

an b

lue

(%)

Itra (μg/mL), 72 h

Eca-109

Figure 1.

A. B.

C. D.

0

20

40

60

80

100

120

Ctrl Itra Ctrl Itra Ctrl Itra (3.0 μg/mL), 24 h

TE-1Primary cancer cells HEEC

* *

Brd

U O

D (%

vs.

“C

trl”

)

E. F.

20

40

60

80

100

120

Ctrl 0.1 0.3 1.0 3.0Itra (μg/mL), 24 h

Brd

U O

D (%

vs.

“C

trl”

)

Eca-109

** *




0

0.3

0.6

0.9

Ctrl 0.1 0.3 1.0 3.0 C6 Cer

Apo

ptos

is E

LISA

OD

*

P > 0.05

Itra (μg/mL), 48 h 10 μg/mL

0

4

8

12

16

Ctrl 0.1 0.3 1.0 3.0 C6 Cer

P > 0.05

*


Ann

exin

V (%

)

0

5

10

15

20

25

TUN

EL (%

)

Ctrl 0.1 0.3 1.0 3.0 C6 Cer


*

Figure 2.

Eca-109 Eca-109 Eca-109A. B. C.

0

20

40

60

80

100

120 DMSODEVDIETDVAD

MTT

OD

(% v

s. “

Ctr

l”)

Ctrl Itra (3.0 μg/mL), 72 h

P > 0.05

Eca-109 Eca-109

0

10

20

30DMSODEVDIETDVAD

Tryp

an b

lue

(%)


P > 0.05

0

1

2

3

4

5

6

Ctrl Itra Ctrl Itra Ctrl Itra (3.0 μg/mL), 48 h

TUN

EL (%

)

TE-1

Primary cancer cells

HEEC

D. E. F.

P > 0.05




0

20

40

60

80

100

120DMSOshRNA-CtrlCCshRNA-AMPKα1

MTT

OD

(% v

s. “

Ctr

l”)

*

* *

#


Ctrl Itra Ctrl Itra, 3.0 μg/mL, 12hp-AMPKα1 T172

p-ACCS79

AMPKα1

ACC

DMSO CC DMSOshRNA-Ctrl shRNA-AMPKα1

Ctrl Itra

A. B. C.

*#

0

20

40

60

80

100

120CVectordn-AMPKα1

MTT

OD

(% v

s. “

Ctr

l”)

*#

* *


Figure 3

Vector

ca-A

MPKα1

0

20

40

60

80

100

120

CVec

tor

ca-A

MPKα1

#

MTT

OD

(% v

s. “

C”)

At day-4

p-AMPKα1 T172

p-ACCS79

AMPKα1

ACC

Akt1

ca-AMPKα1

Itra, 3.0 μg/mL, 12hD. E. F. G.

p-AMPKα1 T172

62kD-

p-ACCS79

280kD-

AMPKα1

ACC280kD-

62kD-

1h 3h 6h 12h

Itra, 3.0 μg/ml

Ctrl

Akt160kD-

Eca-109

C Vector

dn-A

MPKα1

p-AMPKα1 T172

p-ACCS79

AMPKα1

ACC

Akt1

dn-AMPKα1

62kD-

280kD-

280kD-

62kD-

60kD-

-LC3B-II

Beclin-1

β-actin

p-Ulk1

p62

55kD-

60kD-

14kD-

150kD-

45kD-

62kD-

Ulk1

-LC3B-I

ATG-5

150kD-

16kD-

Ctrl 12h 24h

Itra (3.0 μg/mL)

0

20

40

60

80

100

120

MTT

OD

(% v

s. “

Ctr

l”)

Itra (3.0 μg/mL), 72 hCtrl

DMSO3-MANH4Cl

*

&&

H. I. J.

0

10

20

30shRNA-Ctrlsh-AMPKα1 dn-AMPKα1

Itra (3.0 μg/mL), 24 hCtrl

LC3B

pun

cta

(%)

&&

*

62kD-

280kD-

280kD-

62kD-

62kD-

280kD-

280kD-

62kD-

60kD-

Akt160kD-




A. B.

C.

Figure 4

p-4E-BP1Ser65

S6

4E-BP1

pS6Ser235/236

20kD-

20kD-

32kD-

32kD-

1.00 0.14 1.09 0.92 0.99 0.86

1.00 0.12 0.93 0.97 1.31 1.12

Ctrl Itra Ctrl Itra, 3.0 μg/mL, 12hDMSO CC DMSOshRNA-Ctrl shRNA-AMPKα1

Ctrl Itra

1.00 0.01 0.89 0.85 0.77 1.06

1.00 0.01 0.93 1.07 1.15 0.89

p-Akt S473

Akt1

p-Akt T308

60kD-

60kD-

60kD-

Ctrl Itra Ctrl Itra, 3.0 μg/mL, 24hDMSO CC DMSOshRNA-Ctrl shRNA-AMPKα

Ctrl Itra

1.00 0.16 0.15

1.00 0.11 0.04

Ctrl

AIC

AR

ca-A

MPK

α1p-Akt S473

Akt1

p-Akt T308

D.

E. F.

p-4E-BP1

p-S6

S6

4E-BP1

Ctrl

AIC

AR

ca-A

MPK

α1

1.00 0.06

1.00 0.16 0.24

0.22

PDGFRα

EGFR

PDGFRβ

β-actin

Ctrl Itra Ctrl Itra, 12hDMSO CC DMSOshRNA-Ctrl shRNA-AMPKα1

Ctrl Itra

1.00 0.00 1.04 1.01 1.28 1.12

1.01 0.11 0.89 0.91 0.93 0.87

1.00 0.06 0.91 0.88 0.90 0.68

190kD-

190kD-

42kD-

170kD-EGFR

β-actin

Ctrl

AIC

AR

ca-A

MPK

α1

PDGFRβ

PDGFRα

1.00 0.26 0.02

1.00 0.11 0.07

1.00 0.04 0.10




175kD-

190kD-

190kD-

100kD-

110kD-

Ctrl+shRNA-C

Itra+shRNA-C

Itra+sh-AMPKα1

1.00 0.26 0.78

1.00 0.27 0.99

1.00 0.31 1.00

175kD-

190kD-

190kD-

100kD-

110kD-

PDGFRα

EGFR

PDGFRβ

LAMP1

Na,K-ATPase

PDGFRα

EGFR

PDGFRβ

LAMP1

Na,K-ATPase

175kD-

190kD-

190kD-

100kD-

110kD-

Plasma membrane Plasma membrane

1.00

1.00

1.00

PDGFRα

EGFR

PDGFRβ

LAMP1

Na,K-ATPase

0.19

0.14

0.07

Lysosome Lysosome

0

20

40

60

80

100

120

Ctrl Itra Ctrl Itra

P > 0.05

MTT

OD

(% v

s. “

Ctr

l” o

f “sc

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)

shRNA-C

*

sh-PDGFRα/β+sh-EGFR

A. B.

Figure 5

C. D. E.

EGFR

PDGFRα

PDGFRβ

Tubulin

Itra

+NH4C

l

+3-M

ACtrl

175kD-

190kD-

190kD-

55kD-

175kD-

190kD-

190kD-

60kD-

60kD-

PDGFRα

EGFR

PDGFRβ

Akt1

pAkt

shRNA-C sh-PDGFRα/β+sh-EGFR

Ctrl+shRNA-C

Itra+shRNA-C

Itra+sh-AMPKα1

Vectorca-AMPKα1

Vectorca-AMPKα1

175kD-

190kD-

190kD-

100kD-

110kD-




A. B. C.Figure 6

D.E. F.

0

5

10

15

20

25

30

35

Estim

ated

tum

or g

row

th

(mm

per

day

)3

* *

n=10

Veh+shRNA-C

Itra+shRNA-C

Itra+sh-AMPKα1

0

400

800

1200

Tum

or w

eigh

ts (m

g)

* *

n=10

Veh+shRNA-C

Itra+shRNA-C

Itra+sh-AMPKα1

15

17

19

21

23

D0 D7 D14 D21 D28 D35 D42

Ani

mal

wei

ght (

g)

n=10

Veh+shRNA-CItra+shRNA-CItra+sh-AMPKα1

0

300

600

900

Tum

or w

eigh

ts (m

g)

*

n=10

Veh Itra0

400

800

1200

D0 D7 D14 D21 D28 D35 D42

Tum

or v

olum

es (m

m )3 Veh

Itra

n=10

*

G. H. I. J.

0

5

10

15

20

25

Estim

ated

tum

or g

row

th

(mm

per

day

)

*

n=10

Veh Itra

30

400

800

1200

1600

D0 D7 D14 D21 D28 D35 D42

Veh+shRNA-CItra+shRNA-CItra+sh-AMPKα1

n=10

Tum

or v

olum

es (m

m )3

*

#

Eca-109 tumors

Primary tumors

16

18

20

22

24

D0 D7 D14 D21 D28 D35 D42

VehItra

n=10

Ani

mal

wei

ght (

g)

PDGFRα

EGFR

PDGFRβ

β-actin

Veh+shRNA-C

Itra+shRNA-C

Itra+sh-AMPKα1

175kD-

190kD-

190kD-

45kD-

D1

p-AMPKα1 T172

p-ACCS79

AMPKα

ACC

Tubulin

Veh+shRNA-C

Itra+shRNA-C

Itra+sh-AMPKα1

62kD-

280kD-

280kD-

62kD-

55kD-




Published OnlineFirst March 28, 2018.Mol Cancer Ther Min-Bin Chen, Yuan-Yuan Liu, Zhao-yu Xing, et al. cell growth requires AMPK activationItraconazole-induced inhibition on human esophageal cancer

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Itraconazole-induced inhibition on human esophageal cancer cell … · 2018-03-28 · AMP-activated...

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