0 hrs 48 hrs0246

Du145

AC

h (p

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6 cells

)

*

0 hrs 48 hrs0

1020304050

22Rv1

AC

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6 cells

)

*

CHAT VACHT CHAT VACHT0

10

20

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40

Normalized

Expression

0 hrs24 hrs48 hrs72 hrs

******

*****

* ** *

22Rv1 Du145

AA

• 1 in 9 men will develop prostate cancer (PCa) sometime throughout their lifetime.

• Chemotherapy-resistant PCa remains a leading cause of cancer-related deaths in advanced PCa patients.

• Acetylcholine (ACh) and the muscarinic cholinergic receptor 1 (CHRM1) signaling has been shown to play a role in PCa growth but little is known on its role in chemotherapy resistance1,2.

• The ERK/CREB cascade is known to play a role in PCa growth but it is currently unknown if ACh signaling plays a role in its activation3,4.

Background

Methods

Conclusions

• Cell culture: The prostate cancer cell lines 22Rv1 and Du145 were grown in RPMI media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Docetaxel-resistant cell lines were maintained in 3 nM DTX.

• RT-qPCR: Total RNA was isolated using Trizol and converted to cDNA with M-MLV reverse transcriptase. All genes were amplified using PowerUp SYBR green and normalized to GAPDH expression.

• Acetylcholine Secretion: Cells were grown in serum starved media supplemented with neostigmine (100 µM) for 36 hrs before media was collected and frozen. Media was lyophilized and reconstituted with 1/5 volume water. Total ACh was measured with the QuickDetect ACh ELISA Kit.

• Crystal Violet Assay: Cells were treated as described in the figure legends and then stained with crystal violet and absorbance was measured at 570 nm.

• Western blot: Cells were treated as described in the figure legends. Phosphorylated proteins were normalized to the total protein intensity and non-phosphorylated proteins were normalized to actin.

• Drug synergy assay: Cells were treated with varying concentrations of inhibitor followed by DTX. Cells were stained with crystal violet and absorbances measured at 570 nm

• Docetaxel treatment increases production and secretion of ACh which persists in docetaxel resistance

• Docetaxel treatment increases the expression of CHRM1 and inhibition of this receptor enhances DTX-induced cell death. This is enhanced in docetaxel-resistant cells

• ACh signaling activates the ERK/CREB cascade which is mediated by muscarinic receptor signaling.

• Inhibition of ERK or CREB enhances DTX-induced cell death in a synergistic manner

• Inhibition of CHRM1 signaling may be useful for sensitizing and even reversing chemotherapy resistance in PCa.

AcknowledgementsDepartment of Defense PCRP grant W81XWH-15-1-0493, a Concern Foundation CONquer CanCER Now Award, and the WSU start-up fund (to BW) We thank Dr. Milena Rizzo for the kind gift of 22Rv1 and Du145 DTX-resistant cells.

CHRM1-mediated acetylcholine signaling protects prostate cancer cells from the chemotherapy agent docetaxel

Tyler Bland, Karen Vo, Baron Bechtold, Boyang WuDepartment of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, WA 99202, USA

Choline

CHAT VACHT

ACh

CHRM1

B

Dic (µM): - 10 25 - 10 25

DTX: - - - + + +

c-Caspase 3c-PARP1

Actin

CCh: - + - +DTX: - - + +

C

D

Figure 1: Acetylcholine synthesis and secretion in response to docetaxel: A,C, Expression of ACh synthesis and secretion enzymes in response to various exposure times of DTX (1nM)(A) and DTX-resistant (DTXR) cell lines (C) measured by qPCR. B, ACh secretion by PCa cells treated with DTX (1nM, 48hrs)(B) or in DTXR cells (D) measured by ELISA assay. All data is represented as the average ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001

ACh Production

CHRM1 Signaling

ERK/CREB Cascade

Figure 2: CHRM1 protects cells from DTX-induced cell death: A, Expression of CHRM1 in response to various exposure times of DTX (1nM) and DTX-resistant cell lines (DTXR) measured by qPCR. B-C, Crystal violet survival assay of 22Rv1 parental and DTXR cell lines treated with DTX (48 hrs) or first pre-treated with dicyclomine (Dic: 25 µM, 24 hrs). C-D, Representative Western Blot of 22Rv1 parental cell treated with DTX (48 hrs) or first pre-treated with Dic (24 hrs) (C) or carbachol (CCh: 10 µM, 24 hrs) (D). Cleaved caspase 3 (c-Caspase 3) and cleaved PARP1 (c-PARP1) intensity was normalized to actin intensity. All data is represented as the average ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001

B

C

D

ERK1/2 CREB Gene Expression

c-Caspase 3

c-PARP1

Actin

CCh (min): 0 5 10 15 30 60

CREB

p-CREB

ERK1/2

Actin

p-ERK1/2

p-ERK1/2

ERK1/2

CCh: - + - - - +Pilo: - - + - - -

Atropine: - - - + + -

Tubo: - - - - - +

Actin

Figure 3: Acetylcholine activates the ERK/CREB pathway in PCa: A, Representative Western Blot of 22Rv1 cells serum starved overnight and stimulated with CCh (10 µM). B, Representative Western Blot of 22Rv1 cells serum starved overnight and stimulated with CCh (10 µM, 30 min), pilocarpine (Pilo: 10 µM, 30 min) or first pre-treated with atropine (10 µM, 30 min) or tubocurarine (Tubo: 10 µM, 30 min). C, Chou-Talalay synergy plot of 22Rv1 cells stimulated with DTX (48 hrs), UO126, 666-15 (72 hrs) or a combination of DTX and UO126 or 666-15.

Antagonism

Synergy

DTX + UO126 DTX + 666-15

A

B

C

2064

References1.Wang, N. et al. Autocrine activation of

CHRM3 promotes prostate cancer growth and castration resistance via CaM/CaMKK-mediated phosphorylation of Akt. Clin. Cancer Res. (2015).

2.Baig, A. M. et al. Differential receptor dependencies: Expression and significance of muscarinic M1 receptors in the biology of prostate cancer. Anticancer. Drugs (2017).

3.Rodriguez-Berriguete, G. et al. MAP Kinases and Prostate Cancer. J. Signal Transduct. (2012)

4.Kim, J., Jia, L., Stallcup, M. R. & Coetzee, G. A. The role of protein kinase A pathway and cAMP responsive element-binding protein in androgen receptor-mediated transcription at the prostate-specific antigen locus. J. Mol. Endocrinol. (2005).

22Rv1 Du1450

1

2

3

Nor

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CH

RM

1E

xpre

ssio

n

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**

*

A

0.1 1 10 1000

50

100

22Rv1

DTX (nM)%

Sur

viva

l

DTX: 0.862 nMDTX + Dic:0.349 nM

IC50

**0.1 1 10 1000

50

100

22Rv1 DTXR

DTX (nM)

% S

urvi

val

DTX: 18.441 nMDTX + Dic: 3.81 nM

***

IC50

Control

10 µM

25 µM

Control

10 µM

25 µM

01234

Nor

mal

ized

Inte

nsity

c-Caspase 3 c-PARP1

* * *

CHAT VACHT05101520

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Expression

RV_DTXSRV_DTXRDU_DTXSDU_DTXR

**

***

DTXSDTXR

DTXSDTXR

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10152025

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22Rv1 Du145

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22Rv1 Du1450

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2

3

Nor

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ized

CH

RM

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xpre

ssio

n DTXSDTXR

***