Inhibition of Isoprenylcysteine Carboxylmethyltransferase Induces

Published OnlineFirst February 6, 2017; DOI: 10.1158/1535-7163.MCT-16-0703

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Inhibition of Isoprenylcysteine Carboxylmethyltransferase Induces Cell-Cycle Arrest and Apoptosis through p21 and p21- Regulated BNIP3 Induction in Pancreatic Cancer Kanjoormana Aryan Manu1, Tin Fan Chai1, Jing Tsong Teh1, Wan Long Zhu1, Patrick J. Casey1,2, and Mei Wang1,3

Abstract

Pancreatic cancer remains one of the most difficult to treat tion induced mitochondrial respiratory deficiency and cellular human cancers despite recent advances in targeted therapy. Inhi- energy depletion, leading to significant upregulation of p21. bition of isoprenylcysteine carboxylmethyltransferase (ICMT), an Furthermore, we characterized the role of p21 as a regulator and enzyme that posttranslationally modifies a group of proteins coordinator of cell signaling that responds to cell energy deple- including several small GTPases, suppresses proliferation of some tion. Apoptosis, but not autophagy, that is induced via p21- human cancer cells. However, the efficacy of ICMT inhibition on activated BNIP3 expression accounts for the efficacy of ICMT human pancreatic cancer has not been evaluated. In this study, we inhibition in sensitive pancreatic cancer cells in both in vitro and have evaluated a panel of human pancreatic cancer cell lines and in vivo models. In contrast, cells resistant to ICMT inhibition identified those that are sensitive to ICMT inhibition. In these demonstrated no mitochondria dysfunction or p21 signaling cells, ICMT suppression inhibited proliferation and induced changes under ICMT suppression. These findings not only identify apoptosis. This responsiveness to ICMT inhibition was confirmed pancreatic cancers as potential therapeutic targets for ICMT sup- in in vivo xenograft tumor mouse models using both a small- pression but also provide an avenue for identifying those subtypes molecule inhibitor and shRNA-targeting ICMT. Mechanistically, that would be most responsive to agents targeting this critical we found that, in sensitive pancreatic cancer cells, ICMT inhibi- enzyme. Mol Cancer Ther; 16(5); 914–23. 2017 AACR.

Introduction sion of cell anabolism and proliferation in some cancers (10–12). However, the role of ICMT in human pancreatic cancer tumor- Pancreatic carcinomas are among the most difficult cancers to igenesis has not been directly addressed, despite confusing genetic treat, and the 5-year survival rate remains as low as 5% (1). studies in mice (6, 13, 14). Isoprenylcysteine carboxylmethyltransferase (ICMT) is the CDKN1A (often referred to as p21Cip1/Waf1 or p21) belongs to enzyme that catalyzes the last step of posttranslational prenyla- the Cip and Kip family of CDK inhibitors that bind to and inhibit tion-dependent modification of proteins. Most ICMT substrate the function of G cyclin/CDK complexes (15, 16). p21 has been proteins contain a C-terminal CAAX consensus motif. The car- 1 mostly studied as a factor that mediates the downstream signaling boxylmethylation by ICMT is essential for the proper function of of wild-type p53 as tumor suppressor, particularly in response to CAAX proteins by regulating their subcellular localization, pro- DNA damage, to cause cell-cycle arrest (17). However, a number tein–protein interactions, and/or protein stability (2–5). ICMT of studies have suggested that p21 performs tumor-suppressive has shown promise as a therapeutic target, as suggested by genetic functions independent of p53 (18–23). Apart from inhibiting and inhibitor studies (6–9). Recent investigations have indicated cell-cycle progression as a CDK inhibitor, p21 also regulates gene that inhibition of ICMT leads to metabolic disarray and suppres- transcription and apoptosis (18). Many cancers have altered or loss of p53 function, and p21 tumor suppressor functions are of particular interest in these cancers, among which many are of 1 Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, pancreatic origin (24). Singapore, Singapore. 2Department of Pharmacology and Cancer Biology, Duke Intrinsic or acquired resistance to apoptosis is a major reason University School of Medicine, Durham, North Carolina. 3Department of Bio- chemistry, National University of Singapore, Singapore, Singapore. for treatment failure of pancreatic cancers, and this has been frequently associated with the dysregulation of BCL2 family Note: Supplementary data for this article are available at Molecular Cancer – Therapeutics Online (http://mct.aacrjournals.org/). proteins (25 27). BNIP3 is a mitochondrial member of proapoptotic BCL2 family protein containing a motif similar to the K.A. Manu and T.F. Chai contributed equally to this study. BH3 domain (26, 28–30). BNIP3 can interact with the prosurvival Corresponding Author: Mei Wang, Duke-NUS Medical School, 8 College Road, BCL2 family members BCL2 and BCLXL and thereby facilitates the Singapore 169857, Singapore. Phone: 65-6516-8608; E-mail: induction of apoptosis (31–33). In addition to its reported role in [email protected] apoptosis, BNIP3 has been identified as a regulator of autophagy doi: 10.1158/1535-7163.MCT-16-0703 (34, 35). Supporting its role in cancer survival, BNIP3 expression 2017 American Association for Cancer Research. was found to be downregulated in various types of cancers,

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including in pancreatic adenocarcinoma (PDAC), in comparison sequences used are: p53-1, 50-GUAAUCUACUGGGACGGAATT- to normal tissues (36, 37). Loss of BNIP3 correlates with poorer 30 and p53-2, 50-GGUGAACCUUAGUACCUAATT-30; ATG5: 50- survival, and downregulation of BNIP3 results in increased resis- AUUCCAUGAGUUUCCGAUUGAUGGC-30. tance of pancreatic cancer to cytotoxic drug treatment (33, 38). Furthermore, BNIP3 expression was found to sensitize pancreatic Cloning and virus production cancer cells to apoptosis (39). BNIP3 shRNA was designed and cloned into pSuper retroviral In this study, we investigated the impact of ICMT inhibition on vector according to the User Manual (OligoEngine). The targeting human pancreatic cancer cells. We found that a subset of these sequences for BNIP3 were: (i) 50-CACGAGCGTCATGAAGAAA-30 cells are sensitive to ICMT inhibition through the inhibition of and (ii) 50-TACTGCTGGACGCACAGCA-30. MCherry-expressing mitochondria function and induction of an energy-depleted state, p21 shRNA retroviral vector with the target sequence 50- which results in the elevation of p21 and p21-dependent BNIP3 CTAGGCGGTTGAATGAGAG-30 was a gift from Dr. Mathijs Voor- expression, leading to cell-cycle arrest and apoptosis. hoeve. ICMT shRNA–expressing plasmids were constructed in lentiviral vector PLL3.7. The ICMT target sequences were: (i) 50- Materials and Methods CCCTGTCATTGTTCCACTATT-30 and (ii) 50-CTTGGTTTCGGCA- 0 Cells and ICMT inhibitor TCCTTCTT-3 . For expression, BCL-XL cDNA was cloned into ATCC pancreatic cancer cell lines MiaPaCa2, AsPC-1, PANC-1, retroviral vector pMSCV. HEK293T transfection and viral produc- BxPC-3, PANC-10.05, CAPAN-2, and HPAF-II were obtained tion were done according to the standard calcium phosphate from Duke University Tissue Culture Facility (Durham, NC) in protocol (42). 2011. These cell lines have been cultured per ATCC guidelines. The ICMT inhibitor cysmethynil was synthesized by the Duke Mouse xenograft studies Small Molecule Synthesis Facility via established methods (7, 40). MiaPaCa2 xenograft tumors were developed by subcutaneous Cysmethynil treatment of cells was performed as described in injection of 10 million MiaPaCa2 cells into the flanks of SCID prior publications from the laboratory (8, 10). mice that were 6 to 10 weeks old and weighed 18 to 20 g. Drug treatment, tumor measurement and euthanization were per- Cell culture, viability study, and soft-agar colony formed as described previously (8), in accordance with IACUC formation assay guidelines. Cells were seeded in standard DMEM containing 10% FBS and allowed to attach overnight. Cells were treated with various agents Statistical analysis and collected for protein and mRNA analysis at the time points GraphPad Prism (GraphPad) and Instat (GraphPad) software fi indicated in the respective gure legends. For glucose starvation were used for data analysis and presentation. To calculate the studies, culture media were replaced with glucose-free DMEM statistical significance, experimental groups were compared to the (GIBCO) supplemented with 10% FBS. Cell viability was deter- control group using Dunnet test one-way ANOVA to generate P mined using CellTiter 96 AQueous One Solution cell proliferation values. All experimental data are presented as mean SD. assay (Promega) according to the manufacturer's instructions. Differences were considered statistically significant at P < 0.05. Soft-agar colony formation was determined as described (7). Results Flow cytometric analysis Saturating propidium iodide/DAPI staining was used for ana- Suppression of ICMT inhibits proliferation and induces lyzing cell cycle and apoptotic cell death as described (8). Flow apoptosis in human pancreatic cancer cells cytometry was done using Miltenyi Biotec MACSQuant VYB Flow Treatment of a panel of pancreatic cancer cell lines including instrument, and data were analyzed by FlowJo software (FlowJo). MiaPaCa2, AsPC-1, PANC-1, BxPC-3, CAPAN-2, PANC-10.05, and HPAF-II with the ICMT inhibitor cysmethynil resulted in Oxygen consumption rate analysis dose-dependent inhibition of proliferation and reduction of Cell and mitochondrial respiration was determined using Sea- viability (Fig. 1A, top; Supplementary Fig. S1). The study identi- horse XF24 analyzer (Seahorse Bioscience), as previously fied ICMT inhibition–sensitive cell lines and those relatively described (12). Briefly, cells were seeded in XF24 cell culture resistant, with MiaPaCa2 being the most responsive line (Fig. plates at densities appropriate for each cell line and incubated 1A, top; Supplementary Fig. S1). The steep apparent dose– under standard cultural condition. One hour before performing response curves are the result of serum-binding property of the oxygen consumption rate (OCR), the media were replaced cysmethynil (43). The inhibition of ICMT enzymatic activity can with XF assay medium (Seahorse Bioscience) and incubated at be followed by analyzing the accumulation of prelamin A (Fig. 1A, 37 CinaCO2-free environment. Oligomycin, FCCP, and rote- bottom), which directly correlates with the unmethylated pre- none plus antimycin A were added sequentially to measure OCR, lamin A level (44). Indeed, the range of cysmethynil concentra- as described. tions causing progressively increasing prelamin A levels is consistent with that of decreasing cell viability in MiaPaCa2 cells (Fig. Quantitative RT-PCR, Western blot, siRNA knockdown, and 1A, top), supporting the ICMT-specific action of cysmethynil. cloning Immunoblot analysis of sensitive cells MiaPaCa2 and AsPC-1 These procedures are described in our previous report (41). treated with increasing concentrations of cysmethynil showed Antibodies for p21 (2947), p-p53, cleaved caspase-7 (9492), increased levels of cyclin kinase inhibitor p21 and apoptosis cyclin D1 (2978), p-Rb (S795) (9301), and PARP (9542) were marker cleaved caspase-7 and decreased level of cyclin D1 (Fig. from Cell Signalling. The anti-LC3 and anti-p53 were from Abgent 1B), suggesting the induction of cell-cycle arrest and apoptosis. In (Ab1802a) and Santa Cruz (sc-s28), respectively. The siRNA contrast, no similar molecular changes were observed in the

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A B C 150 Mia-2 AsPC1 PANC1 BXPC3 30 CAPAN-2 PANC10.05 HPAFII Mia-2 AsPC1 CAPAN2 HPAFII 100 Cysm 20

GAPDH (%Total)

50 1 p21 10

CyclinD1 Sub G % Viability to control 0 1.0 1.2 1.4 1.6 C-PARP 0 Log [Cysm] (mmol/L) Cysm – +

Sensitive cells Resistant cells [Cysm] 0 12.5 15 17.5 20 22.5 25

Prelamin A

GAPDH

PreA/GAPDH 1 1.3 1.3 1.7 2.1 2.4 3.3

D Mia-2 HPAFII E F shICMT - +++- + 1.2 GAPDH Cysm - 12.5 mmol/L 25 mmol/L shCtrl shIcmt 0.8 LC3

PARP 0.4

C-casp7 ICMT Exp. (fold chg) 0.0 p21 shCtrl shICMT shICMT knock- Mia-2 HPAFII down eﬃciency 99% 87%

G HI

Vehicle Low dose High dose 1.2 4 shICMT shCtrl 2,500 )

3 Control shRNA Start treatment 3 2,000 Icmt shRNA 0.8 ( *** 1,500 2 *** *** 0.4 1,000 1 500 ICMT Exp. (fold chg) 0.0 Tumor volume (mm Relative tumor volume trl 0 0 14 17 20 23 26 29 32 35 38 41 44 47 shCtrlshC –6 –4 –2 0 2 4 shICMTshIC Day Day

Figure 1. Suppression of ICMT inhibits proliferation and induces apoptosis and autophagy in multiple pancreatic cancer cells. A, Top, Cell viability curve of MiaPaCa2, AsPC-1, PANC-1, BxPC-3, PANC-10.05, CAPAN-2, and HPAF-II pancreatic cancer cells treated with cysmethynil at concentrations range from 10 to 40 mmol/L with increments of 2.5 mmol/L for 48 hours. Bottom, Immunoblot study on MiaPaCa2 cell lysates for prelamin A and loading control GADPH. The calculated ratios of prelamin A and GAPDH are shown. B, Immunoblot analysis on lysates of MiaPaCa2, AsPC-1, CAPAN-2, and HPAF-II cells prepared after 48 hours of treatment with 0, 20, 22.5,

and 25 mmol/L cysmethynil. C, Flow cytometric quantiﬁcation of apoptotic (sub-G0) population of MiaPaCa2 cells after 24-hour cysmethynil treatment at 0 or 22.5 mmol/L. D, Immunoblot analysis of the lysates of MiaPaCa2 and HPAF-II cells expressing either control shRNA or that targeting ICMT, 96 hours after infection by shRNA expressing lentiviruses (top). The ICMT knockdown efﬁciency is assessed by qPCR analysis (bottom). E, Colony formation assay of MiaPaCa2 cells treated with the indicated concentration of cysmethynil for 14 days. F, Colony formation assay of MiaPaCa2 cells expressing either control shRNA or that targeting ICMT; qPCR analysis of ICMT expression levels are presented on the right. G, Growth of MiaPaCa2 xenograft tumors under every other day treatment by vehicle, 100 or 150 mg/kg cysmethynil. n ¼ 6 for each group. , P < 0.001 between different groups. H, Image to demonstrate the contralateral growth of xenograft MiaPaCa2 tumors derived from cells expressing either control shRNA (right side of mouse, pointed by black arrow) or that targeting ICMT (left side of mouse, pointed by red arrow). I, Analysis of xenograft data on tumors expressing control shRNA or that targeting ICMT. n ¼ 3 for each dosing group. , P < 0.001 between treatment groups and control. The qPCR analysis of ICMT expression for the 2 groups of cells used for the study was done before implantation and presented on the right side of I. A–F, Data shown are from a single experiment that has been repeated 3 times with similar results.

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resistant cell lines HPAF-II and CAPAN-2 under the same treat- genetic and pharmacologic suppression of ICMT showed efficacy ment by cysmethynil (Fig. 1B). Consistent with the viability study against MiaPaCa2 pancreatic cancer cells in vitro and in vivo. (Fig. 1A) and the immunoblot apoptosis markers (Fig. 1B), flow fi cytometric analysis on MiaPaCa2 cells showed signi cant increase Upregulation of p21, induced by ICMT inhibition, in apoptotic cell population (i.e., the sub-G1 population) under plays an active role in the induction of apoptosis in inhibitor treatment (Fig. 1C). These responses to ICMT inhibition a p53-independent manner fi were con rmed by use of shRNA to target ICMT in the sensitive As noted above, treatment of several pancreatic cancer cell lines MiaPaCa2 cells and resistant HPAF-II cells. ICMT knockdown with cysmethynil markedly increased p21 protein levels, similarly resulted in increased levels of p21, cleaved PARP, and caspase-7 observed with ICMT knockdown (Fig. 1), which suggests a specific and LC3 autophagy marker only in MiaPaCa2, but not HPAF-II link between ICMT inhibition and p21 induction. While the cells (Fig. 1D). inhibitory function of p21 in cell-cycle progression has been To further evaluate the role of ICMT in tumorigenesis, we extensively studied (15), its role in apoptosis is not well under- assessed the impact of ICMT inhibition on MiaPaCa2 cell growth stood (18). To investigate the functional role of p21 elevation that in soft agar and on xenograft tumor formation in mice. ICMT occurs with ICMT inhibition, 2 MiaPaCa2 clones with stable inhibition by either cysmethynil or by the expression of shRNA knockdown of p21 were generated by introducing a retroviral targeting ICMT reduced soft agar colony formation of MiaPaCa2 vector expressing shRNA targeting p21. As expected, these cells (Fig. 1E and F). In the xenograft study, low- and high-dose exhibited significantly reduced p21 levels both at baseline cysmethynil treatment led to tumor growth inhibition and tumor and upon cysmethynil treatment, compared with parental cells regression, respectively (Fig. 1G). Similarly, when MiaPaCa2 cells (Fig. 2A). Importantly, flow cytometric analysis demonstrated expressing either control shRNA or shRNA targeting ICMT were that apoptosis was significantly attenuated in the p21-knockdown fi injected into contralateral sides of the same mice, signi cant cells when subjected to cysmethynil treatment, in comparison to suppression of xenograft tumor formation was observed in the the parental cells similarly treated (Fig. 2B). Consistent with these ICMT-knockdown group (Fig. 1H and I). In summary, both findings, viability assays showed that p21-knockdown clones

A B C EV sh-p21B sh-p21Q EV sh-p21 B sh-p21 Q EV sh-p21 B sh-p21 Q 120 Cysm Sub G1 Sub G1 Sub G1 *** 1.17% 1.56% 1.26% GAPDH -Cysm 80 p21

Sub G1 Sub G1 Sub G1 40 34.5% 7.02% 12.6% +Cysm Viability (% of control) 0 Cysm

D E F EV sh-p21 Mia-2 Expresses empty vector Mia-2 Expressses p21 shRNA 4 120 8 Vehicle Vehicle 7 DMSO 3 Cysmethynil Cysmethynil 80 6 5 2 4 40 *** 3 1 2 Cysm % of CFU to control 0 Cysm - + - + Relative tumor volume Relative tumor volume 1 -- sh-p21 ++ 0 0 97531 151311 97531 151311 Day Day

Figure 2. p21, induced by suppression of ICMT, promotes apoptosis of MiaPaCa2 cells in vitro and inhibits tumor formation in vivo. A, p21 levels were assessed in parental MiaPaCa2 cells (EV) and 2 derived clones expressing shRNA targeting p21 (sh-p21 B and Q), after 48 hours of treatment with concentrations of cysmethynil of 0, 17.5, 20, and 22.5 mmol/L. B, Flow cytometric analysis of parental and p21 shRNA expressing MiaPaCa2 cells, after 48-hour treatment with vehicle or 22.5 mmol/L of cysmethynil. C, Viability of MiaPaCa2 parental and p21-knockdown clones after 48-hour treatment of cysmethynil at concentrations of 0, 17.5, 20, and 22.5 mmol/L. D, Soft-agar colony formation was evaluated for parental MiaPaCa2 and a stable p21-knockdown clone (sh-p21), under the treatment by vehicle or 20 mmol/L cysmethynil for 14 days. Samples from technical repeats were analyzed and the data are presented on the right of D. E and F, In vivo efﬁcacy study of cysmethynil treatment in xenograft tumor model of parental MiaPaCa2 cells (E) and stable p21-knockdown cells (sh-p21; F). Animals were dosed with vehicle or cysmethynil at 150 mg/kg every other day. , P < 0.001. A–D, Data shown are from a single experiment that has been repeated 3 times with similar results.

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survived better under cysmethynil treatment (Fig. 2C). Further from cysmethynil treatment (Supplementary Fig. S2B). Hence, the investigation showed that p21-knockdown cells were also much induction of p21 and its link to cell responsiveness to ICMT more resistant to ICMT inhibition in the soft-agar colony forma- suppression are independent of p53 function. tion assay compared with control cells (Fig. 2D). In xenograft mouse model studies, while the tumors from cells expressing p21 elevation induced by ICMT suppression promotes the control shRNA were responsive to cysmethynil treatment (Fig. expression of autophagy and apoptosis genes 2E), similar to the parental MiaPaCa2 cells (Fig. 1G), tumors from We next evaluated the impact of ICMT inhibition on apoptosis cells expressing shRNA targeting p21 were resistant to cysmethynil and autophagy in MiaPaCa2 cells. We found that cysmethynil treatment (Fig. 2F), despite their faster growth rate due to the loss treatment increased p21, LC3, ULK1, and BNIP3 mRNA levels of p21. Hence, both in vitro and in vivo studies indicate that p21 (Fig. 3A). Similarly, MiaPaCa2 cells expressing shRNA targeting elevation is functionally important for the efficacy of ICMT ICMT demonstrated elevated p21, LC3, ULK1, and BNIP3 mRNA inhibition in sensitive pancreatic cancer cells. levels, albeit at more modest levels compared with that from Although p21 production is subject to multiple modes of inhibitor treatment (Fig. 3B). The differences are likely due to the regulation, p53 is considered the most prominent regulator requirement for high efficiency and prolonged knockdown to (17). However, cysmethynil treatment, while inducing p21 and completely suppress the cellular function of ICMT. Worth noting, autophagosome protein LC3 in a dose-dependent fashion, did analysis of mRNA levels in the MiaPaCa2 xenografts consistently not elicit significant changes in either the total amount of p53 or showed higher expression of p21, LC3, ULK1, and BNIP3 in the its Ser15-phosphorylated nuclear form in MiaPaCa2 cells (Sup- tumors exposed to cysmethynil, in a dose-dependent fashion, plementary Fig. S2A). Furthermore, knockdown of p53 had no compared with those treated with control vehicle (Fig. 3C). effect on either the increases in the levels of p21 or LC3 resulting Furthermore, the xenograft tumors derived from cells expressing

AB p21 LC3 ULK1 BNIP3 ICMT p21 LC3 ULK1 BNIP3 30 6 8 15 2 4 2 2 3

6 3 20 4 10 2 4 1 2 1 1 10 2 5 1 2 1 (fold change) (fold change) Gene expression Gene expression 0 0 0 0 0 0 0 0 0 Cysm sh-ICMT - + - + - + - + - + C D 16 p21 6 LC3 30 ULK1 30 BNIP3 ICMT p21 LC3 ULK1 BNIP3 2 3 3 3 5 12 4 4 20 20 2 2 2 3 8 1 2 2 10 10 1 4 1 1 (fold change) (fold change) 1 Gene expression Gene expression 0 0 0 0 0 0 0 0 0 Cysm sh-ICMT - + - + - + - + - +

E 20 p21 6 LC3 4 ULK1 20 BNIP3

15 3 15 4 10 2 10 2 5 1 5 Gene expression 0 0 0 0 Cysm sh-p21 - + - + - + - +

Figure 3. ICMT inhibition–induced p21 promotes transcription of autophagy and apoptosis genes. All panels show qPCR expression analysis of the indicated genes. RNA samples were prepared from (A) MiaPaCa2 cells after 48-hour treatment with 0, 20, or 22.5 mmol/L cysmethynil, (B) MiaPaCa2 cells 96 hours after infection with lentivirus expressing either control shRNA or that targeting ICMT, (C) xenograft tumor samples obtained from mice treated every other day with either vehicle or cysmethynil at 100 and 150 mg/kg, respectively, (D) xenograft tumor samples derived from MiaPaCa2 cells expressing control shRNA or that targeting ICMT, and (E) MiaPaCa2 cells selected to express either control shRNA () or shRNA targeting p21 (þ) after 48-hour treatment with cysmethynil at 0, 17.5, 20, or 22.5 mmol/L. All studies have been repeated 3 times with similar results.

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shRNA targeting ICMT also demonstrated higher levels of expres- silencing of BNIP3 may be an important step in tumorigenesis and sion of these genes compared with tumors containing control tumor maintenance in several human cancer types, most notably shRNA (Fig. 3D). These in vitro and in vivo results strongly support pancreatic cancers (36, 37). To assess the importance of upregula- the notion that ICMT inhibition induction of these genes is tion of BNIP3 and it role in apoptosis and autophagy in the functionally important for the antitumor efficacy. LC3, ULK1, sensitivity of pancreatic cancer cells to ICMT inhibition, we and BNIP3 proteins promote cellular autophagy (45), and BNIP3 studied the responsiveness of MiaPaCa2 cells to cysmethynil also functions as a proapoptosis BCL family member. As the when BNIP3 expression was silenced. Knockdown of BNIP3 by evidence demonstrated the importance of p21 in ICMT inhibition shRNA (Fig. 4A) significantly reduced cysmethynil-induced apo- in targeting tumor cell survival and proliferation (Fig. 2), we ptosis, as assessed by the sub-G1 population from flow cytometry investigated the role of p21 in these autophagy and apoptosis (Fig. 4B) and consistent reduction of the apoptosis markers gene expression. We found that p21 knockdown significantly cleaved PARP and caspase-7 (Fig. 4C). attenuated the induction of ULK1, LC3, and BNIP3 transcription Consistent with the notion that BNIP3 is a positive regulator of arising from cysmethynil treatment (Fig. 3E), placing p21 as an cellular autophagy (34, 35, 48), suppression of BNIP3 expression upstream regulator for the transcription of these genes, which is an also reduced the level of autophagy marker LC3 (Fig. 4C). Autop- underexplored role for p21, in contrast to its well-known function hagy is known to be double edged in cancer cell survival. Studies in regulating cell cycle (15). have demonstrated that excessive stimulation of autophagy can also lead to cell death (8). We then investigated the role of Induction of BNIP3 plays an essential role in pancreatic cancer autophagy in the sensitivity of MiaPaCa2 cells to ICMT inhibition cell apoptosis induced by ICMT inhibition by concurrently suppressing autophagy using Atg5 siRNA and Among the genes whose expression is induced by ICMT inhi- treating with cysmethynil. As expected, cellular autophagy was bition, LC3 and ULK1 have well-established roles in promoting impaired in Atg5 knockdown cells (Fig. 4D). However, the reduc- cellular autophagy. BNIP3, on the other hand, has been reported tion of cell viability and induction of apoptosis resulted from to have dual roles of promoting autophagy and apoptosis (46, ICMT inhibition were not significantly different between cells 47). Furthermore, recent studies suggest that downregulation or transfected with control or Atg5 siRNA (Fig. 4D–F), suggesting

B -Cysm+Cys +Cysmyym C A 2.5 sh-Ctrll sh-BNIP3 Sub G1 Sub G1 Cysm 2.0 3.24% 29.6% GAPDH

1.5 si-Ctrl LC3

1.0 C-PARP Sub G1 Sub G1 0.5 3.28% 14.9% GAPDHA BNIP3 Exp. (fold chg) C-casp7 0.0 12 si-BNIP3 Cysm sh-BNIP3 - +

D si-control si-Atg5 E 120 F –Cysm +Cysm Cysm Sub G Sub G 100 1 1 GAPDH 1.58% 228.1% 80 Atg5-Atg12AA 60 si-control LC3 40 Sub G1 Sub G1 PARP 20 1.40% 33.9% % Viability of control 0 C-casp7 Cysm si-Atg5 si-Atg5 - +

Figure 4. BNIP3-stimulated apoptosis, but not autophagy, promotes cell death resulted from ICMT inhibition. A, qPCR analysis of BNIP3 expression in MiaPaCa2 cells expressing either control shRNA () or shRNA targeting BNIP3 (þ) after 48-hour treatment with 0, 20, or 22.5 mmol/L cysmethynil. B, Flow cytometric analysis of MiaPaCa2 cells with or without BNIP3 knockdown after 48-hour treatment with either vehicle or 22.5 mmol/L cysmethynil. C, Immunoblot analysis of the autophagy and apoptosis markers on cell lysates from (A). D, Immunoblot analysis of autophagy and apoptosis markers on MiaPaCa2 cells treated with cysmethynil at 0, 20, and 22.5 mmol/L, with or without Atg5 knockdown as indicated. E, Viability of MiaPaCa2 cells treated with 0, 17.5, 20, and 22.5 mmol/L cysmethynil for 48 hours, with or without Atg5 knockdown as indicated. F, Flow cytometric analysis of MiaPaCa2 cells treated with vehicle or 22.5 mmol/L cysmethynil for 48 hours, with or without of Atg5 knockdown as indicated. For all studies, data shown are from a single experiment that has been repeated 3 times with similar results.

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that autophagy induction is not a pro-death mechanism in tion of 4EBP1 and S6 (Fig. 5B). Together, these data suggest that MiaPaCa2 cells under ICMT inhibition. In contrast, overexpres- ICMT inhibition activated catabolic and suppressed anabolic sion of BCL-XL confers significant resistance of MiaPaCa2 cells to signaling, consistent with the observation of elevated autophagy. cysmethynil treatment (Supplementary Fig. S3), supporting the In contrast, HPAF-II cells showed no significant changes in these role of apoptosis in the cell death induced by ICMT inhibition. In markers. Finally, while apoptosis markers such as cleaved PARP summary, these data indicate that induction of p21 and p21- and caspase-7 were elevated in MiaPaCa2 cells under cysmethynil stimulated expression of proapoptotic BNIP3 mediates the pan- treatment, they were not observed in HPAF-II cells (Fig. 5B), creatic cancer cell apoptosis induced by ICMT inhibition. consistent with the outcome of viability study (Fig. 1A; Supple- mentary Fig. S1). It is important to note that these signaling ICMT inhibition–sensitive, but not the resistant, pancreatic changes were also observed in MiaPaCa2 cells under the condi- cancer cells demonstrated characteristic alterations of AMPK- tion of shRNA knockdown of ICMT, including cell-cycle and mTOR, cell proliferation, and apoptosis signaling profiles in apoptosis marker changes (Fig. 1D) and AMPK and mTOR sig- response to ICMT inhibition naling changes (Fig. 5C). Further testing of the signaling profile Cell viability assays have identified MiaPaCa2 and AsPC-1 cells demonstrates that ICMT inhibition induced similar changes in the to be more sensitive (Fig. 1A; Supplementary Fig. S1), while other sensitive cell line such as AsPC-1 but not in the more HPAF-II, CAPAN-2, and BxPC-3 are more resistant, to cysmethynil resistant BxPC-3 cells (Supplementary Fig. S4), consistent with treatment (Fig. 1A; Supplementary Fig. S1). To assess determi- their sensitivity profile. nants of sensitivity to ICMT inhibition in pancreatic cancer cells, we compared the molecular responses of MiaPaCa2 and HPAF-II Activated p21/BNIP3 signaling, in response to mitochondria cells to ICMT inhibition. MiaPaCa2 cells responded to ICMT respiratory deficiency and metabolic stress, leads to apoptotic inhibition with increased p21 and BNIP3 expression (Fig. 5A, cell death top), whereas HPAF-II showed little response (Fig. 5A, bottom). We recently discovered that ICMT inhibition leads to mito- Analysis of major regulatory pathways showed different cell chondria dysfunction in PC3 prostate and MDA-MB-231 breast signaling profiles between MiaPaCa2 and HPAF-II cells in several cancer cells, which in turn results in cell metabolic distress (12). categories, including cell-cycle regulators, mTOR and AMPK sig- We were particularly interested in determining whether sensitive naling, and apoptosis indicators (Fig. 5B). MiaPaCa2 cells pre- and resistant pancreatic cancer cells respond differently to ICMT sented with increased level of p21 and decreased levels of Cyclin inhibition in terms of mitochondrial respiration and cell metab- D1 and pRb, indications for G1 cell-cycle arrest. In contrast, these olism, which seemed a likely scenario based on the differences markers were unchanged in the resistant HPAF-II cells under the observed in AMPK and mTOR signaling in these lines as described same cysmethynil treatment. above. To this end, OCRs were measured for MiaPaCa2 and MiaPaCa2 cells also showed increased phosphorylation of HPAF-II cells treated with either vehicle or cysmethynil. Mia- AMPK and ACC under cysmethynil treatment, suggesting that PaCa2 cells responded to cysmethynil with dose-dependent the cells may be under metabolic stress. Consistent with this reduction of cell respiration, mitochondria basal respiration, and notion, mTOR signaling was suppressed by cysmethynil treat- maximum respiratory capacity (Fig. 6A). HPAF-II cells, in contrast, ment in MiaPaCa2 cells, as demonstrated by hypophosphoryla- demonstrated no changes in mitochondria respiration under the

A 10 MiaPaca2 B Mia-2 HPAF-II C Cysm - + - + sh-Icmt - + 8 GAPDH GAPDH 6 p21 pAMPK 4 Figure 5.

(fold of DMSO) Cyclin D1 GAPDH Suppression of ICMT inhibits cell-cycle

Gene expression 2 AMPK progression and induces metabolic stress 0 pRb and cell death in sensitive pancreatic cancer cells. A, qPCR analysis of BNIP3 and pACC P21 p21 mRNA levels in MiaPaCa2 (top) and BNIP3 pAMPK HPAF-II (bottom) cells after 48-hour 10 HPAF-II p4EBP1 treatment with DMSO control or 22.5 AMPK mmol/L cysmethynil. B and C, Immunoblot 8 pS6 analysis of the indicated proteins in pACC MiaPaCa2 and HPAF-II cells following 48- 6 hour treatment with DMSO control ()or p4EBP1 22.5 mmol/L (þ) of cysmethynil (B), or 4 MiaPaCa2 cells 96 hours after infection with lentivirus-expressing either control (fold of DMSO) 2 pS6 þ

Gene expression shRNA ( ) or that targeting ICMT ( )(C).

0 PARP

P21 BNIP3 C-Casp7 DMSO Cysmethynil

920 Mol Cancer Ther; 16(5) May 2017 Molecular Cancer Therapeutics

ICMT Inhibition Induces Apoptosis in Pancreatic Cancer

A B C D DMSO Gluc + - - BNIP3 p21 DMSO Gluc + Low dose cysm 5 8 140 Low dose cysm GAPDH GAPDH 120 Ligh dose cysm High dose cysm 4 120 pAMPK 6 100 C-casp7 100 AMPK 3 80 PARP 4 80 pS6 60 2 60 2 40 1 40 LC3 Gene expression 20 20 0 0

O2 Consum (% control) Gluc + - + - O2 Consum (% control) 0 0 Cyclin D1 Intact Basal Max ROX Intact Basal Max ROX Resp Resp Resp Resp Resp Resp pRb S795 p21

E No ICMT Suppression With ICMT suppression ICMT ICMT

Reduced mitochondria respiration mito mito

ATP ATP Macromolecules Macromolecules

P21 P21

ULK1, LC3 BNIP3 ULK1, LC3 BNIP3

AUTOPHAGY APOPTOSIS AUTOPHAGY APOPTOSIS

Figure 6. Metabolic stress–induced p21 elevation inhibits cell proliferation and promotes autophagy and apoptosis. A and B, Cell respiration analysis of MiaPaCa2 (A)and

HPAF-II (B) cells for intact cell, basal mitochondrial, and maximum mitochondrial OCR (O2 consumption) and non-mitochondrial oxygen consumption (ROX) following 24 hours of treatment with 0 mmol/L (DMSO), 20 mmol/L (low-dose cysm), or 22.5 mmol/L (high-dose cysm) cysmethynil. C, Immunoblot analysis of the indicated proteins in MiaPaCa2 cells after growth in normal (þ) or glucose-deprived () media. D, qPCR analysis of mRNA levels of BNIP3 and p21 in the cells from (C). A–D, Data shown are from a single experiment that has been repeated 3 times with similar results. E, Schematic model summarizing the findings that energy depletion, induced by ICMT inhibition or nutrient deprivation, leads to transcriptional activation of p21 and p21-dependent inhibition of cell proliferation and induction of ULK1, LC3, and BNIP3, which results in further induction of autophagy and apoptosis in susceptible pancreatic cancer cells. same treatment conditions (Fig. 6B). Notably, non-mitochondri- (ETC) that can be supplied in growth media. Indeed, pAMPK and al oxygen consumption was not changed under ICMT inhibition LC3II levels were increased, and pS6 level was reduced in cells in either cell line (Fig. 6A and B), confirming that the reduction of growing in glucose-deprived medium, consistent with energy cellular respiration is of mitochondria origin. stress (Fig. 6C), and these cells showed significant upregulation The studies described above suggested that the induction of p21 of p21 and BNIP3 expression (Fig. 6D). In the same cells, is an important mediator of the responses of pancreatic cancer increased p21 and reduced cyclin D1 and pRb protein levels were cells to energy depletion and metabolic distress arising from ICMT observed, suggesting inhibition of cell proliferation (Fig. 6C). inhibition–induced mitochondria dysfunction. We hypothesized Finally, there was consistent increase in the levels of cleaved PARP that p21 and apoptosis induction would also occur in other and caspase-7 under glucose deprivation, demonstrating the energy-depleted metabolic stress. To investigate whether the induction of apoptosis (Fig. 6C). regulation by p21 and BNIP3 represented a cellular response In summary, our findings identified a previously undescribed mechanism not limited to ICMT inhibition but rather to energy regulatory function of p21, as modeled in Fig. 6E, to be a depletion in general, we compared cell signaling in MiaPaCa2 coordinator for the control of cell proliferation, catabolism, and cells grown either under glucose deprivation or normal growth cell death in response to metabolic stress, which can result from condition. Glucose is a fuel molecule for electron transport chain nutrient deprivation or mitochondria dysfunction as in the case of

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Manu et al.

ICMT inhibition. These perturbations can activate p21 and its particular cancer cells to ICMT inhibition will depend on further downstream signaling, leading to cell catabolism, proliferation studies, the signatures we have identified of sensitive cells— inhibition, and apoptosis. reduction of ETC function, energy and metabolic distress, p21 elevation and subsequently signaling—should be useful in the Discussion identification of cancer types that can be targeted by ICMT inhibition. In this study, we provide a strong case for suppressing ICMT as a method to inhibit cell proliferation and induce cell death in pancreatic cancer cells. In sensitive cells, suppression of ICMT Disclosure of Potential Conflicts of Interest resulted in mitochondria dysfunction and metabolic stress, induc- No potential conflicts of interest were disclosed. tion of p21 independent of p53, followed by a constellation of changes that included cell-cycle inhibition, autophagy, and apo- Authors' Contributions ptosis. The consistent finding that p21 accumulates in these Conception and design: K.A. Manu, T.F. Chai, M. Wang sensitive cells points to its involvement not only in the cell-cycle Development of methodology: K.A. Manu, T.F. Chai, P.J. Casey, M. Wang arrest but also in autophagy and apoptosis induction. It makes Acquisition of data (provided animals, acquired and managed patients, biologic sense that coordinated cell adaptations to energy deple- provided facilities, etc.): K.A. Manu, J.T. Teh, W.L. Zhu Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tion would include (i) cessation of growth and proliferation that computational analysis): K.A. Manu, T.F. Chai, J.T. Teh, M. Wang depend on energy-rich condition and anabolic activity such as Writing, review, and/or revision of the manuscript: K.A. Manu, T.F. Chai, protein synthesis and lipid synthesis, (ii) compensatory increase P.J. Casey, M. Wang in catabolic activity, such as autophagy, which provides emergent Administrative, technical, or material support (i.e., reporting or organizing production of fuel and building block molecules, and (iii) initi- data, constructing databases): K.A. Manu, T.F. Chai, W.L. Zhu, M. Wang ation of cell death process when the energy-depleted state pro- Study supervision: M. Wang Other (secured grant support for the project): M. Wang longs or gets more severe. This study provides strong evidence that p21 is likely a coordinator for these adaptations. We also identified BNIP3, a BH3 domain–containing BCL2 family protein that Acknowledgments promotes both autophagy and apoptosis, as a downstream effec- The authors would like to thank Drs. Koji and Yoko Itahana of Duke-NUS Medical School for the selection of p53 antibodies and PCR primers. tor of p21 to regulate autophagy and apoptosis. It remains unclear why some cancer cells are more sensitive to ICMT inhibition–induced respiratory suppression and resultant Grant Support energy depletion. One possible explanation for the differences can Financial support for this work is from Singapore Ministry of Health and be that the expression, and therefore the function, of particular Ministry of Education awarded to M. Wang. The costs of publication of this article were defrayed in part by the payment of ICMT substrate(s) that regulate mitochondria function vary page charges. This article must therefore be hereby marked advertisement in between different cells types. Another possibility is that mito- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. chondria function in different cancer cells have different vulner- ability to ICMT suppression mediated by the enzyme's substrates. Received October 23, 2016; revised January 24, 2017; accepted January 24, While the exact mechanism that accounts for the sensitivity of 2017; published OnlineFirst February 6, 2017.

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www.aacrjournals.org Mol Cancer Ther; 16(5) May 2017 923

Inhibition of Isoprenylcysteine Carboxylmethyltransferase Induces Cell-Cycle Arrest and Apoptosis through p21 and p21-Regulated BNIP3 Induction in Pancreatic Cancer

Kanjoormana Aryan Manu, Tin Fan Chai, Jing Tsong Teh, et al.

Mol Cancer Ther 2017;16:914-923. Published OnlineFirst February 6, 2017.

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