ormeloxifene suppresses desmoplasia and enhances...
TRANSCRIPT
1
Ormeloxifene Suppresses Desmoplasia and Enhances Sensitivity of Gemcitabine in 1
Pancreatic Cancer 2
Sheema Khan1, Mara C. Ebeling2, Neeraj Chauhan1, Paul A. Thompson3, Rishi K. Gara1, Aditya 3
Ganju1, Murali M. Yallapu1, Stephen W. Behrman4, Haotian Zhao2, Nadeem Zafar5, Man Mohan 4
Singh6, Meena Jaggi1, Subhash C. Chauhan1* 5
1Department of Pharmaceutical Sciences and Center for Cancer Research, University of 6
Tennessee Health Science Center, Memphis, Tennessee, USA. 7
2Cancer Biology and Sanford Children’s Health Research Center, Sanford Research, Sioux Falls, 8
South Dakota, USA. 9
3Methodology and Data Analysis Center, Sanford Research, Sioux Falls, South Dakota, USA. 10
4Department of Surgery, University of Tennessee Health Science Center, Memphis, Tennessee, 11
USA. 12
5Department of Pathology, University of Tennessee at Memphis, Memphis, Tennessee, USA. 13
6Saraswati Dental College, Lucknow, Uttar Pradesh, India 14
Running title 15
Ormeloxifene suppresses desmoplasia in pancreatic cancer 16
Key words 17
Pancreatic Cancer, PDAC, Ormeloxifene, Desmoplasia, Stroma, Gemcitabine, Therapeutics 18
Financial support: 19
Subhash C. Chauhan: This work was partially supported by grants from Department of 20
Defense (PC130870); the National Institute of Health (RO1 CA142736 and UO1 CA162106A) 21
and financial support from Kosten Foundation for pancreatic cancer research (UT 14-0558). 22
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
2
Meena Jaggi: Department of Defense (PC130870); the National Institutes of Health (UO1 1
CA162106A) and the College of Pharmacy Dean's Seed Grant of the University of Tennessee 2
Health Science Center. 3
*Corresponding Author: 4
Subhash C. Chauhan, Ph.D., Professor, Department of Pharmaceutical Sciences, University of 5
Tennessee Health Science Center, 19 South Manassas, Cancer Research Building, Memphis, TN, 6
38163. Phone: (901) 448-2175. Fax: (901)-448-1051. E-mail: [email protected] 7
Potential conflicts of interest 8
The authors declare that there are no financial and non-financial competing interests. 9
Word count 10
5000 words 11
Total number of figures and tables 12
Seven figures 13
14
15
16
17
18
19
20
21
22
23
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
3
Abstract 1
The management of pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to lack of 2
an efficient therapy and development of chemoresistance to the current standard therapy, 3
gemcitabine (GEM). Recent studies implicate the intimate reciprocal interactions between 4
epithelia and underlying stroma due to paracrine Sonic hedgehog (SHH) signaling in producing 5
desmoplasia and chemoresistance in PDAC. Herein, we report for the first time that a 6
nonsteroidal drug, ormeloxifene (ORM), has potent anti-cancer properties and depletes tumor-7
associated stromal tissue by inhibiting the SHH signaling pathway in PDAC. We found that 8
ORM inhibited cell proliferation and induced death in PDAC cells, which provoked us to 9
investigate the combinatorial effects of ORM with GEM at the molecular level. ORM caused 10
potent inhibition of the SHH signaling pathway via downregulation of SHH and its related 11
important downstream targets such as Gli-1, SMO, PTCH1/2, NFκB, p-AKT and Cyclin D1. 12
ORM potentiated the anti-tumorigenic effect of GEM by 75% in PDAC xenograft mice. Further, 13
ORM depleted tumor-associated stroma in xenograft tumor tissues by inhibiting the SHH cellular 14
signaling pathway and mouse/human collagen I expression. Xenograft tumors treated with ORM 15
in combination with GEM restored the tumor suppressor miR-132, and inhibited stromal cell 16
infiltration into the tumor tissues. Additionally, invasiveness of tumor cells co-cultivated with 17
TGFβ-stimulated human pancreatic stromal cells was effectively inhibited by ORM treatment 18
alone or in combination with GEM. We propose that ORM has high therapeutic index and in a 19
combination therapy with GEM it possesses great promise as a treatment of choice for 20
PDAC/pancreatic cancer. 21
22
23
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
4
Introduction 1
Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis largely due to its propensity for 2
early local invasion, distant metastasis and lack of effective therapies. Many chemotherapeutic 3
regimens have failed and the current standard-of-care therapy, gemcitabine (GEM), extends 4
patient survival by only a few months (1). Newer treatment options for PDAC patients are 5
FOLFIRINOX and nab-paclitaxel/GEM, which improved overall survival by 4.3 and 1.8 months 6
over GEM therapy, respectively (2, 3). However, safety profile of these drugs is less favorable 7
than GEM therapy, accounting for myelosuppression and peripheral neuropathy (2-4). Despite 8
these advances, the overall outcome remains miserable for this patient population. Thus, 9
investigations on alternative approaches for PDAC therapy are a high research priority. 10
Activation of oncogenes such as Kras and/or inactivating mutations or loss of expression of 11
tumor suppressor genes (including DPC4, p16, p53, and SMAD4) is known in PDAC (5). It has 12
been shown that extensive desmoplasia is one of the underlying causes of pancreatic cancer’s 13
poor prognosis and chemoresistance (6). Desmoplasia is typically characterized by excessive 14
production of extracellular matrix (ECM) and collagen I and is associated with proliferation of 15
stromal cells, myofibroblasts and pancreatic stellate cells. A profound role of Sonic hedgehog 16
(SHH) pathway is implicated in desmoplasia (7) and cancer progression (8), including PDAC 17
(9). This developmental pathway, dormant in the adult pancreas, becomes reactivated early in 18
PDAC development (13). SHH is a member of the Hedgehog (Hh) family of secreted signaling 19
proteins, having diverse functions during vertebrate development (10). In pancreatic tumors, 20
intimate reciprocal interactions occur between epithelia and underlying stroma due to paracrine 21
Hh signaling that lead to desmoplasia and form a barrier to chemotherapy drug(s) penetration 22
(11). Depletion of tumor stroma leads to the increasing functional vasculature that provides a 23
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
5
feasible avenue for efficient therapeutic drug delivery (12). Additionally, Hh signaling plays a 1
key role in the maintenance of pancreatic cancer stem cells (CSCs) that are involved in drug 2
resistance, cancer recurrence and poor clinical outcome (13). Therefore, molecular and/or 3
chemical intervention to target Hh signaling and disrupt the microenvironment in tumors could 4
be an interesting therapeutic approach for pancreatic cancer (12). Some of the well-known Hh 5
signaling antagonists such as vismodegib (GDC-0449) have been investigated alone or as an 6
adjuvant to the traditional anti-cancer drugs but have not yielded clinically meaningful results 7
(14, 15) and have shown notable adverse effects including teratogenic properties (16, 17). Thus, 8
identification of novel therapies with high therapeutic index that can target Hh and tumor 9
progression signaling pathways with no or minimal adverse effects is required. 10
Repurposing of established drugs as anti-cancer agents is a current active investigative approach. 11
Ormeloxifene (ORM) is a non-hormonal, non-steroidal oral contraceptive molecule (18). Recent 12
studies suggested that ORM may be effective in inhibiting breast cancer, head and neck cancer, 13
and chronic myeloid leukemia cells (18). Moreover, ORM is reported to have an excellent 14
therapeutic index and is safe for chronic administration (19). This study demonstrates the 15
inhibitory role of ORM on the SHH signaling pathway, and describes inhibitory patterns of this 16
drug on pancreatic tumor progression using bidirectional tumor stromal interactions. This 17
inhibitory effect was either more pronounced or comparable to a known SMO inhibitor, GDC-18
0449, in PDAC cells. ORM disrupts the stroma of fibrotic pancreatic tumors and inhibits the 19
proliferating stellate and myeloid cells involved in the development of pancreatic fibrosis. 20
Further, the combinatorial effects of ORM with GEM induce increased GEM sensitivity. 21
Additionally, these studies also suggest wide use of ORM in PDAC patients due to its intended 22
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
6
safe use in fertile women, considering the teratogenic potential of other Hh pathway inhibitors 1
such as Cyclopamine and GDC-0449 (20, 21). 2
Materials and Methods 3
Cell culture, growth conditions and treatments 4
Cell lines were purchased from the American Type Cell Culture collection (ATCC) and were 5
maintained at 37ºC/5% CO2 in recommended growth medium with 10% FBS (RPMI, DMEM 6
and DMEM/Ham’s F12) (Hyclone Laboratories). Human CSCs (CD133+/CD44 +/CD24+/ESA+) 7
were obtained from Celprogen Inc. They were isolated from primary tumors and have been 8
described previously (22). ORM was generously synthesized and provided by Fathi Halaweish, 9
(South Dakota State University) as described earlier (23). GEM was purchased from Sigma 10
Aldrich (catalog number G6423) and GDC-0449 from Sellekchem (catalog number S1082). 11
Cells were treated with indicated doses of ORM, GEM and GDC-0449 after completely 12
solubilized in ethanol, PBS and DMSO, respectively. 13
Cell proliferation by MTS assay 14
The anti-proliferative effect of ORM was determined after 48 hours using the CellTiter 96 AQeous 15
One solution assay (catalog number G5421, Promega) using a microplate reader (BioMate 3 UV-16
Vis spectrophotometer, Thermo Electron Corporation). Ethanol- or PBS-containing medium 17
served as the vehicle control. Additionally, the anti-proliferative effect of ORM was determined 18
at 24 and 48 hours using Cell Counting Kit-8 (Mayflower Bioscience) and the percentage 19
viability of Panc-1 and BxPC-3cells was determined after treatment with GDC-0449 and ORM. 20
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
7
The anti-proliferative effect of each treatment was calculated as a percentage of cell growth with 1
respect to the vehicle control. 2
Cell proliferation by xCELLigence assay 3
PDAC cells (10,000 cells per well) were seeded in E-plate (Roche) following the xCELLigence 4
Real Time Cell Analyzer (RTCA) DP instrument manual as provided by the manufacturer 5
(Roche) (24). After 24 hours, ORM or the vehicle control was added and the experiment was 6
allowed to run for 100 hours. Average baseline cell index for ORM treated cells compared to 7
control cells was calculated for at least two measurements from three replicated experiments. 8
Flow cytometric analysis of apoptosis and necrosis 9
BxPC-3 and Panc-1 cells (1 x 106) were treated for 24 hours with ORM (15 µM) and GEM (100 10
nM) alone and in combination. Cells were stained with Annexin V-FITC and propidium iodide 11
(PI). The apoptotic and necrotic populations were detected as described earlier (25). Cells were 12
scanned in FL-1 (FITC) versus FL-2 (PI) channels and analyzed using an Accuri C6 flow 13
cytometer (Accuri Cytometers, Inc.). 14
Cell cycle analysis 15
Cells were exposed to ORM (15 µM) and GEM (100 nM) alone or in combination for 24 hours 16
and stained with Telford Reagent containing propidium iodide (catalog number P-4170, Sigma 17
Aldrich). Cells were analyzed with an Accuri C6 flow cytometer. Cells with hypodiploid DNA 18
(content less than G0-G1) were deemed apoptotic (sub-G0/G1). 19
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
8
Dual-luciferase reporter assay 1
Dual-luciferase reporter assay was carried out to investigate the effect of treatments on Gli-1 and 2
NFκB transcriptional activity using a luciferase assay kit (catalog number E2940; Promega) 3
according to the manufacturer's protocol. BxPC-3 and Panc-1 cells were transfected with 4
luciferase reporter constructs (NFκB, gift from Dr. Ajay Singh, Mitchell Cancer Institute; Cignal 5
GLI Reporter (luc) Kit, catalog number CCS-6030L, Qiagen) and treated with ORM and GEM 6
alone or in combination for 24 hours. The normalized luciferase activity was expressed as a ratio 7
of firefly luciferase to Renilla luciferase units. 8
Indirect co-culture of PDAC cells and pancreatic stromal cells 9
Human pancreatic stromal cell (PSC) fibroblasts and stellate cells were attained from an islet 10
transplant program and maintained in CMRL-1066 medium (catalog number 15110, Corning) 11
supplemented with 10% FBS, penicillin sodium and streptomycin sulfate at 37ºC in humidified 12
atmosphere containing 5% CO2. Human PSCs (3 x 106 cells/culture insert) were seeded into the 13
culture inserts of 1.0 µM pore size (BD Biosciences) in CMRL-1066 media. On day 2, the 14
culture inserts were placed into 6-well plates containing Panc-1 cells (0.8 x 106 cells/well), 15
followed by treatment with ORM (10 µM) and GEM (100 nM) and incubated up to 2 days in 16
DMEM medium. As previous studies have shown TGF-β to be a potent inducer of epithelial-17
mesenchymal transition (EMT) in several cancer cells including pancreatic cancer cells (26, 27), 18
we used recombinant TGF-β (2 ng/ml) to stimulate the stromal cells as a mediator of PSC-19
induced EMT in cells. 20
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
9
Clonogenic assay 1
For the clonogenic assay, 500 cells were treated with indicated concentrations of ORM for 12 2
days. The visible colonies (≥ 50 cells) were counted following hematoxylin staining (Fisher 3
Scientific) and the percent of colonies was calculated as compared to control, as described earlier 4
(28). 5
Cell motility, migration and invasion assays 6
Cell motility was analyzed with a Boyden's chamber assay (28). For cell invasion assays, BD 7
Biocoat Matrigel Invasion Chambers (BD Biosciences) were used as per manufacturer's 8
suggestions. After 48 hours incubation, the invading cells were stained and counted in 10 fields 9
of view. Additionally, a wound healing migration assay was also used to evaluate the effect of 10
ORM on the migratory ability of cancer cells. The cell monolayer was scraped using a 11
micropipette tip and 48 to 72 hours after treatment, the residual gap length was calculated from 12
photomicrographs. To further confirm these findings, real-time migration and proliferation were 13
performed by the xCELLigence system, which is an electrical impedance-based method that 14
allows for the measurement of cell migration and proliferation in real-time (24). Briefly, 4 × 104 15
cells were seeded per chamber of CIM (cell invasion and migration) plate and the cells was 16
analyzed in xCELLigence instrument at 37ºC, 5% CO2 for migration and invasion assays. 17
Immunoblot analysis 18
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
10
Human PDAC cells (1x106) were treated with ORM (15 µM) and GEM (100 nM) alone 1
and in combination for 24 hours. Total cell lysates were prepared followed by immunoblotting 2
for various indicated proteins as described earlier (25). 3
Reverse transcription–quantitative real-time polymerase chain reaction (Q-RT-PCR) 4
Total RNA was extracted using TRIZOL reagent (catalog number AM 9738, Invitrogen) and 5
integrity was checked with an RNA 6000 Nano Assay kit and 2100 Bioanalyzer (Agilent 6
Technologies). The mRNA expression levels were determined by Q-RT-PCR using Taqman 7
PCR master mixture and Taqman specific probes (Applied Biosystems). The expression of genes 8
was normalized to the 18S rRNA gene. 9
Tumorsphere assay 10
Pancreatic CSCs were plated on ultra-low attachment plates (Corning) at a density of 11
1×103/100µl well/96 well plate and treated with ORM or GDC-0449 (2.5-10 µM). The plates 12
were allowed to grow for 7 days in 0.5% serum free medium (Cellprogen) to form primary 13
spheres. Following the incubation, the primary spheres were dissociated into single cell 14
suspension and plated at a density of 1×104/2 ml/6 well ultra-low attachment plate. Secondary 15
spheres were counted after 7 to 10 days in culture. 16
17
In vivo tumor xenograft model 18
Six-week-old female athymic nude (nu/nu) mice were purchased from Charles River 19
Laboratories International, Inc., and maintained in a pathogen-free environment. The mice were 20
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
11
injected with BxPC-3 cells intraperitonally (i.p.) (3×106) and (5×106) cells in 200 µl 1
PBS/matrigel subcutaneously. On day 15, the mice were treated with vehicle (ethanol), ORM 2
(200 µg), GEM (500 µg), or their combination via intraperitoneal injections, thrice a week, for 3
six subsequent weeks. Mice were weighed twice a week to monitor their health and tumor 4
growth. Tumor volume (V) was estimated from the length (l), width (w), and height (h) of the 5
tumor using the formula V = ¼ 0.52 (l x w x h), as described previously (28) (Fig. S4). 45 days 6
after the first drug injection, mice were euthanized and tumor burden (wet weight) and 7
metastases were noted. The organs, including pancreas, were harvested and checked for 8
metastases. The data were modeled with time (discrete), group (control, ORM, GEM and 9
ORM+GEM), and the interaction between them. Primary analyses involved planned comparisons 10
(separately for each time point) between control and ORM/GEM vs. ORM+GEM. Animal care 11
was performed in accordance with institutional guidelines and all animal experiments were done 12
using protocols approved by the Sanford Research Institutional Animal Care and Use Committee 13
(IACUC). 14
In situ hybridization for microRNA detection and expression 15
We detected the expression of miR-132 in formalin fixed paraffin embedded (FFPE) tissues of 16
control and treated xenograft mice. We employed an in situ hybridization technique and used a 17
Biochain kit (catalog number K2191050, Biochain IsHyb In Situ hybridization kit) as previously 18
described (29). Briefly, tissues were hybridized with hybridization buffer and digoxigenin-19
labeled probe (EXIQON) at 45oC overnight followed by incubation with the AP-conjugated anti-20
digoxingenin antibody and NBT/BCIP (Pierce) and nuclear fast red counterstaining. 21
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
12
Immunoflorescence and Immunohistochemical analyses 1
Immunoflorescence and Immunohistochemical analyses were used to analyze the 2
untreated and treated xenograft tumor tissues to detect changes associated with the expression of 3
important proteins involved in tumor-stromal interactions as described previously (30). The 4
slides were stained with specific antibodies following heat-induced antigen retrieval techniques 5
and imaged using a laser scanning confocal microscope (Nikon TIRF) with a 20X Apochromat 6
objective for immunoflorescence. For immunohistochemistry, the slides were stained using 7
Biocare’s MACH4 Universal HRP-Polymer kit (Biocare Medical) and analyzed as previously 8
described (29, 30). 9
Statistical analyses 10
Statistical significance of the studies was analyzed by Student's t test. Differences with P values 11
of <0.05 are considered significant. Tumor size values were examined at the Day 50 point, using 12
an analysis of variance approach. Tests of main effects (differences between treatments) and 13
contrasts were performed. 14
Results 15
ORM treatment suppresses tumorigenic features of PDAC cells 16
ORM was found to have an anti-cancer effect on all tested PDAC cells (Fig. 1A and S1A). To 17
confirm these results, we measured the growth in real time for duration of 100 hours using the 18
xCELLigence System (Fig. 1B). This assay monitors cell growth in real time by measuring 19
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
13
changes in electric impedance between two golden electrodes embedded in the bottom of the cell 1
culture wells. The impedance, which is converted to a cell index value, is directly proportional to 2
the number of cells and also reflects the cells’ viability, morphology and adhesion strength (31). 3
The growth curve, which is presented as a baseline cell index, showed that ORM significantly 4
reduced the baseline cell index compared to the control cells (Fig. 1B). Further, ORM treatment 5
inhibited the clonogenic potential of PDAC cells (BxPC-3, Panc-1, AsPC-1, MiaPaca and 6
HPAF-II) as evident by the decreased number of colonies after ORM treatment (Fig. 1C). 7
Moreover, ORM was also found to inhibit cellular motility (Fig. 2A) and invasion (Fig. 2B) of 8
PDAC cells. The inhibition of the migratory ability of cells is demonstrated by wound healing 9
assay (Fig. S1B) and cellular invasion by Matrigel invasion assay (Fig. S1C), which was further 10
confirmed using the xCELLigence method (Fig. 2C). 11
Additionally, we sought to compare the anticancer potential of ORM with a known SMO 12
inhibitor (GDC-0449) in human PDAC cells. ORM showed more pronounced or comparable 13
inhibitory effect on cell proliferation, clonogenicity and invasion than GDC-0449 at equal 14
indicated concentrations (Fig. S2A, B and C). Inhibition of cell viability and invasion was 15
observed within 48 hours following exposure to these drugs. 16
17
ORM treatment inhibits tumorsphere formation in pancreatic stem cells 18
We observed a significant effect of ORM on tumorsphere formation in CSCs as reflected by a 19
reduction in size and number of tumorspheres in cells upon treatment suggesting the clonogenic 20
depletion of the CSCs. In contrast, GDC-0449 did not show a significant effect on secondary 21
tumorsphere formation (Fig. S2D). 22
23
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
14
ORM inhibits SHH signaling in PDAC cells 1
The SHH signaling pathway has been implicated in the development of pancreatic cancer (9). 2
Therefore, PDAC cells were treated with ORM and changes in the SHH signaling pathway were 3
evaluated by Western blot and qRT-PCR analyses. ORM treatment effectively inhibited SHH 4
expression at protein and mRNA levels at indicated concentrations (Fig. 2D and S3A and B). 5
ORM treatment also inhibited the expression of Gli-1, SMO, cyclin D1 and p-AKT, the key 6
downstream proteins that drive the oncogenic signaling of SHH signaling pathway in BxPC-3 7
and MiaPaca cells (11) (Fig. 2D and S3A). ORM treatment also increased the expression of 8
tumor suppressor SUFU, which interacts directly with Gli-1 proteins to repress SHH signaling 9
(32) (Fig. 2D and S3A). 10
Importantly, ORM treatment caused a marked (~70%) decrease in the expression of the SHH 11
transcription factor NFκB-65 (33) and its downstream target, Cyclin D1 (34), within 24 hours 12
(Fig. 2D and S3C). Cyclin D1 is the important mediator of SHH-induced cell proliferation and 13
carcinogenesis. These data suggest that ORM treatment effectively inhibits tumorigenic 14
phenotypes via modulation of SHH and its downstream signaling molecules. 15
ORM and GEM in combination induce apoptosis in PDAC cells 16
We investigated if ORM treatment enhanced the apoptotic index in GEM-resistant PDAC cells 17
(Panc-1 and BxPC-3). Our data show that when combined, ORM (15 µM) and GEM (100 nM) 18
induced a significantly higher (21%) apoptotic population in 24 hours as compared to ORM and 19
GEM treatment alone (Fig. 3A). However, PI-positive post-apoptotic/necrotic cell population 20
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
15
was relatively small, suggesting that the induced cytotoxicity was predominantly through 1
activation of apoptotic pathways. This data suggests that ORM-alone induced cell death does not 2
involve the release of phosphatidylserine onto the outer leaflet, indicative of Annexin V positive 3
apoptotic cells and mitochondrial apoptotic signaling. Instead, it may involve death receptor-4
mediated extrinsic apoptotic signaling. Therefore, we sought to investigate the effect of ORM on 5
cell cycle phase distribution. Typically, D-type cyclins are required for the progression of cells 6
from the G1 phase of the cell cycle to S phase (35). ORM treatment decreased the expression of 7
cyclin D in BxPC-3 and MiaPaca cells (Fig. 2D and S3A). ORM treatment led to cell cycle arrest 8
at sub-G0-G1 phase in Panc-1 and BxPC-3 cells. Cells in sub-G1 phase increased up to 74% 9
after ORM treatment (15 µM), while cells in the S phase decreased from 19 to 5%. However, 10
GEM treatment did not show an additional effect on cell cycle phases (Fig. 3B). Similar effects 11
in cell cycle phase distribution were observed in BxPC-3 cells, which showed significant 12
inhibition of the G2M phase upon treatment with ORM and GEM in combination. 13
ORM and GEM combination targets SHH signaling pathway and inhibits cell invasion 14
and migration in PDAC cells 15
Additionally, we investigated combinatorial effects of ORM and GEM on SHH and downstream 16
signaling molecules. Treatment with ORM and GEM has relatively more pronounced inhibitory 17
effects on the expression of SHH, Gli-1 and SMO as compared to ORM or GEM alone (Fig. 3D). 18
This reveals the potentiated effects of ORM in combination with GEM. We also confirmed these 19
results by qRT-PCR analysis and observed an apparent decrease in the mRNA levels of main 20
effectors of the SHH signaling pathway in response to ORM alone or in combination with GEM. 21
This included decreased expression of SHH (four-fold), SMO (five-fold) and patched 1/2 22
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
16
(PTCH1/2) compared to the control (Fig. 3C). ORM alone or in combination with GEM also 1
showed a marked (~40%) decrease in the level of anti-apoptotic, Bcl-xL protein (Fig. 3D). The 2
Bcl-xL protein is also an important mediator of SHH and is transcriptionally regulated by SHH 3
through the Gli-1 transcription factor (34). Additionally, ORM alone or in combination with 4
GEM inhibited the Gli-1 and NFκB-65 transcriptional activity in PDAC cells (Fig. 4A and S3C). 5
These results present first evidence that ORM inhibits the SHH–Gli-1 signaling pathway in 6
PDAC. 7
Moreover, we evaluated the ability of ORM and GEM to inhibit tumor progression and found 8
that ORM inhibited motility (Fig. 4B) and the migratory ability of PDAC cells as demonstrated 9
by wound healing (Fig. 4C). 10
ORM and GEM combination efficiently abrogates TGF-β induced SHH signaling 11
The interactions among the stromal and tumor cells and the various cytokines embedded in the 12
extracellular matrix (ECM) contribute to the neoplastic phenotype (36). In addition to the 13
activated tumor-stromal myofibroblasts (characterized by the expression of contractile genes 14
such as smooth-muscle actin, αSMA) (37), the activated pancreatic stellate cells (PSCs) that are 15
characterized by expression of the stellate cell activation-associated protein (cygb/STAP) are 16
identified as the major source of the excessive stromal ECM production in pancreatic tumors (6). 17
Here, we show for the first time that indirect co-culture of PDAC cells with PSCs that are 18
stimulated with TGF-β induce increased secretion of SHH and chemokine CXCL12 (stromal 19
cell-derived factor-1, SDF1) was abrogated by ORM alone or in combination with GEM. GEM 20
treatment alone did not show any effect as observed through ELISA of the conditioned media in 21
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
17
which the PDAC cells were cultured (Fig. 4D). CXCL12 is abundantly produced by the stromal 1
cells that induce SHH expression, which promotes progression, metastasis and chemoresistance 2
of PDAC cells (38). Additionally, treatment with ORM alone or in combination inhibited the 3
proliferation of pancreatic stromal cells as depicted by the decreased expression of αSMA and 4
cygb/STAP in the immunofluorescence of PSCs (Fig. 4D; lower panel). These results suggest 5
that ORM not only reduces the number of stromal cells involved in the development of 6
pancreatic fibrosis but also inhibits the paracrine SHH signaling between cancer and stromal 7
cells that leads to desmoplasia and causes chemoresistance. 8
Combined ORM and GEM treatment effectively inhibits tumor burden in mice model 9
To investigate the anti-cancer effects of ORM, we used a subcutaneous (for solid tumor) and 10
intraperitoneal (metastatic) pre-clinical murine xenograft model generated with GEM resistant 11
BxPC-3 cells. Both ORM and GEM, administered alone, inhibited overall tumor burden, but 12
combination treatment of the two was more efficacious than either of them alone (Fig. 5A and 13
B). When compared to the control mice, mice treated with ORM (p = 0.0301) or GEM (p = 14
0.0009) or ORM+GEM in combination (p < 0.0001) showed a marked reduction in tumor weight 15
(Fig. 5B). Moreover, in the intraperitoneal model, tumors barely developed in the ORM+GEM 16
treated group. Upon further examination, we also found there were fewer or no metastases in the 17
mice treated with ORM alone or in combination with GEM (Fig. 5B, inset table). miR-132 is 18
downregulated in pancreatic cancer, which contributes to pancreatic cancer development (39). 19
Treatment of ORM alone or in combination with GEM leads to increased levels of miR-132 in 20
xenograft tumors (Fig. 5C). This data further confirms that ORM treatment along with GEM 21
could be an effective therapeutic modality for pancreatic cancer. 22
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
18
ORM inhibits tumor desmoplasia and the host cells invading the tumor 1
To elucidate the basis of the potentiated anti-tumorigenic effects of ORM in combination with 2
GEM in mice, we analyzed the FFPE tumor tissues through tumor histopathology, 3
immunofluorescence (IF) and immunohistochemical (IHC) analyses. We observed a clear 4
inhibition of Gli-1 expression in tumor tissues from mice treated with either ORM alone or in 5
combination with GEM (Fig. 5D). In contrast to vehicle or GEM treated mice, which exhibited 6
profuse desmoplastic tumor stroma, mice treated with ORM showed markedly depleted 7
desmoplastic stroma. This was evidenced by a decrease in collagen I content in tumor xenografts 8
and the invading host mice cells migrating into the tumors (Fig. 6A). It was found that only 9
ORM, but not GEM, reduced the amount of collagen I deposition. Interestingly, these differences 10
were apparent in mice treated with ORM or ORM+GEM. Additionally, ORM alone or in 11
combination with GEM treatment showed decreased number of activated stromal cell 12
populations as identified by the reduced expression of αSMA and Fibroblast surface protein 13
(FSP) positive stromal myofibroblasts (Fig. 6A) and cygb/STAP positive activated stellate cells 14
(Fig. 6B) (37). GEM alone treatment did not show any effects on these parameters. This decrease 15
in proliferation was accompanied by a decrease in SHH expression in ORM and ORM+GEM 16
treated tumor tissues (Fig. 6B). Further, we analyzed these tumor tissues for the presence of 17
tumor infiltrating macrophages and found a large increasing population of macrophages in 18
ORM-treated mice tumor tissues (Fig. 6B) that might become tumoricidal and facilitate the 19
depletion of the tumor stroma (40, 41). This signifies that ORM alone or in combination with 20
GEM inhibits the host cells invading the tumor tissue and disrupts the desmoplastic stroma that 21
can facilitate the delivery and enhance the efficacy of GEM. 22
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
19
Discussion 1
Pancreatic tumors are typically characterized by a high desmoplastic reaction (42). Desmoplasia 2
plays an important role to initiate cross-talk between stromal-cancer cells, limit the delivery and 3
effectiveness of chemotherapy, and induce chemoresistance. The Sonic hedgehog (SHH) 4
pathway is a major player for desmoplasia and is activated in both stromal and cancer cells in 5
PDAC (7, 43). Therefore, suppression of the Hh pathway and desmoplasia may limit the 6
molecular/clinical course of PDAC and improve drug(s) access in tumors (12). Currently 7
available Hh pathway antagonists, including GDC-0449, have been investigated as a single agent 8
or in combination with conventional chemotherapies for cancer treatment (14, 15). GDC-0449 is 9
an SMO (Hh) inhibitor approved by the FDA for the treatment of locally advanced and 10
metastatic basal cell carcinomas. But severe toxicity issues and adverse effects (fatigue, nausea, 11
asthenia, mucositis, peripheral sensory neuropathy, dysgeusia, muscle spasms, and dehydration) 12
and the lack of strong efficacy, limits its use in cancer therapy (44). Additionally, no significant 13
improvement in survival of pancreatic, colon and ovarian cancer patients is noticed in recent 14
clinical trials of Hh signaling inhibitors. As other signaling pathways (such as PI3K or TGFβ 15
signaling) (45, 46) are also known to activate transcriptional activity of Gli in addition to SMO, 16
the therapeutic efficacy of SMO inhibitors is compromised in cancer. This is a probable rationale 17
for shifting interest from SMO inhibitors towards more specific Gli inhibitors in order to 18
effectively suppress the Hh signaling pathway. In this endeavor, we have identified ORM, a non-19
steroidal triphenylethylene compound that effectively blocks the Hh signaling pathway by 20
inhibiting the important effectors of this pathway, such as SHH, SMO, Gli-1, and SDF-1 21
(CXCL12). ORM disrupts multiple paracrine factors that are important for the maintenance of 22
Hh signaling, and thus inhibits stromal and tumor cell cross-talk within the tumor. 23
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
20
1
Experimental investigations indicate that ORM inhibits proliferation, invasion and clonogenicity 2
of PDAC cells (Fig. 1 and Fig. S2), comparable to cells treated with GDC-0449. Additionally, 3
reduced tumorsphere formation of CSCs that were treated with ORM indicates that ORM also 4
inhibits pancreatic CSC proliferation and self renewal. This suggests that the anticancer effects 5
of ORM are greater or comparable to GDC-0449. Investigations of the mechanism of ORM-6
induced cell death showed the induction of cell cycle arrest at G0-G1 phase, suggesting that 7
ORM may induce apoptosis. It was also an intriguing observation that treatment of ORM in 8
combination with GEM showed an increasing population of Annexin V positive cells as 9
compared to when both were used alone. These results indicate that in the presence of ORM, 10
GEM induces higher apoptotic cell death that might be triggered through the mitochondrial 11
pathway. Alternatively, the other possibility is that ORM might involve death receptor-mediated 12
cell death. Altogether, the results indicated that ORM potentiates the anticancer effect of GEM 13
when used in combination. 14
It has been reported that NFκB (33) and SHH (7, 43) signaling pathways play crucial roles in 15
PDAC progression and drug resistance, including GEM. ORM treatment stabilizes IκB-α, which 16
inhibits protein and transcriptional activity of NFκB-65, preventing it from binding to the SHH 17
promoter and leading to its transcription (33). This was further confirmed on finding that ORM 18
alone or in combination with GEM inhibits the main downstream targets of SHH, SMO, and 19
PTCH1/2 and downregulates the expression and transcriptional activity of Gli-1 in PDAC cells. 20
No such effects were found in cells when treated with GEM alone. In the absence of SHH, cells 21
have small amounts of PTCH1/2 and Gli and therefore, the high concentrations of these 22
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
21
transcripts generally indicate involvement of the SHH pathway in PDAC (47). The aberrantly 1
activated SHH binds to its receptor PTCH1/2 and inhibits the suppressive effect of PTCH1/2 on 2
SMO, which activates Gli-1 to transcribe Hh oncogenic target genes (43). ORM inhibits AKT 3
phosphorylation, which is known to activate Gli-1 (48). The observations collected from co-4
culturing the PDAC cells with stromal cells indicate the inhibition of paracrine stromal cell 5
signaling through the inhibition of their proliferation and secretion of SHH and SDF1. All these 6
results confirm that ORM inhibits hedgehog signaling in PDAC cells; thus, we hypothesized that 7
it might also disrupt the stroma of pancreatic tumors and alter the desmoplastic reaction. 8
Recent studies implicate the profound role of stroma in drug resistance in numerous tumor types 9
(49). Thus, treatment paradigms targeting both neoplastic cells and stromal components are 10
emerging for PDAC (50). The enhanced anti-tumor effect of ORM and GEM combination 11
treatment was observed in xenograft mouse models when compared to their treatment alone. An 12
abundant stromal component was observed in the control and GEM treated tumor tissues while 13
mice treated with ORM alone or ORM + GEM combination showed markedly less stromal 14
component and invaded stromal tissue. This was indicated by the presence of reduced numbers 15
of stroma myofibroblasts infiltrating the tumor tissue, as indicated by reduced PSCs, αSMA, FSP 16
and cygb/STAP expression in the tumor tissue. This might be well supported by the observations 17
showing the inhibition of both mouse and human collagen I in tissues, as activated PSCs are the 18
predominant source of collagen in the desmoplastic reaction in pancreatic cancers (6). 19
Interestingly, we also observed an increased number of macrophages in the tumors obtained from 20
ORM treated mice. The increasing macrophage recruitment to tumor site can be explained as the 21
emergence of tumor immunity that serves as a part of immune surveillance for targeting tumor 22
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
22
stroma in the treatment of cancer. The activated macrophages may rapidly infiltrate the tumors, 1
become tumoricidal and facilitate the depletion of tumor stroma. 2
3
These findings suggest that ORM inhibits desmoplastic reaction in PDAC. Due to the toxicity 4
and morbidity of available drugs, there is an urgent need for effective therapies that could target 5
both tumor and stromal compartments to regulate pancreatic tumor growth. Therefore, ORM 6
might be a drug of choice as it is very safe for chronic use in humans. Our results signify that 7
ORM is effective in targeting Hh and tumor progression signaling pathways. Our results 8
emphasize that ORM interrupts the tumor-stromal interactions to inhibit the reciprocal 9
relationship between these two components, leading to reduction of tumor progression, invasion, 10
metastasis and chemoresistance (Fig. 7). This facilitates the anti-cancer effects of ORM and 11
potentiates the chemotherapeutic effects of GEM for pancreatic cancer treatment. Our results 12
have important implications towards the development of effective therapy for pancreatic cancer 13
treatment. 14
Conclusion 15
In summary, our study provides new evidence regarding the anti-cancer effects of ORM in 16
PDAC. This study demonstrates novel role of an existing drug, ORM to inhibit the SHH pathway 17
and desmoplasia, resulting in tumor growth inhibition and potentiation of the anti-tumor effect of 18
GEM. This suggests that a combination of ORM and GEM may have the capacity to inhibit the 19
SHH signaling cascade in PDAC cells and alter the behavior of surrounding stromal cells so that 20
cancer progression is repressed. Therefore, this study provides evidence that ORM in 21
combination with GEM could serve as a novel therapeutic intervention for pancreatic cancer. 22
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
23
Acknowledgements 1
This work was partially supported by grants from Department of Defense (PC073887 to SCC 2
and PC073643 MJ), the National Institutes of Health (RO1 CA142736 to SCC), Pilot grant to SK 3
(8P20GM103548-02) and UO1 CA162106A (to SCC and MJ) and the College of Pharmacy 4
2013 Dean's Seed Grant of the University of Tennessee Health Science Center (to MJ and 5
MMY). Authors also acknowledge the Kosten Foundation for pancreatic cancer research 6
support. The authors are also thankful to Cathy Christopherson (Sanford Research) for editorial 7
assistance. 8
References 9
1. Burris HA, 3rd, Moore MJ, Andersen J, Green MR, Rothenberg ML, Modiano MR, et al. 10
Improvements in survival and clinical benefit with gemcitabine as first-line therapy for 11
patients with advanced pancreas cancer: a randomized trial. J Clin Oncol. 1997;15:2403-13. 12
2. Moorcraft SY, Khan K, Peckitt C, Watkins D, Rao S, Cunningham D, et al. FOLFIRINOX 13
for Locally Advanced or Metastatic Pancreatic Ductal Adenocarcinoma: The Royal 14
Marsden Experience. Clin Colorectal Canc. 2014;13:232-8. 15
3. Conroy T, Desseigne F, Ychou M, Bouche O, Guimbaud R, Becouarn Y, et al. 16
FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med. 17
2011;364:1817-25. 18
4. Von Hoff DD, Ervin T, Arena FP, Chiorean EG, Infante J, Moore M, et al. Increased 19
survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N Engl J Med. 20
2013;369:1691-703. 21
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
24
5. Han H, Bearss DJ, Browne LW, Calaluce R, Nagle RB, Von Hoff DD. Identification of 1
differentially expressed genes in pancreatic cancer cells using cDNA microarray. Cancer 2
Res. 2002;62:2890-6. 3
6. Apte MV, Park S, Phillips PA, Santucci N, Goldstein D, Kumar RK, et al. Desmoplastic 4
reaction in pancreatic cancer: role of pancreatic stellate cells. Pancreas. 2004;29:179-87. 5
7. Bailey JM, Swanson BJ, Hamada T, Eggers JP, Singh PK, Caffery T, et al. Sonic hedgehog 6
promotes desmoplasia in pancreatic cancer. Clin Cancer Res. 2008;14:5995-6004. 7
8. Pasca di Magliano M, Hebrok M. Hedgehog signalling in cancer formation and 8
maintenance. Nat Rev Cancer. 2003;3:903-11. 9
9. Thayer SP, di Magliano MP, Heiser PW, Nielsen CM, Roberts DJ, Lauwers GY, et al. 10
Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature. 11
2003;425:851-6. 12
10. Varjosalo M, Taipale J. Hedgehog: functions and mechanisms. Gene Dev. 2008;22:2454-72. 13
11. Kelleher FC. Hedgehog signaling and therapeutics in pancreatic cancer. Carcinogenesis. 14
2011;32:445-51. 15
12. Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, et al. 16
Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of 17
pancreatic cancer. Science. 2009;324:1457-61. 18
13. Merchant AA, Matsui W. Targeting Hedgehog--a cancer stem cell pathway. Clin Cancer 19
Res. 2010;16:3130-40. 20
14. Kim EJ, Sahai V, Abel EV, Griffith KA, Greenson JK, Takebe N, et al. Pilot Clinical Trial 21
of Hedgehog Pathway Inhibitor GDC-0449 (Vismodegib) in Combination with Gemcitabine 22
in Patients with Metastatic Pancreatic Adenocarcinoma. Clin Cancer Res. 2014;20:5937-45. 23
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
25
15. LoRusso PM, Rudin CM, Reddy JC, Tibes R, Weiss GJ, Borad MJ, et al. Phase I trial of 1
hedgehog pathway inhibitor vismodegib (GDC-0449) in patients with refractory, locally 2
advanced or metastatic solid tumors. Clin Cancer Res. 2011;17:2502-11. 3
16. Juhasz ML, Marmur ES. Systematic review of vismodegib toxicity profile in the treatment 4
of advanced Basal cell carcinomas compared to other systemic therapies in dermatology. J 5
Drugs Dermatol. 2014;13:729-33. 6
17. Sheikh A, Alvi AA, Aslam HM, Haseeb A. Hedgehog pathway inhibitors - current status 7
and future prospects. Infect Agent Cancer. 2012;7:29. 8
18. Gara RK, Sundram V, Chauhan SC, Jaggi M. Anti-cancer potential of a novel SERM 9
ormeloxifene. Curr Med Chem. 2013;20:4177-84. 10
19. Singh MM. Centchroman, a selective estrogen receptor modulator, as a contraceptive and 11
for the management of hormone-related clinical disorders. Med Res Rev. 2001;21:302-47. 12
20. Lipinski RJ, Hutson PR, Hannam PW, Nydza RJ, Washington IM, Moore RW, et al. Dose- 13
and route-dependent teratogenicity, toxicity, and pharmacokinetic profiles of the hedgehog 14
signaling antagonist cyclopamine in the mouse. Toxicol Sci. 2008;104:189-97. 15
21. LoRusso PM, Piha-Paul SA, Mita M, Colevas AD, Malhi V, Colburn D, et al. Co-16
administration of vismodegib with rosiglitazone or combined oral contraceptive in patients 17
with locally advanced or metastatic solid tumors: a pharmacokinetic assessment of drug-18
drug interaction potential. Cancer chemoth Pharm. 2013;71:193-202. 19
22. Shankar S, Nall D, Tang SN, Meeker D, Passarini J, Sharma J, et al. Resveratrol inhibits 20
pancreatic cancer stem cell characteristics in human and KrasG12D transgenic mice by 21
inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition. PLoS 22
One. 2011;6:e16530. 23
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
26
23. Ji X MY, Liu Y, Jin T, Song P. Journal of Chinese Pharmaceutical Sciences. 1998;7:69-71. 1
24. Limame R, Wouters A, Pauwels B, Fransen E, Peeters M, Lardon F, et al. Comparative 2
analysis of dynamic cell viability, migration and invasion assessments by novel real-time 3
technology and classic endpoint assays. PLoS One. 2012;7:e46536. 4
25. Khan S, Kaur R, Shah BA, Malik F, Kumar A, Bhushan S, et al. A Novel cyano derivative 5
of 11-Keto-beta-Boswellic acid causes apoptotic death by disrupting PI3K/AKT/Hsp-90 6
cascade, mitochondrial integrity, and other cell survival signaling events in HL-60 cells. 7
Mol Carcinogen. 2012;51:679-95. 8
26. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 9
2009;119:1420-8. 10
27. Berna MJ, Seiz O, Nast JF, Benten D, Blaker M, Koch J, et al. CCK1 and CCK2 receptors 11
are expressed on pancreatic stellate cells and induce collagen production. J Biol Chem. 12
2010;285:38905-14. 13
28. Chauhan SC, Ebeling MC, Maher DM, Koch MD, Watanabe A, Aburatani H, et al. MUC13 14
mucin augments pancreatic tumorigenesis. Mol Cancer Ther. 2012;11:24-33. 15
29. Khan S, Ebeling MC, Zaman MS, Sikander M, Yallapu MM, Chauhan N, et al. MicroRNA-16
145 targets MUC13 and suppresses growth and invasion of pancreatic cancer. Oncotarget. 17
2014;5:7599-609. 18
30. Yallapu MM, Ebeling MC, Khan S, Sundram V, Chauhan N, Gupta BK, et al. Novel 19
curcumin-loaded magnetic nanoparticles for pancreatic cancer treatment. Mol Cancer Ther. 20
2013;12:1471-80. 21
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
27
31. Abassi YA, Xi B, Zhang W, Ye P, Kirstein SL, Gaylord MR, et al. Kinetic cell-based 1
morphological screening: prediction of mechanism of compound action and off-target 2
effects. Chem Biol. 2009;16:712-23. 3
32. Dunaeva M, Michelson P, Kogerman P, Toftgard R. Characterization of the physical 4
interaction of Gli proteins with SUFU proteins. J Biol Chem. 2003;278:5116-22. 5
33. Kasperczyk H, Baumann B, Debatin KM, Fulda S. Characterization of sonic hedgehog as a 6
novel NF-kappaB target gene that promotes NF-kappaB-mediated apoptosis resistance and 7
tumor growth in vivo. FASEB J. 2009;23:21-33. 8
34. Morton JP, Mongeau ME, Klimstra DS, Morris JP, Lee YC, Kawaguchi Y, et al. Sonic 9
hedgehog acts at multiple stages during pancreatic tumorigenesis. PNAS. 2007;104:5103-8. 10
35. Matsushime H, Roussel MF, Ashmun RA, Sherr CJ. Colony-stimulating factor 1 regulates 11
novel cyclins during the G1 phase of the cell cycle. Cell. 1991;65:701-13. 12
36. Shekhar MP, Pauley R, Heppner G. Host microenvironment in breast cancer development: 13
extracellular matrix-stromal cell contribution to neoplastic phenotype of epithelial cells in 14
the breast. Breast cancer Res. 2003;5:130-5. 15
37. Liu M, Xu J, Deng H. Tangled fibroblasts in tumor-stroma interactions. Int J Cancer. 16
2011;129:1795-805. 17
38. Singh AP, Arora S, Bhardwaj A, Srivastava SK, Kadakia MP, Wang B, et al. 18
CXCL12/CXCR4 protein signaling axis induces sonic hedgehog expression in pancreatic 19
cancer cells via extracellular regulated kinase- and Akt kinase-mediated activation of 20
nuclear factor kappaB: implications for bidirectional tumor-stromal interactions. J Biol 21
Chem. 2012;287:39115-24. 22
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
28
39. Zhang S, Hao J, Xie F, Hu X, Liu C, Tong J, et al. Downregulation of miR-132 by promoter 1
methylation contributes to pancreatic cancer development. Carcinogenesis. 2011;32:1183-9. 2
40. Beatty GL, Chiorean EG, Fishman MP, Saboury B, Teitelbaum UR, Sun W, et al. CD40 3
agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and 4
humans. Science. 2011;331:1612-6. 5
41. Mantovani A, Sica A. Macrophages, innate immunity and cancer: balance, tolerance, and 6
diversity. Curr Opin Immunol. 2010;22:231-7. 7
42. Merika EE, Syrigos KN, Saif MW. Desmoplasia in pancreatic cancer. Can we fight it? 8
Gastroenterol Res Pract. 2012;2012:781765. 9
43. Bailey JM, Mohr AM, Hollingsworth MA. Sonic hedgehog paracrine signaling regulates 10
metastasis and lymphangiogenesis in pancreatic cancer. Oncogene. 2009;28:3513-25. 11
44. Berlin J, Bendell JC, Hart LL, Firdaus I, Gore I, Hermann RC, et al. A randomized phase II 12
trial of vismodegib versus placebo with FOLFOX or FOLFIRI and bevacizumab in patients 13
with previously untreated metastatic colorectal cancer. Clin Cancer Res. 2013;19:258-67. 14
45. Javelaud D, Pierrat MJ, Mauviel A. Crosstalk between TGF-beta and hedgehog signaling in 15
cancer. FEBS letters. 2012;586:2016-25. 16
46. Ramaswamy B, Lu Y, Teng KY, Nuovo G, Li X, Shapiro CL, et al. Hedgehog signaling is a 17
novel therapeutic target in tamoxifen-resistant breast cancer aberrantly activated by 18
PI3K/AKT pathway. Cancer Res. 2012;72:5048-59. 19
47. Elamin MH, Shinwari Z, Hendrayani SF, Al-Hindi H, Al-Shail E, Khafaga Y, et al. 20
Curcumin inhibits the Sonic Hedgehog signaling pathway and triggers apoptosis in 21
medulloblastoma cells. Mol Carcinogen. 2010;49:302-14. 22
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
29
48. Stecca B, Mas C, Clement V, Zbinden M, Correa R, Piguet V, et al. Melanomas require 1
HEDGEHOG-GLI signaling regulated by interactions between GLI1 and the RAS-2
MEK/AKT pathways. PNAS. 2007;104:5895-900. 3
49. Straussman R, Morikawa T, Shee K, Barzily-Rokni M, Qian ZR, Du J, et al. Tumour micro-4
environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature. 5
2012;487:500-4. 6
50. Heinemann V, Haas M, Boeck S. Systemic treatment of advanced pancreatic cancer. Cancer 7
Treat Rev. 2012;38:843-53. 8
9
Legends 10
Figure 1. Determination of proliferation, clonogenicity and cytotoxicity profiles of ORM in 11
PDAC cells. (A) Structure of ormeloxifene (IUPAC name: 1-[2-[4-[(3S,4R)-7-methoxy-2,2- 12
dimethyl-3-phenyl-chroman-4-yl] phenoxy] ethyl] pyrrolidine) and its effect on cell growth was 13
monitored by MTS assay for 48 hours and is shown as percentage. (B) Clonogenicity assay was 14
performed to determine the ability of cells to form colonies (percent inhibition) following 15
treatment. Cells were photographed and counted using AlphaEaseFC™ (Alpha ImagerHP AIC) 16
software analysis tool. Bars represent mean ± SD; n=3; *p<0.05 and **p<0.001. 17
Figure 2. ORM targets Sonic hedgehog signaling pathway and inhibits PDAC cell invasion 18
and migration. Effect of ORM on (A) cell invasion through matrigel invasion assay (B) cell 19
migration ability through migration assay. Cells were photographed and counted using an 20
imaging system. Bars represent mean ± SD; n=3; *p<0.0001. (C) Effect of ORM on cell invasion 21
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
30
and migration ability was confirmed using β-actin as an internal control. Flow cytometric 1
analysis of Annexin V positive cells and cells in G0–G1 stage after treatment. Bars represent 2
mean ± SD; n=3; *p<0.01, **p<0.001 and ***p<0.0001 as compared to CT. (C) Relative fold 3
change in the mRNA levels of key molecules involved in Sonic hedgehog pathway by qRT-PCR. 4
Bars represent mean ± SD; n=3; *p<0.01, **p<0.001 and ***p<0.0001. (D) Western blotting 5
analysis indicating the effect of ORM and GEM on the important proteins in Sonic hedgehog 6
pathway. Data are representative of one of three similar experiments. 7
Figure 4. ORM and GEM combination inhibits Gli-1 transcriptional activity and inhibits 8
pancreatic cancer invasiveness. (A) Treatment of ORM alone or with GEM inhibited Gli-1 9
transcriptional activity and (B) inhibited cell migration. (C) Wound healing assay. The initial (0 10
hours) and the residual gap length, 48 hours after wounding, were analyzed from 11
photomicrographs. (D) Indirect co-culture of PDAC and stromal cells and treatment with ORM 12
alone and in combination with GEM. ELISA was performed to observe the effect on the 13
secretion of key proteins (SHH and SDF1) involved in tumor stromal interactions. Bars represent 14
mean ± SD; n=3; *p<0.01 and **p<0.001. Immunofluorescence indicates that treatment with 15
ORM and GEM in the presence of pancreatic stromal cells (PSCs) reduces the number of 16
myofibroblasts expressing Cygb/STAP and αSMA. 17
Figure 5. ORM and GEM in combination inhibit tumor growth in pancreatic xenograft 18
mice. (A) Photographs of xenograft mice from each treatment group. (B) Average tumor volume 19
and average tumor weight was determined. Bars represent mean ± SD; *p<0.01 and **p<0.0001. 20
The corresponding inset table shows the effect of ORM on tumor development and 21
dissemination. (C) In situ hybridization for tumor suppressor miR-132 was performed on the 22
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
31
excised tumor tissues from treated mice. (D) Immunohistochemical staining showing the 1
inhibition of Gli-1 expression in tumor tissues from mice treated with ORM alone or with GEM. 2
Bars represent mean ± SD; *p<0.01 and **p<0.0001. 3
Figure 6. Representative photomicrographs of immunofluorescence studies on excised 4
xenograft tumor tissues using confocal microscopy. (A and B) Treatment of ORM alone or in 5
combination with GEM inhibited both human and mouse collagen I, FSP, and therefore reduced 6
the number of total stroma cells within the tumor which is indicated by reduced myofibroblasts 7
expressing αSMA and cygb/STAP. This was observed using a laser scanning confocal 8
microscope (Nikon TIRF), original magnifications 20X. Additionally, tissues were stained for 9
F4/80 that indicated increased number of macrophages infiltrating into the tumor. 10
Figure 7. Diagrammatic representation of the ORM modulation of the Sonic hedgehog 11
signaling pathway. 12
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397
Correction
Correction: Ormeloxifene SuppressesDesmoplasia and Enhances Sensitivity ofGemcitabine in Pancreatic Cancer
In the original version of this article (1), the loading controls for theWestern blots inFigs. 2D and 3D were missing for all different percentages of gels. These have beenadded in the recent online HTML and PDF versions of the article. The authors regretthis omission.
Reference1. Khan S, Ebeling MC, Chauhan N, Thompson PA, Gara RK, Ganju A, et al. Ormeloxifene sup-
presses desmoplasia and enhances sensitivity of gemcitabine in pancreatic cancer. Cancer Res2015;75:2292–304.
Published online May 1, 2018.doi: 10.1158/0008-5472.CAN-18-0427�2018 American Association for Cancer Research.
CancerResearch
Cancer Res; 78(9) May 1, 20182444
Published OnlineFirst April 3, 2015.Cancer Res Sheema Khan, Mara C Ebeling, Neeraj Chauhan, et al. Sensitivity of Gemcitabine in Pancreatic CancerOrmeloxifene Suppresses Desmoplasia and Enhances
Updated version
10.1158/0008-5472.CAN-14-2397doi:
Access the most recent version of this article at:
Material
Supplementary
http://cancerres.aacrjournals.org/content/suppl/2015/04/04/0008-5472.CAN-14-2397.DC1
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://cancerres.aacrjournals.org/content/early/2015/04/03/0008-5472.CAN-14-2397To request permission to re-use all or part of this article, use this link
on May 17, 2018. © 2015 American Association for Cancer Research. cancerres.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 April 3, 2015; DOI: 10.1158/0008-5472.CAN-14-2397