Ormeloxifene Suppresses Desmoplasia and Enhances ...cancerres.aacrjournals.org/content/canres/early/2015/04/...4 Ganju1, Murali M. Yallapu1, Stephen W. Behrman4, Haotian Zhao2, Nadeem

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





http://cancerres.aacrjournals.org/



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

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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

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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




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




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




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




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




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




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




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




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




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

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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




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




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




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




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




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




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




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




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




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

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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




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




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

























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

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-18-0427&domain=pdf&date_stamp=2018-4-27

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

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