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Published OnlineFirst April 1, 2014; DOI: 10.1158/1940-6207.CAPR-13-0362

Cancer Prevention Research Article Research

Inhibition of the Transition of Ductal Carcinoma In Situ to Invasive Ductal Carcinoma by a Gemini Vitamin D Analog

Joseph Wahler1, Jae Young So1, Yeoun Chan Kim1, Fang Liu1,2,3, Hubert Maehr1, Milan Uskokovic1, and Nanjoo Suh1,3

Abstract Ductal carcinoma in situ (DCIS) is a nonmalignant lesion of the breast with the potential to progress to invasive ductal carcinoma (IDC). The disappearance and breakdown of the myoepithelial cell layer and basement membrane in DCIS have been identified as major events in the development of breast cancer. The MCF10DCIS.com cell line is a well-established model, which recapitulates the progression of breast cancer from DCIS to IDC. We have previously reported that a novel Gemini vitamin D analog, 1a,25-dihydroxy- 20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124) is a potent inhibitor of the growth of MCF10DCIS.com xenografted tumors without hypercalcemic toxicity. In this study, we utilized the MCF10DCIS.com in vivo model to assess the effects of BXL0124 on breast cancer progression from weeks 1 to 4. Upon DCIS progression to IDC from weeks 3 to 4, tumors lost the myoepithelial cell layer and basement membrane as shown by immunofluorescence staining with smooth muscle actin and laminin 5, respectively. Administration of BXL0124 maintained the critical myoepithelial cell layer as well as basement membrane, and animals treated with BXL0124 showed a 43% reduction in tumor volume by week 4. BXL0124 treatment decreased cell proliferation and maintained vitamin D receptor levels in tumors. In addition, the BXL0124 treatment reduced the mRNA levels of matrix metalloproteinases starting at week 3, contributing to the inhibition of invasive transition. Our results suggest that the maintenance of DCIS plays a significant role in the cancer preventive action of the Gemini vitamin D BXL0124 during the progression of breast lesions. Cancer Prev Res; 7(6); 617–26. 2014 AACR.

Introduction the transition from DCIS to invasive ductal carcinoma Breast cancer is one of the most common cancers, and the (IDC) is a crucial event in the advancement of breast tumors. second highest cancer-related cause of death among women The human breast ductal structure is made up of an inner (1). Regardless of its high prevalence in society, the etiology layer of luminal epithelial cells, surrounded by an outer and pathogenesis of breast cancer have yet to be elucidated layer of myoepithelial cells and is separated from the stroma (2–5). Genetic, hormonal, and environmental factors have by the basement membrane (13). In order for tumor cells to been suggested as causes of breast cancer (6, 7). Early invade and metastasize, they must penetrate these 2 pro- development of breast cancer consists of noninvasive tective layers (14). The disappearance and breakdown of the lesions such as ductal carcinoma in situ (DCIS), and approx- myoepithelial cell layer and basement membrane in DCIS imately 25% of breast abnormalities detected during screen- have been identified as major events in the progression from ing are DCIS (8). DCIS is defined by enhanced proliferation DCIS to IDC (15). Thus, it is important to find chemo- of intraductal epithelial cells (9). DCIS is a precancerous preventive agents, such as vitamin D, that could inhibit the lesion and if left untreated, approximately 30% to 50% of progression to later stages of breast cancer. cases will progress to invasive disease (10–12). Therefore, Exposure to sunlight has shown an inverse relationship to breast cancer risk (16). This is linked to the increased production of vitamin D3 upon exposure to ultraviolet light Authors' Affiliations: 1Department of Chemical Biology, Ernest Mario (17–20). Since this discovery, many studies have linked 2 School of Pharmacy; Center for Advanced Biotechnology and Medicine, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active Rutgers, The State University of New Jersey, Piscataway; and 3Rutgers Cancer Institute of New Jersey, New Brunswick, New Jersey metabolite of vitamin D, and vitamin D analogs to the suppression of cancer cell invasion, proliferation, and Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/). metastasis (21–24). However, the pharmacologic dose of 1,25(OH)2D3 required to elicit a response on breast tumors Corresponding Author: Nanjoo Suh, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New can induce hypercalcemic toxicity (24, 25). Therefore, non- Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854. Phone: 848-445- calcemic vitamin D analogs have been of interest in the 8030; Fax: 732-445-0687; E-mail: [email protected] inhibition of breast cancer (21, 25–27). We have previously doi: 10.1158/1940-6207.CAPR-13-0362 demonstrated that a Gemini vitamin D analog BXL0124 2014 American Association for Cancer Research. significantly decreased tumor growth of MCF10DCIS.com

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xenografts in vivo without hypercalcemic toxicity (25, 28). Xenograft animal studies However, the preventive effects of BXL0124 on the early All animal studies were approved by the Institutional transition from DCIS to IDC have not been elucidated. We Review Board for the Animal Care and Facilities Com- utilized the MCF10DCIS.com progression model to inves- mitteeofRutgersUniversity.Femalenudemice(5–6 tigate whether BXL0124 blocks or delays the early transition weeks old) were purchased from Charles River Labora- of DCIS to IDC in vivo. tories. They were allowed to acclimate to the facilities for The MCF10DCIS.com cell line is one of the human 2 weeks, at which time they were injected (7–8 weeks old) MCF10A series of cell lines, which is unique in the fact that with human MCF10DCIS.com cells into the dorsal flank it is representative of breast cancer progression in vitro and in with 106 cells per site and treatment began the following vivo (29, 30). The MCF10DCIS.com cell line consistently day. Mice were treated with DMSO control or BXL0124 produces comedo DCIS-like lesions in animal xenografts, (0.1 mg/kg body weight) intraperitoneally 6 times per which highly resemble the histopathology of human DCIS weekoverthecourseof4weeks. Tumors were palpated in xenograft models. The DCIS-like lesions have been twice per week and total body weights were measured shown to reproducibly progress to invasive tumors, pro- weekly. Tumors were measured with a vernier caliper and viding a unique in vivo model to investigate the transition tumor volume (V,cm3) was calculated using the equation of DCIS to IDC (28, 29). The MCF10DCIS.com xenograft V ¼ Dd2/2, where D (cm) and d (cm) are the largest and model forms histology that resembles human DCIS, how- smallest perpendicular diameters. Animals were sacrificed ever this is not classical DCIS, which are premalignant cells 1 to 4 weeks after injection, at which time tumors were confined to the mammary ducts. Therefore, from this point excised and blood drawn for further analysis. Tumors forward when we refer to DCIS we are referring to the DCIS- from 1 to 4 weeks were fixed in 10% formalin for 15 hours like histology that is observed in the subcutaneous xeno- for immunohistochemical analysis. Tumors from 3 and grafts. In addition, Hu and colleagues showed similarities of 4 weeks were snap frozen in liquid nitrogen for qPCR cell type–specific expression profiles between human DCIS analysis. samples and MCF10DCIS.com xenografts by comparing myoepithelial and epithelial cell gene expression profiles Determination of serum calcium level (15). The study showed a statistically significant enrichment The determination of calcium concentration in serum of genes involved in the extracellular matrix, basement was carried out by using the calcium reagent set from Pointe membrane structure, and development in the myoepithelial Scientific, Inc., following the manufacturer’s protocol. Brief- cell populations of both the human DCIS samples and ly, serum (4 mL) was mixed with the diluted reagent (200 MCF10DCIS.com xenografts. Because the enrichment pat- mL) in the wells of a 96-well plate and incubated at room terns of genes were highly similar from human to the temperature for 1 minute and absorbance read at 570 nm MCF10DCIS.com xenograft tumors, this is an excellent using a Tecan infinite M200 plate reader (Tecan). The model to test the effects of chemopreventive agents on the calcium concentrations were calculated from calcium stan- structural, molecular, and phenotypic changes during the dards provided by the manufacturer. progression of DCIS to IDC. In this report, we investigated the effects of BXL0124 treatment on MCF10DCIS.com Immunohistochemical and immunofluorescence xenografts, and found that BXL0124 maintained the integ- analysis rity of critical structures related to noncancerous breast Subcutaneous tumors were fixed, embedded in paraffin, lesions, which are typically lost during the progression to and sectioned at 5-mm thickness. Individual tumors were malignant disease. analyzed histopathologically by hematoxylin and eosin (H&E) staining. For immunohistochemistry, sections Materials and Methods were stained as previously described (32) with antibodies Reagents and cell culture to proliferating cell nuclear antigen (PCNA; 1:8000; Dako; Gemini vitamin D analog, 1a,25-dihydroxy-20R-21(3- M0879) and vitamin D receptor (VDR; 1:200, Santa Cruz hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne- Biotechnology; sc-13133). The sections were counter- 26,27-hexafluoro-cholecalciferol (BXL0124, >95% purity; stained with Harris hematoxylin (Sigma Aldrich). The ref. 31) was provided by BioXell, Inc. and dissolved in PCNA nuclear intensity and VDR total pixel intensity were dimethyl sulfoxide (DMSO). For in vivo animal experiments, quantified by blinding the samples and having a third party BXL0124 was diluted in Cremophor EL: PBS (1:8, v/v). carry out the analysis using a Scan Scope (Aperio). For MCF10DCIS.com human breast cancer cell line was pro- immunofluorescence staining, the slides were blocked in vided by Dr. F. Miller at the Barbara Ann Karmanos Cancer 10% goat serum, and then incubated sequentially overnight Institute (Detroit, MI). The MCF10DCIS.com cell line was at 4C with the indicated combination of primary anti- authenticated by short tandem repeat profiling at American bodies to smooth muscle actin (SMA; 1:200; Abcam; Type Culture Collection. MCF10DCIS.com cells were main- ab5694), laminin 5 (1:50; Santa Cruz Biotechnology; tained in monolayer cell culture in DMEM/F12 medium sc13587), pancytokeratin (panCK; 1:50; Dako; M3515), supplemented with 5% horse serum, 1% penicillin/strepto- and TO-PRO-3 iodide nuclear antibody (Invitrogen; 1 mycin, and 1% 4-(2-hydroxyethyl)piperazine-1-ethanesul- mmol/L). Fluorophore-conjugated secondary antibodies fonic acid (HEPES) solution at 37 C, 5% CO2. (Alexa Fluor 488 or 546, 1:100; Invitrogen) were incubated

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at room temperature for 30 minutes. The images were taken tary Fig. S1B). The subcutaneous xenografts show a clear using confocal microscopy with lasers at 488, 546, and 633 point DCIS to IDC transition so it is easy to determine nm (TO-PRO-3). Immunofluorescence was visualized whether pharmacologic agents, such as BXL0124, inhibits using a Nikon Eclipse C1 Plus confocal microscope system. this transition (Supplementary Fig. S2A), whereas the mammary fat pad xenografts it is not clear when or if the Western blot analysis transition is blocked (Supplementary Fig. S2B). Therefore, The procedures have been described previously (32). Five we proceeded with the MCF10DCIS.com subcutaneous tumor samples from each group were homogenized and xenograft model for the clarity of studying the effects of pooled for analysis. Primary antibodies against VDR (1:200; pharmacologic agents on DCIS progression to IDC. Using Thermoscientific; MA1-710) and b-actin (1:2,000; Sigma- MCF10DCIS.com subcutaneous xenografts, we investigated Aldrich; A1978) were used for analysis. Secondary antibo- the histopathologic and molecular changes that occurred dies were from Santa Cruz Biotechnology. Western blots during the growth of tumors in nu/nu mice over the course were quantified by using ImageJ software (US National of 4 weeks. Animals treated with BXL0124 showed a reduc- Institutes of Health, Bethesda, MD) and calculating tion in average tumor volume over the first 3 weeks the relative density of the bands using the gel analyzer and significant repression was observed by week 4 with a command. 43% reduction in tumor size (P < 0.05; Fig. 1A). BXL0124 treatment did not cause any significant changes in body- qPCR analysis weight or serum calcium levels, indicating that there was These procedures have been reported previously (33, 34). no observed hypercalcemic toxicity associated with the The Taqman probe-based gene expression system from given dose (Fig. 1B and C). H&E staining showed that Applied Biosystems was used to detect the genes of interest. MCF10DCIS.com cells subcutaneously xenografted into nu/nu mice formed lesions histologically resembling that DCIS quantification of human DCIS. In the control group, DCIS lesions started Hematoxylin and eosin images of DCIS were quantified to escape to an invasive-like stage at week 3 and the tumors using ImageJ software (US National Institutes of Health). advanced to IDC rapidly by week 4, at which time the The total tumor area was selected, and then the areas of tumors were 80% invasive (Fig. 1D). The tumors treated DCIS within the tumor were calculated. The percentage with BXL0124 formed DCIS lesions by week 3 and unlike of DCIS within each tumor was presented as the area of control tumors, maintained these DCIS lesions through DCIS divided by the total tumor area. week 4, showing approximately 30% invasive histology (P < 0.05; Fig. 1D). Statistical analysis Statistical significance was evaluated using the Student BXL0124 treatment decreases the proliferation of t-test. MCF10DCIS.com tumors at week 4 Cell proliferation was determined by measuring Results PCNA by immunohistochemistry staining. The cell pro- Gemini vitamin D analog, BXL0124, inhibits the liferation in MCF10DCIS.com tumors remained relatively transition of ductal carcinoma in situ to IDC in low through week 3. As control tumors progressed to an MCF10DCIS.com xenografts invasive phenotype at week 4, there is a marked increase Previous studies have shown that BXL0124 has an inhib- in the level of PCNA expression compared with previous itory effect on the growth of MCF10DCIS.com xenograft weeks. Treatment with BXL0124 showed a significant mammary tumors at 5 weeks (28). However, the effect of decrease in PCNA levels compared with the control at BXL0124 on DCIS progression to IDC has not been inves- week 4 (Fig. 2A). Four tumors from each group were tigated. The early stages of breast cancer, specifically the blinded and were analyzed for PCNA staining intensity. DCIS to IDC transition, are of utmost importance from a The intensities were scored from 0þ (negative staining) to prevention standpoint. Therefore, in this study, we evalu- 3þ (strongest staining) for each individual cell. The sum ated the effects of BXL0124 on the early transition of DCIS of all positive staining, including 1þ,2þ,and3þ,was to IDC. In our preliminary studies, we first compared the used to calculate the percentage of PCNA-positive cells. DCIS progression of MCF10DCIS.com xenografts between Quantification showed that 52% of the cells were PCNA- the mammary fat pad and subcutaneous injections. The positive in the control group at week 4, whereas only 32% subcutaneous xenografts formed a higher number of com- of cells were positive in the BXL0124-treated group at edo DCIS lesions and more consistently produced DCIS week 4 (P < 0.05; Fig. 2B). lesions compared with the mammary fat pad xenografts (Supplementary Fig. S1A and S1B), in agreement with a BXL0124 treatment maintains VDR levels in previous report by Hu and colleagues (15). Comparison of MCF10DCIS.com tumors 4 separate lesions from subcutaneous or mammary fat pad MCF10DCIS.com xenograft tumors express VDR. From at week 3 xenografts shows that subcutaneous xenografts weeks 1 to 3, VDR levels were similar between control and reproducibly form a higher number of DCIS lesions com- BXL0124 treatment groups. VDR was lost upon the rapid pared with the mammary fat pad xenografts (Supplemen- progression from DCIS to invasive tumors in the control

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A B C ) 3 0.7 30 14 0.6 25 12 0.5 20 10 0.4 * 15 8 0.3 6 10 (mg/dL) 0.2 4 5 Body weight (g) Body 0.1 Serum calcium 2 0.0 0 0 Tumor volume (cm Tumor volume

Control BXL0124 Control BXL0124 Control BXL0124

D Week 1 Week 2 Week 3 Week 4 Control H&E BXL0124

Week 1 Week 2 Week 3 Week 4 100 100 100 100 * 75 75 75 75 50 50 50 50

% DCIS % 25 25 25 25 0 0 0 0

Figure 1. BXL0124 inhibits tumor growth and inhibits progression to IDC in MCF10DCIS.com subcutaneous xenografts in nu/nu mice. A, average tumor volume at weekly time points is shown, P < 0.05 (n ¼ 5 per group). Tumor volume (V; cm3) was calculated using the equation V ¼ Dd2/2, where D (centimeters) and d (centimeters) are the largest and smallest perpendicular diameters. B, average ﬁnal bodyweight at autopsy is shown (n ¼ 5 per group). C, serum calcium determination to assess hypercalcemic toxicity is shown (n ¼ 5 per group). D, a representative H&E staining showing the progression of MCF10DCIS.com subcutaneous xenografts in nu/nu mice from weeks 1 to 4 (40). DCIS quantiﬁcation, P < 0.05 (n ¼ 4 per group).

group in week 4. However, treatment with BXL0124 not (strongest staining) based on pixel intensity of staining. The only maintained DCIS histology but also retained VDR sum of all positive staining, including 1þ,2þ, and 3þ, was levels at week 4 (Fig. 3A). It is interesting to note that VDR used to calculate the percentage of VDR-positive cells. expression is lost where epithelial and stromal cells come in Quantiﬁcation showed 66% VDR-positive staining in the contact, suggesting that this cell-to-cell interaction could control tumors, compared with 81% positive staining in the potentially reduce VDR levels. Four tumors from each group BXL0124 group at week 4 (P < 0.05; Fig. 3B). VDR levels were blinded and analyzed for VDR staining intensity. The were also analyzed in xenograft tumors by Western blot intensities were scored from 0þ (negative staining) to 3þ analysis, showing a 60% increase in the xenografts from

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A Week 1 Week 2 Week 3 Week 4 Control PCNA BXL0124

B Control BXL0124

Week 1 Week 2 Week 3 Week 4 100 100 100 100

80 80 80 80 * 60 60 60 60 40 40 40 40 20 20 20 20 % PCNA staining PCNA % * 0 0 0 0 0+ 1+ 2+ 3+ 0+ 2+1+ 3+ 0+ 1+ 2+ 3+ 1+0+ 2+ 3+ Intensity Intensity Intensity Intensity

Figure 2. BXL0124 treatment decreases the proliferation of MCF10DCIS.com tumors at week 4. A, a representative immunohistochemical analysis of PCNA in tumor samples from weeks 1 to 4 is shown (100). PCNA-positive staining is found in the nucleus of the cells. B, 4 tumors from each group were blinded and quantiﬁed, 3 representative areas from each tumor were quantiﬁed for the intensity of PCNA staining, mean SEM. The staining intensities were scored from 0þ (negative staining) to 3þ (the strongest staining), P < 0.05.

BXL0124-treated mice compared with the control xenograft progressed to IDC. However, the course of BXL0124 treat- tumors (Fig. 3C). ment showed the formation of the myoepithelial cell layer in week 3 and maintained organization of the myoepithelial Treatment with BXL0124 inhibits progression to IDC cell layer at week 4 (Fig. 4). by maintaining the myoepithelial cell layer The main diagnostic feature that distinguishes DCIS from invasive lesions is the loss of the critical myoepithelial cell Treatment with BXL0124 maintains the basement layer and basement membrane (15). To investigate the membrane integrity of the cell layers throughout progression, we ana- To assess the effects of BXL0124 on the integrity of the lyzed the myoepithelial marker SMA and the epithelial basement membrane, co-immunoﬂuoresence staining was marker panCK in xenograft tumors. Tumors at weeks 1 and carried out with a basement membrane marker, laminin 5, 2 were composed primarily of epithelial cells as noted by and a myoepithelial marker, SMA. The basement mem- panCK staining. There was a gradual establishment of the brane formed by week 2, and was maintained in week 3 myoepithelial cell layer starting at week 2, fully forming in both the control and BXL0124-treated groups. At week 4, around the epithelial cells in both the control and BXL0124- the basement membrane is disrupted in the invasive-like treated group by week 3 (Fig. 4). In the control group tumors of the control group, as indicated by reduced and tumors, the myoepithelial cell layer spontaneously disso- fragmented staining of laminin 5. This structure remained ciated from its organized structure by week 4 as the tumors intact in the BXL0124-treated tumors at week 4 (Fig. 5).

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A Week 1 Week 2 Week 3 Week 4 Week 4 Control VDR BXL0124

Control BXL0124 B C Week 4 pooled Week 1 Week 2 Week 3 Week 4 samples 100 100 100 100 BXL0124 – + 80 80 80 80 60 60 60 60 VDR 40 40 40 40 1.0 1.6 * 20 20 20 20 b-Actin

% VDR staining VDR % 0 0 0 0 3+2+1+0+ 0+ 3+2+1+ 0+ 2+1+ 3+ 2+1+0+ 3+ 1.0 1.0 Intensity Intensity Intensity Intensity

Figure 3. BXL0124 treatment maintains VDR levels in MCF10DCIS.com tumors. A, a representative immunohistochemical analysis for VDR from weeks 1 to 4 is shown (40). VDR-positive staining is found in the cytoplasm and nucleus of the cells. Whole tumor mounts of VDR expression from week 4 is shown as a contracted view. B, 4 tumors from each group were blinded and quantiﬁed and 3 representative areas from each tumor were quantiﬁed for the intensity of VDR staining, mean SEM. The staining intensities were scored from 0þ (negative staining) to 3þ (the strongest staining), P < 0.05. C, the protein level of VDR was increased in the tumors of week 4 BXL0124-treated mice as shown by Western blot analysis. Five xenograft tumors from each group were combined for pooled samples. b-Actin was used as a loading control.

BXL0124 inhibits the mRNA expression of the matrix with the myoepithelial marker SMA. VDR staining did not metalloproteinases during DCIS to IDC progression colocalize with SMA staining in any of the samples that were Matrix metalloproteinases (MMP) have been implicated analyzed, indicating that VDR is not expressed in myoe- in increased tumor growth, invasion, and metastasis (34, pithelial cells (Fig. 6B). In addition, the loss of VDR is 35). Consequently, we assessed the mRNA levels of specific evident in week 4 control xenografts, whereas VDR levels are MMPs in MCF10DCIS.com tumor xenografts at weeks 3 and maintained upon BXL0124 treatment at week 4 (Fig. 6B). 4. Analysis of MCF10DCIS.com tumors showed a significant decrease in the mRNA levels of MMP2, 9, 14, and 15 upon BXL0124 treatment at week 3 whereas MMP16 did not Discussion change (Fig. 6A). The modulation of MMP2 and MMP14 DCIS progression to IDC is defined by the escape of inner expression by BXL0124 treatment persisted to week 4. These luminal epithelial cells through the outer layer of myoe- observations suggest that reduction of MMP expression by pithelial cells and the basement membrane, ultimately BXL0124 contributes to the inhibition of transition from coming in contact with the stromal cell population (13). DCIS to invasive carcinomas. Interestingly, despite the Previous reports have shown that myoepithelial cells can changes in protein levels of VDR we did not see a significant arise from the luminal cell population, but not vice versa change in mRNA expression of the VDR gene at weeks 3 or 4 (13, 36–38). In our study, the MCF10DCIS.com xenografts upon BXL0124 treatment (Fig. 6A). We did not detect show growth of the epithelial cells in the early weeks significant biologic changes in the vitamin D metabolizing followed by the formation of the myoepithelial cell layer genes CYP24A1 (catabolism) or CYP27B1 (synthesis) in that is consistent with those reports. BXL0124 does not week 3 and 4 xenograft samples (Supplementary Fig. S3). To seem to affect the rate of formation of the myoepithelial determine if VDR is expressed in myoepithelial cells, we layer (Fig. 4; weeks 1 to 3), however it does significantly assessed the immunofluorescence staining of VDR together reduce the rate at which the myoepithelial cell layer is

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Week 1 Week 2 Week 3 Week 4 Week 4 Control ToPro3 / panCK / SMA BXL0124

Figure 4. Treatment with BXL0124 inhibits progression to IDC by maintaining the myoepithelial cell layer. A representative immunofluorescence staining for tumor samples from weeks 1 to 4 with the myoepithelial cell marker SMA (shown in red) and the epithelial cell marker panCK (shown in green) is shown (200). Nuclei were stained with TO-PRO-3 (blue). Expanded magnification is shown for specific areas from week 4 tumors. Scale bars represent 100 mm. broken down (Fig. 4; week 4), suggesting that BXL0124 pithelial cell layer observed in week 4 control tumors. inhibits the transition from DCIS to invasive carcinoma. Analyzing the effects of BXL0124 on the progression of When the staining of laminin 5 in the DCIS and IDC lesions DCIS to IDC (Fig. 1D), we found that the treatment sustains are examined histologically, it is clear that BXL0124 treat- DCIS lesions and prevents progression to IDC through ment helps to maintain an intact, organized structure of maintenance of the critical myoepithelial cell layer and the the critical basement membrane (Fig. 5; week 4). It is also basement membrane. interesting to note that the basement membrane forms first The progression from DCIS to IDC is believed to be in week 2 followed by the myoepithelial cell layer at week 3. provoked largely by the production of proteolytic This suggests that the basement membrane might act as a enzymes (39). MMPs degrade proteins involved in extra- scaffold for the formation of the myoepithelial cell layer, cellular matrix structure and molecules involved in cell– and the loss of this scaffold through enzymatic degradation cell adhesion, which releases epithelial cells from their could partially account for the disorganization of the myoe- ordered layers and deregulates cell signaling, ultimately

Week 1 Week 2 Week 3 Week 4 Week 4 Control / Laminin5 ToPro3 SMA BXL0124

Figure 5. Treatment with BXL0124 maintains the basement membrane. A representative immunofluorescence staining with the myoepithelial cell marker SMA (shown in red) and the basal membrane marker laminin 5 (shown in green) on tumors from weeks 1 to 4 is shown (200). Nuclei were stained with TO-PRO-3 (blue). Expanded magnification is shown for specific areas from week 4 tumors. Scale bars represent 100 mm.

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A Control BXL0124 B Week 3 Week 4 MMP2 MMP9 MMP14 1.5 3.0 1.5

1.0 2.0 1.0 *** **

mRNA mRNA 0.5 * 1.0 0.5 * Control (fold change) 0.0 0.0 0.0 ToPro3 / VDR MMP16 MMP15 VDR / 1.5 1.5 1.5

1.0 1.0 1.0 SMA * 0.5 0.5 0.5 mRNA mRNA BXL0124

(fold change) 0.0 0.0 0.0

Figure 6. BXL0124 inhibits the mRNA expression levels of the MMPs during DCIS to IDC progression. A, qPCR analysis of MMPs and VDR mRNA levels in MCF10DCIS.com tumor samples from weeks 3 and 4 is shown, mean SEM. Cycle numbers are shown in parenthesis: MMP2 (25), MMP9 (28), MMP14 (23), MMP15 (28), MMP16 (25), and VDR (26). Statistical signiﬁcance refers to the respective week control, P < 0.05, P < 0.01, P < 0.001 (n ¼ 3–5 per group). B, a representative immunoﬂuorescence staining with the myoepithelial cell marker, SMA (shown in red), and VDR (shown in green) on tumors from weeks 3 and 4 are shown (200). Nuclei were stained with TO-PRO-3 (blue). Scale bars represent 100 mm.

leading to extensive changes in gene transcription (40). the control group at week 4. However, mRNA expression of MMP2 and MMP9, the gelatinases, are responsible for VDR was unchanged in weeks 3 or 4, suggesting that loss of the degradation of type IV collagen as well as laminin 5, VDR in week 4 control tumors is likely because of protein components of the basement membrane (39, 41). MMP2 is degradation or possible posttranslational regulation. Inter- secreted in a latent form and requires activation by MMP14 estingly, loss of DCIS architecture seems to be a determining to its pro-MMP2 form (42). Aside from its activating func- factor in the loss of VDR expression, suggesting that the tion, MMP14 as well as MMP15 and MMP16 have been interaction with the surrounding stromal cells could play a shown to directly affect cell invasion by remodeling the major role in the downregulation of VDR. Whether adjacent basement membrane in vivo (43). Thus, reduction of MMP2, stromal-to-epithelial cell contact or factors secreted from MMP9, MMP14, and MMP15 by BXL0124 in week 3 before stromal cells contribute to the loss of VDR needs to be the transition of DCIS to IDC likely contributes to the further investigated. Analysis of VDR and SMA colocaliza- maintenance of the basement membrane in the tumor tion studies show that VDR is not expressed in the myoe- xenografts. This suggests that the downregulation of MMPs pithelial cell layer or stroma but within the luminal cells. In by BXL0124 treatment could play a major role in the invasive tumors, VDR negative cells include both tumor and preservation of DCIS histology. stroma cells. Taken together, it seems that VDR is expressed As tumors progress to IDC, there is a significant increase in the luminal cell population and this expression is lost in cell proliferation, which is consistent with previous upon progression to IDC. This further suggests that findings that stromal and tumor epithelial cell interactions BXL0124 does not act directly on the myoepithelial cell can enhance proliferation (44). The significant reduction of layer but may exert its antitumor effects through a paracrine the proliferation rate in tumors from BXL0124-treated mechanism from the luminal cells. The analysis of DCIS animals at week 4 demonstrates the potential of BXL0124 revealed increased VDR levels and decreased cell prolifer- to slow the growth of DCIS epithelial cells. Because the ation, providing a prevention strategy to inhibit the early effects of BXL0124 are known to be dependent on VDR progression of DCIS to IDC with the treatment of vitamin (28, 34), we analyzed VDR levels over the course of tumor D or its analogs. These data suggest a novel mechanism progression. It was previously shown that a decrease in in which downregulation of VDR is synchronized with the protein levels of VDR have been correlated with the pro- loss of the critical myoepithelial cell layer and basement gression from benign to malignant breast lesions (45). In membrane. our study, the BXL0124 treatment not only maintained the Breast cancer progression is indicated by the loss of integrity of the DCIS structure, but also retained VDR levels normal tissue architecture allowing invasion into surround- in the epithelial cells (Fig. 3A; week 4). VDR protein levels ing tissue (46). Our study shows that BXL0124 has the were higher in BXL0124-treated group when compared with potential to exert its effects indirectly on the myoepithelial

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Inhibition of Breast Cancer Progression with a Vitamin D Analog

cell population and directly on the luminal cell population Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Wahler, Y.C. Kim, N. Suh to inhibit tumorigenesis. In this study, BXL0124 inhibited Analysis and interpretation of data (e.g., statistical analysis, biosta- the progression of DCIS to IDC by maintaining the integrity tistics, computational analysis): J. Wahler, F. Liu, N. Suh of the myoepithelial cell layer and basement membrane. Writing, review, and or revision of the manuscript: J. Wahler, J.Y. So, F. Liu, H. Maehr, N. Suh The inhibitory activity of BXL0124 on MMP mRNA expres- Administrative, technical, or material support (i.e., reporting or orga- sion is likely a contributor to the maintenance of DCIS nizing data, constructing databases): J. Wahler, M. Uskokovic, N. Suh Study supervision: N. Suh organization and inhibition of tumor progression. This Other: H. Maehr contributed synthesis of drug substance. study with a novel Gemini vitamin D analog BXL0124 in the MCF10DCIS.com model suggests a unique treatment Grant Support modality preventing the progression of DCIS to an aggres- This work was supported in part by the NIH National Cancer Institute R01 sive, invasive-like disease. CA127645 (N. Suh), the National Institute of Environmental Health Sciences Grant ES005022 (H. Zarbl), and The Trustees Research Fellowship Program at Rutgers, The State University of New Jersey (N. Suh). Disclosure of Potential Conﬂicts of Interest The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked No potential conﬂicts of interest were disclosed. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: J. Wahler, J.Y. So, F. Liu, N. Suh Received October 18, 2013; revised March 4, 2014; accepted March 25, Development of methodology: J. Wahler, N. Suh 2014; published OnlineFirst April 1, 2014.

References 1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer 16. Grant WB. An ecologic study of dietary and solar ultraviolet-B links to J Clin 2014;64:9–29. breast carcinoma mortality rates. Cancer 2002;94:272–81. 2. Amir E, Freedman OC, Seruga B, Evans DG. Assessing women at high 17. Garland CF, Garland FC, Gorham ED, Lipkin M, Newmark H, Mohr SB, risk of breast cancer: a review of risk assessment models. J Natl et al. The role of vitamin D in cancer prevention. Am J Public Health Cancer Inst 2010;102:680–91. 2006;96:252–61. 3. Troester MA, Swift-Scanlan T. Challenges in studying the etiology of 18. Garland CF, Garland FC. Do sunlight and vitamin D reduce the breast cancer subtypes. Breast Cancer Res 2009;11:104. likelihood of colon cancer? Int J Epidemiol 1980;9:227–31. 4. Almendro V, Fuster G. Heterogeneity of breast cancer: etiology and 19. Garland CF, Gorham ED, Mohr SB, Grant WB, Giovannucci EL, Lipkin clinical relevance. Clin Transl Oncol 2011;13:767–73. M, et al. Vitamin D and prevention of breast cancer: pooled analysis. 5. Pare R, Yang T, Shin JS, Tan PH, Lee CS. Breast cancer precursors: J Steroid Biochem Mol Biol 2007;103:708–11. diagnostic issues and current understanding on their pathogenesis. 20. Garland FC, Garland CF, Gorham ED, Young JF. Geographic variation Pathology 2013;45:209–13. in breast cancer mortality in the United States: a hypothesis involving 6. Hu X, Stern HM, Ge L, O'Brien C, Haydu L, Honchell CD, et al. Genetic exposure to solar radiation. Prev Med 1990;19:614–22. alterations and oncogenic pathways associated with breast cancer 21. El Abdaimi K, Dion N, Papavasiliou V, Cardinal PE, Binderup L, Goltz- subtypes. Mol Cancer Res 2009;7:511–22. man D, et al. The vitamin D analogue EB 1089 prevents skeletal 7. Walsh T, Lee MK, Casadei S, Thornton AM, Stray SM, Pennil C, et al. metastasis and prolongs survival time in nude mice transplanted with Detection of inherited mutations for breast and ovarian cancer using human breast cancer cells. Cancer Res 2000;60:4412–8. genomic capture and massively parallel sequencing. Proc Natl Acad 22. Krishnan AV, Feldman D. Mechanisms of the anti-cancer and anti- Sci U S A 2010;107:12629–33. inﬂammatory actions of vitamin D. Annu Rev Pharmacol Toxicol 8. Brinton LA, Sherman ME, Carreon JD, Anderson WF. Recent trends in 2011;51:311–36. breast cancer among younger women in the United States. J Natl 23. Matthews D, LaPorta E, Zinser GM, Narvaez CJ, Welsh J. Genomic Cancer Inst 2008;100:1643–8. vitamin D signaling in breast cancer: insights from animal models and 9. Virnig BA, Tuttle TM, Shamliyan T, Kane RL. Ductal carcinoma in situ of human cells. J Steroid Biochem Mol Biol 2010;121:362–7. the breast: a systematic review of incidence, treatment, and outcomes. 24. Swami S, Krishnan AV, Wang JY, Jensen K, Horst R, Albertelli MA, et al. J Natl Cancer Inst 2010;102:170–8. Dietary vitamin D(3) and 1,25-dihydroxyvitamin D(3) (calcitriol) exhibit 10. Page DL, Dupont WD, Rogers LW, Jensen RA, Schuyler PA. Continued equivalent anticancer activity in mouse xenograft models of breast and local recurrence of carcinoma 15–25 years after a diagnosis of low prostate cancer. Endocrinology 2012;153:2576–87. grade ductal carcinoma in situ of the breast treated only by biopsy. 25. Lee HJ, Paul S, Atalla N, Thomas PE, Lin X, Yang I, et al. Gemini vitamin Cancer 1995;76:1197–200. D analogues inhibit estrogen receptor-positive and estrogen receptor- 11. Collins LC, Tamimi RM, Baer HJ, Connolly JL, Colditz GA, Schnitt SJ. negative mammary tumorigenesis without hypercalcemic toxicity. Outcome of patients with ductal carcinoma in situ untreated after Cancer Prev Res 2008;1:476–84. diagnostic biopsy: results from the Nurses' Health Study. Cancer 26. So JY, Wahler JE, Yoon T, Smolarek AK, Lin Y, Shih WJ, et al. Oral 2005;103:1778–84. administration of a gemini vitamin D analog, a synthetic triterpenoid 12. Sanders ME, Schuyler PA, Dupont WD, Page DL. The natural history of and the combination prevents mammary tumorigenesis driven by low-grade ductal carcinoma in situ of the breast in women treated by ErbB2 overexpression. Cancer Prev Res 2013;6:959–70. biopsy only revealed over 30 years of long-term follow-up. Cancer 27. Anzano MA, Smith JM, Uskokovic MR, Peer CW, Mullen LT, Letterio 2005;103:2481–4. JJ, et al. 1a,25-Dihydroxy-16-ene-23-yne-26,27-hexaﬂuorocholecal- 13. Gudjonsson T, Adriance MC, Sternlicht MD, Petersen OW, Bissell MJ. ciferol (Ro24–5531), a new deltanoid (vitamin D analogue) for preven- Myoepithelial cells: their origin and function in breast morphogenesis tion of breast cancer in the rat. Cancer Res 1994;54:1653–6. and neoplasia. J Mammary Gland Biol Neoplasia 2005;10:261–72. 28. So JY, Lee HJ, Smolarek AK, Paul S, Wang CX, Maehr H, et al. A novel 14. Pandey PR, Saidou J, Watabe K. Role of myoepithelial cells in breast Gemini vitamin D analog represses the expression of a stem cell marker tumor progression. Front Biosci (Landmark Ed) 2010;15:226–36. CD44 in breast cancer. Mol Pharmacol 2011;79:360–7. 15. Hu M, Yao J, Carroll DK, Weremowicz S, Chen H, Carrasco D, et al. 29. Miller FR, Santner SJ, Tait L, Dawson PJ. MCF10DCIS.com xenograft Regulation of in situ to invasive breast carcinoma transition. Cancer model of human comedo ductal carcinoma in situ. J Natl Cancer Inst Cell 2008;13:394–406. 2000;92:1185–6.

www.aacrjournals.org Cancer Prev Res; 7(6) June 2014 625

Wahler et al.

30. So JY, Lee HJ, Kramata P, Minden A, Suh N. Differential expression of 38. Smalley MJ, Titley J, O'Hare MJ. Clonal characterization of mouse key signaling proteins in MCF10 cell lines, a human breast cancer mammary luminal epithelial and myoepithelial cells separated by progression model. Mol Cell Pharmacol 2012;4:31–40. ﬂuorescence-activated cell sorting. In Vitro Cell Dev Biol Anim 1998; 31. Maehr H, Lee HJ, Perry B, Suh N, Uskokovic MR. Calcitriol derivatives 34:711–21. with two different side chains at C-20. V. Potent inhibitors of mammary 39. Duffy MJ, Maguire TM, Hill A, McDermott E, O'Higgins N. Metallopro- carcinogenesis and inducers of leukemia differentiation. J Med Chem teinases: role in breast carcinogenesis, invasion and metastasis. 2009;52:5505–19. Breast Cancer Res 2000;2:252–7. 32. Lee HJ, Ju J, Paul S, So JY, DeCastro A, Smolarek A, et al. Mixed 40. Radisky ES, Radisky DC. Matrix metalloproteinase-induced epithelial- tocopherols prevent mammary tumorigenesis by inhibiting estrogen mesenchymal transition in breast cancer. J Mammary Gland Biol action and activating PPAR-gamma. Clin Cancer Res 2009;15: Neoplasia 2010;15:201–12. 4242–9. 41. Giannelli G, Falk-Marzillier J, Schiraldi O, Stetler-Stevenson WG, 33. Lee HJ, Liu H, Goodman C, Ji Y, Maehr H, Uskokovic M, et al. Gene Quaranta V. Induction of cell migration by matrix metalloprotease-2 expression proﬁling changes induced by a novel Gemini vitamin D cleavage of laminin-5. Science 1997;277:225–8. derivative during the progression of breast cancer. Biochem Pharma- 42. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, et al. A col 2006;72:332–43. matrix metalloproteinase expressed on the surface of invasive tumour 34. So JY, Smolarek AK, Salerno DM, Maehr H, Uskokovic M, Liu F, et al. cells. Nature 1994;370:61–5. Targeting CD44-STAT3 signaling by Gemini vitamin D analog leads to 43. Hotary K, Li XY, Allen E, Stevens SL, Weiss SJ. A cancer cell metallo- inhibition of invasion in basal-like breast cancer. PLoS ONE 2013;8: protease triad regulates the basement membrane transmigration pro- e54020. gram. Genes Dev 2006;20:2673–86. 35. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators 44. Schnitt SJ. The transition from ductal carcinoma in situ to invasive of the tumor microenvironment. Cell 2010;141:52–67. breast cancer: the other side of the coin. Breast Cancer Res 2009; 36. Gudjonsson T, Villadsen R, Nielsen HL, Ronnov-Jessen L, Bissell MJ, 11:101. Petersen OW. Isolation, immortalization, and characterization of a 45. Lopes N, Sousa B, Martins D, Gomes M, Vieira D, Veronese LA, et al. human breast epithelial cell line with stem cell properties. Genes Dev Alterations in vitamin D signalling and metabolic pathways in breast 2002;16:693–706. cancer progression: a study of VDR, CYP27B1 and CYP24A1 expres- 37. Pechoux C, Gudjonsson T, Ronnov-Jessen L, Bissell MJ, Petersen sion in benign and malignant breast lesions. BMC Cancer 2010;10:483. OW. Human mammary luminal epithelial cells contain progenitors to 46. Bombonati A, Sgroi DC. The molecular pathology of breast cancer myoepithelial cells. Dev Biol 1999;206:88–99. progression. J Pathol 2011;223:307–17.

626 Cancer Prev Res; 7(6) June 2014 Cancer Prevention Research

Inhibition of the Transition of Ductal Carcinoma In Situ to Invasive Ductal Carcinoma by a Gemini Vitamin D Analog

Joseph Wahler, Jae Young So, Yeoun Chan Kim, et al.

Cancer Prev Res 2014;7:617-626. Published OnlineFirst April 1, 2014.

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