MYB Promotes Desmoplasia in Pancreatic Cancer throughDirect Transcriptional Up-regulation and Cooperative Actionof Sonic Hedgehog and Adrenomedullin*

Received for publication, April 14, 2016, and in revised form, May 26, 2016 Published, JBC Papers in Press, May 31, 2016, DOI 10.1074/jbc.M116.732651

Arun Bhardwaj‡1, Sanjeev K. Srivastava‡1, Seema Singh‡§, Nikhil Tyagi‡, Sumit Arora‡, James E. Carter¶,Moh’d Khushman�, and Ajay P. Singh‡§2

From the ‡Departments of Oncologic Sciences and �Interdisciplinary Clinical Oncology, Mitchell Cancer Institute, University ofSouth Alabama, Mobile, Alabama 36604 and §Departments of Biochemistry and Molecular Biology and ¶Pathology, College ofMedicine, University of South Alabama, Mobile, Alabama 36688

Extensive desmoplasia is a prominent pathological character-istic of pancreatic cancer (PC) that not only impacts tumordevelopment, but therapeutic outcome as well. Recently, wedemonstrated a novel role of MYB, an oncogenic transcriptionfactor, in PC growth and metastasis. Here we studied its effecton pancreatic tumor histopathology and associated molecularand biological mechanisms. Tumor-xenografts derived fromorthotopic-inoculation of MYB-overexpressing PC cells exhib-ited far-greater desmoplasia in histological analyses comparedwith those derived from MYB-silenced PC cells. These findingswere further confirmed by immunostaining of tumor-xenograftsections with collagen-I, fibronectin (major extracellular-ma-trix proteins), and �-SMA (well-characterized marker of myofi-broblasts or activated pancreatic stellate cells (PSCs)). Likewise,MYB-overexpressing PC cells provided significantly greatergrowth benefit to PSCs in a co-culture system as compared withthe MYB-silenced cells. Interrogation of deep-sequencing datafrom MYB-overexpressing versus -silenced PC cells identifiedSonic-hedgehog (SHH) and Adrenomedullin (ADM) as two dif-ferentially-expressed genes among others, which encode forsecretory ligands involved in tumor-stromal cross-talk. In-silicoanalyses predicted putative MYB-binding sites in SHH andADM promoters, which was later confirmed by chromatin-im-munoprecipitation. A cooperative role of SHH and ADM ingrowth promotion of PSCs was confirmed in co-culture by usingtheir specific-inhibitors and exogenous recombinant-proteins.Importantly, while SHH acted exclusively in a paracrine fashionon PSCs and influenced the growth of PC cells only indirectly,ADM could directly impact the growth of both PC cells andPSCs. In summary, we identified MYB as novel regulator of pan-creatic tumor desmoplasia, which is suggestive of its diverseroles in PC pathobiology.

Pancreatic cancer (PC)3 is one of the most lethal malignan-cies in the United States, with a dismal median survival rate of2– 8 months after diagnosis and a 5-year overall survival rate of�7% (1). According to an estimate by the American CancerSociety, it is expected to overtake breast cancer to become thethird leading cause of cancer-related death in the United States(2). Nearly 53,070 patients are expected to be diagnosed withPC this year and �41,780 will succumb to it (1). This continuedincrease in mortality rates is alarming and necessitates that wegenerate a better understanding of PC biology by identifyingkey molecular determinants of its pathological phenotypes, sothat novel and more effective mechanism-based therapeuticapproaches can be developed.

Pancreatic tumors are highly desmoplastic in nature exhibit-ing the presence of dense, fibrous connective tissue surround-ing the tumor cells (3, 4). Desmoplasia is characterized by anincrease in the presence of �-smooth muscle actin (�-SMA)-positive fibroblasts (myofibroblasts or activated pancreatic stel-late cells) along with extensive deposition of extracellularmatrix (ECM) proteins (5). This desmoplastic reaction not onlygives pancreatic tumors their characteristic histoarchitecture,but is suggested to play important roles in disease progressionand chemoresistance (6). Pancreatic stellate cells (PSCs) are themajor cellular component of the desmoplastic stroma that syn-thesize and secrete several ECM components within tumormicroenvironment (7–10). Pancreatic tumor cell-derived sig-naling ligands have been shown to promote proliferation anddifferentiation of PSCs, and targeting of these signaling nodes issuggested to improve the efficacy of chemotherapy in preclini-cal models (5, 6).

MYB, a cellular progenitor of v-Myb oncogene, encodes anoncogenic transcription factor that is shown to regulate a vari-ety of cellular function by controlling expression of a wide arrayof genes (11). Genetic alterations of MYB in humans were firstreported in acute myelogenous leukemia (12). However, in sub-sequent years, several studies have demonstrated genetic alter-ations or deregulated MYB expression in many other humancancers as well, including pancreatic cancer (13–16). Recently,we demonstrated that MYB serves as a novel regulator of pan-

* This work was supported by funding from National Institutes of Health/NCIGrants CA167137, CA175772, and CA185490 (to A. P. S.) and the USAMCI.The authors declare that they have no conflicts of interest with the con-tents of this article. The content is solely the responsibility of the authorsand does not necessarily represent the official views of the National Insti-tutes of Health.

1 Both authors contributed equally to this manuscript.2 To whom correspondence should be addressed: Dept. of Oncologic Sci-

ences, Mitchell Cancer Institute, University of South Alabama, 1660Springhill Ave., Mobile, AL 36604-1405. Tel.: 251-445-9843; Fax: 251-460-6994; E-mail: asingh@health.southalabama.edu.

3 The abbreviations used are: PC, pancreatic cancer; SMA, smooth muscleactin; ECM, extracellular matrix; PSC, pancreatic stellate cells; SHH, Sonic-hedgehog; ADM, Adrenomedullin.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 31, pp. 16263–16270, July 29, 2016

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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creatic tumor growth and metastasis (17). Its expression wasreported in majority of PC tissues and cell lines, while itremained largely undetectable in normal pancreatic cells. Wealso identified several gene targets of MYB that may mediate itsimpact on PC pathogenesis. However, its in-depth mechanisticinvolvement and diverse effects on pancreatic tumor pheno-types are not yet well understood.

Here, we provide evidence supporting a role of MYB in pan-creatic tumor desmoplasia. We show that the tumors derivedfrom MYB-overexpressing PC cells exhibit greater desmopla-sia, characterized by the expression of myofibroblast marker(�-SMA) and extracellular-matrix proteins (collagen I andfibronectin). We also establish SHH (Sonic hedgehog) and ADM(Adrenomedullin) as direct transcriptional targets of MYB thatmediate its effects on the growth of pancreatic tumor and stel-late cells via autocrine and/or paracrine signaling. Together,our studies establish MYB as a key driver of PC progression andmetastasis by directly impacting the tumor cells as well as byaltering the tumor microenvironment.

Results

Tumors Derived from MYB-overexpressing Pancreatic Can-cer Cells Exhibit High Desmoplasia—We performed histo-pathological analyses of tumor xenografts derived fromrecently developed orthotopic mouse model of pancreatic can-cer (17). Since tumors were derived from MYB-overexpressingand -silenced cells, we observed expected differences in MYBexpression in tumor xenograft sections as well (data notshown). When stained with H&E, tumor sections from highMYB-expressing group exhibited dense desmoplastic regionssurrounding the pancreatic tumor cells, while only minimaldesmoplasia was observed in that of low MYB-expressing group(Fig. 1A). This observation was further confirmed by immuno-

staining the tumor sections for collagen I, fibronectin, and�-SMA, which are well-characterized markers of desmoplasia(5). A dramatic decrease in the expression of collagen I,fibronectin, and �-SMA was detected in tumors generatedfrom MiaPaCa-shMYB cells compared with those derived fromMiaPaCa-NT-Scr cells (Fig. 1, B–D). Likewise, we also observedhigh desmoplasia associated with elevated expression of colla-gen I and �-SMA in tumors derived from MYB-overexpressingBxPC3-MYB cells (data not shown). Together, these findingssuggest that MYB may serve as a novel regulator of pancreatictumor desmoplasia.

MYB Expression in Pancreatic Tumor Cells Is Associated withGrowth Induction and Differentiation of Pancreatic StellateCells in a Co-culture System—Since pancreatic stellate cells(PSCs) are the major cellular component of the stromal com-partment of pancreatic tumors and responsible for desmoplas-tic reaction, we next investigated the functional consequencesof their interaction with high and low MYB-expressing pancre-atic cancer cells (PCCs) in a co-culture system. For this, we usedmatched cell lines, MiaPaCa-NT-Scr/MiaPaCa-shMYB andBxPC3-Neo/BxPC3-MYB that are genetically engineered toserve as control or MYB-knockdown/overexpressing cells (17).Differential MYB expression in these cell lines was confirmedby immunoblot assay prior to any functional assay (data notshown). PSCs co-cultured with BxPC3-MYB and MiaPaCa-NT-Scr cells exhibited significant enhancement in their growth(58.4 and 51.8%, respectively), while it was minimal in PSCsco-cultured with BxPC3-Neo and MiaPaCa-shMYB cells (10.4and 9.2%, respectively) (Fig. 2A). Enhanced growth of PSCs alsocorrelated with increased expression of �-SMA (differentiationmarker of PSCs) (Fig. 2B). In addition, we also monitored thegrowth of high and low MYB expressing PCCs when co-cul-

FIGURE 1. MYB induces desmoplasia in pancreatic tumors. A, tissue sections of orthotopically developed pancreatic tumors from high (MiaPaCa-NT-Scr) andlow (MiaPaCa-shMYB) MYB-expressing PC cells were deparaffinized, rehydrated, and stained with H&E to study their histopathological characteristics. B–D,deparaffinized and rehydrated tumor tissue sections were incubated overnight with (B) collagen-I, (C), fibronectin, and (D) �-SMA specific antibodies. Subse-quently, sections were incubated at room temperature with respective polymer and probe, and immunoreactivity was visualized by incubation with DABChromogen followed by hematoxylin counterstain. Arrows indicate the desmoplastic region surrounding the tumor cells.

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tured with PSCs. Data demonstrate that the growth of MYB-overexpressing BxPC3-MYB and MiaPaCa-NT-Scr cells wasinduced (�53 and 47%, respectively) when co-cultured withPSCs, whereas only little induction was observed (�8%) in lowMYB-expressing BxPC3-Neo and MiaPaCa-shMYB cells (Fig.2C). Taken together, our data suggest that MYB overexpressionin PC cells is of mutual significance for both pancreatic tumorand stellate cells when they co-exist and communicate freelywith each other.

ADM and SHH Are Novel Direct Transcriptional Targets ofMYB in Pancreatic Cancer Cells—To understand the molecularbasis of MYB-potentiated phenotypic changes, we interrogatedour differential gene expression data from deep sequencinganalysis of MYB-expressing and -silenced pancreatic tumorcells (GEO accession number GSE61290).4 Among several dif-ferentially expressed genes, we found a decreased expression ofADM (adrenomedullin) and SHH (sonic hedgehog) in MYB-silenced cells, which have previously been implicated in thedevelopment of desmoplasia in pancreatic tumors (5, 18). Toascertain the effect of MYB silencing on ADM and SHH, weanalyzed their expression in MYB-expressing (MiaPaCa-NT-Scr) and -silenced (MiaPaCa-shMYB) cells as well as MYB-nullBxPC3 and ectopically MYB-expressing BxPC3-MYB cells atboth mRNA (by quantitative RT-PCR) and protein (by immu-noblot) levels. We observed enhanced expression of ADM andSHH in high MYB-expressing cells as compared with low MYBor MYB-null pancreatic cell lines both at the transcript (Fig. 3,A and B) and protein (Fig. 3C) levels. Since both ADM and SHHare secreted proteins, we also measured their levels in culturesupernatants of MYB-overexpressing/silenced PC cells byELISA. As expected, significantly enhanced levels of ADM (3.6-fold; Fig. 3D) as well as SHH (5.3-fold; Fig. 3E) were recorded inthe medium secretions of BxPC3-MYB in comparison to the

BxPC3-Neo cells, while these were decreased considerably(ADM, 5.4-fold; SHH, 6.8-fold) in MYB-silenced MiaPaCa cells(Fig. 3, D and E). Tumor sections derived from mice of high andlow MYB- expressing groups also exhibited similar associationin IHC analyses (Fig. 3F).

To examine if ADM and SHH were direct transcriptionalcontrol of MYB, we performed in silico analyses of �1-kb DNAregion 5� upstream of their coding DNA sequence (CDS)(GenBankTM accession numbers D43639 and AY422195.1,respectively) using web-based applications (ALGGEN-PROMOand TFsearch). Several putative MYB binding sites weredetected within this region of both ADM and SHH (Fig. 4A). Wenext conducted ChIP analyses to confirm the direct binding ofMYB to the ADM and SHH promoters. This was followed byPCR amplification of ChIPed DNA using primers in the flank-ing regions of putative MYB binding sites (Fig. 4A). It wasrevealed that MYB was able to bind to the ADM and SHH pro-moters at least in the regions that are in close proximity totranscriptional initiation sites (Fig. 4, B and C). Furthermore,MYB-silenced MiaPaCa-shMYB cells exhibited significantlyreduced PCR amplification signal for both ADM and SHHupstream regions supporting the specificity of MYB-dependentchromatin pulldown (Fig. 4, B and C, respectively). Together,these findings establish MYB as a novel transcriptional regula-tor of ADM and SHH in pancreatic tumor cells.

ADM and SHH Cooperatively Mediate the Effect of MYB onthe Growth of Pancreatic Tumor and Stellate Cells via Para-crine and/or Autocrine Signaling—To confirm the role of ADMand SHH in MYB-induced growth of PSCs, we subjected theco-culture of PSCs and high MYB-expressing cells to ADMantagonist (ADM-(22–52)) and/or SHH-neutralizing antibody(5E1) treatments. In parallel, the co-culture of PSCs with lowMYB-expressing cells was treated with recombinant humanADM-(1–52) and SHH (r-SHH). Significant growth reductionin BxPC3-MYB and MiaPaCa-NT-Scr cells was observed upontreatment with ADM-(22–52), whereas it was relatively lessin cells treated with 5E1 (Fig. 5A). Likewise, a greater decreasein growth induction of PSCs co-cultured with MYB-overex-pressing BxPC3-MYB and MiaPaCa-NT-Scr cells was observedwhen treated with ADM-(22–52) (�63 and �65%, respec-tively) as compared with that observed upon treatment with5E1 (�48 and �50%, respectively) (Fig. 5B). A more potentinhibition of growth induction of both the PCCs (Fig. 5A) andPSCs (Fig. 5B) was found when the co-culture was treatedsimultaneously with ADM-(22–52) and 5E1. Growth ofBxPC3-Neo cells co-cultured with PSCs was increased by 12.3and 15.3% upon treatment with ADM-(1–52) and r-SHH,respectively, while slightly higher effect (19.8%) was observedupon their co-treatments (Fig. 5C). Likewise, the growth ofPSCs co-cultured with low MYB-expressing cells also increasedsignificantly upon ADM-(1–52) treatment (64.7%, BxPC3-Neo;85.4%, MiaPaCa-shMYB); or r-SHH (77.3%, BxPC3-Neo;76.5%, MiaPaCa-shMYB) (Fig. 5D). A greater effect on thegrowth of PSCs was observed when the co-culture was simul-taneously treated with ADM-(1–52) and r-SHH (84.2%,BxPC3-Neo; 90.8%, MiaPaCa-shMYB) (Fig. 5D).

To ascertain if the impact of ADM and SHH was solely para-crine or involve an autocrine loop as well, we treated high MYB-4 Transcript profiling: GEO accession number GSE61290.

FIGURE 2. MYB promotes growth of pancreatic stellate cells. A, pancreaticstellate cells (PSCs; 2 � 105/well) were seeded in the 6-well plates, and pan-creatic cancer cells (PCCs; 1 � 105/well) were seeded into insert chamber.After 24 of culturing, insert having PCCs was placed over the well containingPSCs and allowed to grow for 144 h with medium replacement after every48 h. Induction of growth in PSCs relative to respective monocultures (PSCs/PSCs) was examined by counting the number of viable cells. B, PCCs (1 �105/well) were seeded in the 6-well plates, and PSCs (2 � 105/well) wereseeded into insert chamber and growth of PCCs was examined by countingthe number of viable cells after 144 h as described above. For control, bothwell and insert chambers were seeded with PCCs. Data presented as mean �S.D., n � 3; *, (p � 0.05). C, total protein from PSCs co-cultured with high(BxPC3-MYB and MiaPaCa-NT-Scr) and low (BxPC3-Neo and MiaPaCa-shMYB)MYB-expressing cells was isolated and the expression of �-SMA was exam-ined by immunoblot assay. �-actin was used as internal control.

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expressing PC cells with ADM-(22–52) and 5E1 alone or incombination. Similarly, we also treated low MYB-expressingcells with ADM-(1–52) and/or r-SHH. We observed that the

growth of high MYB-expressing PC cells was inhibited (22.3%,BxPC3-MYB; 23.9%, MiaPaCa-NT-Scr) when treated withADM-(22–52); however, limited effect was observed upon

FIGURE 3. MYB enhances the expression of SHH and ADM in pancreatic cancer cells. A–B, total RNA was isolated from BxPC3-Neo/BxPC3-MYB andMiaPaCa-NT-Scr/MiaPaCa-shMYB PC cells and expression of (A) ADM and (B) SHH was examined by q-RT-PCR using specific primer sets. GAPDH served as aninternal control. Bars represent mean � S.D., n � 3; *, (p � 0.05). C, total protein from PC cells was isolated, resolved by SDS-PAGE and the expression of ADMand SHH was examined by immunoblot assays. �-actin was used as loading control. D–E, PC cells (1 � 106/well) were grown in regular culture condition for 24 h.Subsequently, medium was replaced with 2% FBS-containing medium and allowed to grow for next 48 h. Thereafter, culture supernatants were collected andcentrifuged to clear the cellular debris and levels of (D) ADM and (E) SHH were measured using respective ELISA kits. Expression levels of ADM and SHH werenormalized with cell number at end point, and data shown as mean � S.D., n � 3; *, (p � 0.05). F, tissue sections of orthotopically developed tumors fromMiaPaCa-NT-Scr/MiaPaCa-shMYB PC cells were incubated overnight with ADM or SHH specific antibodies at 4 °C. Thereafter, sections were incubated at roomtemperature with respective polymer and probe, and immunoreactivity was visualized by using DAB Chromogen followed by hematoxylin counterstain.

FIGURE 4. MYB regulates SHH and ADM via direct binding to their promoter regions. A, localization of putative MYB binding sites (thick black bars) inhuman ADM (hADM) and human SHH (hSHH) promoter region. Arrows indicate the complementary sites for the forward and reverse primers in the flankingregion of MYB binding site(s). B–C, DNA-protein was cross-linked with formaldehyde. Cross-linked chromatin was sheared and subjected to immunoprecipi-tation using anti-MYB or normal rabbit IgG (as control). PCR was performed using specific primers sets flanking the MYB-binding sites within the (B) ADM and(C) SHH promoter regions. Input DNA (without immunoprecipitation) and normal IgG-precipitated DNA were used as positive and negative controls, respec-tively. Bars represent mean � S.D., n � 3; *, (p � 0.05).

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treatment with 5E1 (Fig. 6A). Interestingly, when we treatedlow MYB-expressing cells with either ADM-(1–52) or r-SHHor combination, we did not observe much induction in theirgrowth (7.9, 6.5, 10.6% in BxPC3-Neo and 8.6, 6.4, 13.1% inMiaPaCa-shMYB, respectively) (Fig. 6B). Treatment of PSCswith ADM-(1–52), r-SHH, ADM-(22–52) and 5E1 alone or incombination confirmed their growth response to both ADMand SHH (Fig. 6C). Taken together, data suggest that ADM andSHH cooperatively mediate the effect of MYB on the growth ofpancreatic stellate as well as tumor cells via paracrine and/orautocrine signaling.

Discussion

Although some doubts have been raised regarding the func-tional significance of extensive desmoplasia in PC pathobiol-ogy, the fact remains that it is a unique feature of PC and co-evolves with the malignant disease during the course of itsprogression (6, 9, 10, 19, 20). Therefore, it remains of utmostimportance to understand the molecular basis of its evolutionas well as its biological importance in PC development. Ournovel findings provide compelling data to suggest a role of MYBin pancreatic tumor desmoplasia, and thus further adding to itspathobiological significance in PC.

We recently reported a novel role of MYB in pancreatictumor growth and metastasis (17). These effects were suggestedto involve a positive impact of MYB on cell-cycle progression,apoptosis-resistance, as well as malignant features of pancreatictumor cells via targeting of functionally relevant gene targets.The data presented herein demonstrate that MYB-overex-pressing pancreatic tumors also differ in their histopathology.Tumors derived from MYB-expressing PC cells were highlyfibrotic, as demonstrated by the immunostaining of ECM pro-teins, collagen I, and fibronectin, while only minimal reactivitywas reported in tumors derived from MYB-silenced PC cells.

Both collagen I and fibronectin are not only major componentsof pancreatic tumor-associated stroma, but they have also beenshown to promote cancer development by inducing EMT andinvasiveness of pancreatic tumor cells (21–23). A major sourceof these ECM proteins is myofibroblasts, which originate fromactivated pancreatic stellate cells (PSCs) (7, 8). Accordingly, our

FIGURE 5. ADM and SHH mediate the effects of MYB on growth of pancreatic tumor and stellate cells. A–B, PSCs were co-cultured with high MYB-expressing (BxPC3-MYB and MiaPaCa-NT-Scr) PCCs as described earlier and treated with ADM antagonist (ADM-(22–52)) and/or SHH neutralizing antibody(5E1) for 144 h with replacement of treatment medium after every 48 h. Number of viable (A) PCCs and (B) PSCs was measured by trypan blue dye-exclusionmethod. Cell suspension was diluted 1:1 using a 0.4% trypan blue solution followed by cell counting using Countess� Automated Cell Counter that quantify cellnumber (live, dead, and total cells) and provides percent viability. C–D, co-culture of PSCs and low MYB-expressing (BxPC3-Neo and MiaPaCa-shMYB) PCCswere treated with ADM-(1–52) and/or r-SHH for 144 h with treatment medium replacement after every 48 h and growth change of (C) PCCs and (D) PSCs wasexamined as described above by by trypan blue dye-exclusion method. Data (mean � S.D.; n � 3) shown as change in growth as compared with control.*, p �0.05.

FIGURE 6. MYB-induced and ADM/SHH-mediated growth of pancreatictumor and stellate cells involves autocrine and/or paracrine signaling. A,high MYB-(BxPC3-MYB and MiaPaCa-NT-Scr) were grown (3 � 103 cells/well)in 96 well plate for 24 h. Post-incubation, cells were treated with ADM antag-onist (ADM-(22–52)) or SHH neutralizing antibody (5E1) alone or in combina-tion for 144 h with treatment medium replacement after every 48 h andgrowth examined by WST-1 assay as per the manufacturer’s instructions. B,low MYB- (BxPC3-Neo and MiaPaCa-shMYB)-expressing PC cells were cul-tured in a 96-well plate (3 � 103 cells/well) and treated with ADM-(1–52) andr-SHH, alone or in combination, and effect on the growth induction was exam-ined after 144 h by WST-1 assay. C, PSCs were grown (3 � 103 cells/well) in a96-well plate and treated with ADM-(1–52), r-SHH, ADM-(22–52), or 5E1 alone orin combination for 144 h with replacement of treatment medium after every 48 hand effect on growth was determined by WST-1 assay. Data (mean � S.D.; n � 3)shown as change in growth in comparison to control.*, p � 0.05.

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immunohistochemical data demonstrated high levels ofsmooth muscle �-actin (�-SMA), a well-established marker ofmyofibroblasts, within desmoplastic stroma of MYB-overex-pressing pancreatic tumors. This also suggested that MYB-overexpressing PC cells interact differentially with PSCs to pro-mote desmoplastic reaction during tumor development.

Positive interaction of MYB-overexpressing pancreatictumor cells with PSCs was also confirmed in our in vitro co-cul-ture studies. MYB-overexpressing PC cells not only promotedthe growth of PSCs in co-culture, but also induced the expres-sion of �-SMA suggesting their activation and differentiationinto myofibroblasts. These effects were less pronounced inMYB-silenced or MYB-null cells thus supporting a role of MYBin this tumor-stromal cross-talk. However, stimuli for PSCsshould come from the changes in its microenvironment (24 –26), and specifically in this case from factors secreted by MYB-overexpressing cells. These factors were characterized to beSHH and ADM that exhibited greater expression in MYB-over-expressing cells at RNA, protein and secretome levels. Moreimportantly, our data suggested mostly a paracrine action ofSHH, while the other factor ADM, influenced the growth ofboth PSCs and PC cells and thus likely acted in both paracrineand autocrine manner. These findings were consistent withpublished data that has suggested mostly a paracrine signalingfor pancreatic tumor cell-derived SHH (27). Inhibition of SHHwas shown to decrease desmoplasia through direct activation ofhedgehog signaling in PSCs (5), while it indirectly affected PCmetastasis and lymphangiogenesis due to altered tumormicroenvironment (28). In other reports, ADM has also beenshown to be up-regulated in PC patients (29). It has been sug-gested that ADM acts directly on the tumor cells, but also influ-ences tumor progression through its effects on endothelial cellsand PSCs (18, 30). Moreover, despite some conflicting data sug-gesting a suppressive effect of PSCs on PC progression andsurvival (9, 10), our data indicate mutual benefit of pancreatictumor and stellate cells cross-talk at the molecular level.

Several mechanisms have been reported for tumor-stromalinteractions culminating into ECM remodeling (31, 32). In fact,not only the tumor cells, but also the hypoxic and inflammatorytumor microenvironment could also influence stromal compo-sition of pancreatic tumors (23, 24, 26). Studies on bi-direc-tional tumor-stromal cross-talk in PC have reported signalingpathways that could influence the production of SHH andADM (27, 33). Moreover, hypoxic tumor microenvironmenthas also been shown to promote synthesis and secretion of SHHby pancreatic tumor cells to promote desmoplasia and thusengage in vicious hypoxia-desmoplasia loop (34). Similarly,ADM has also been reported to be up-regulated by hypoxia inpancreatic tumor cells (35). In this regard, our findings estab-lishing MYB as a novel transcriptional regulator of SHH andADM are of great significance and indicative of important rolesof MYB as a mediator in active cross-talk between tumor cellsand a variety of factors within the tumor microenvironment. Itis likely that MYB, either mediate the tumor-tumor microenvi-ronment signaling or act in cooperation to promote positivetumor-stromal interaction, and thus facilitate pancreatic tumordevelopment.

In summary, we have identified important role of MYB in thegrowth promotion and differentiation of pancreatic stellatecells through directly regulating SHH and ADM. We have alsodemonstrated that ADM and SHH cooperatively mediate theeffect of MYB on the growth of pancreatic tumor and stellatecells via paracrine and/or autocrine signaling. These findingsestablishing MYB as a novel regulator of pancreatic tumor des-moplasia are of great significance from the molecular patho-genesis standpoint and provide us novel insight into PCpathobiology.

Experimental Procedures

Cell Lines and Culture Conditions—All the pancreatic cancercell lines used in this study were procured and maintained aspreviously described (17). Pancreatic stellate cells (generouslygifted by Dr. P. K. Singh, Eppley Cancer Institute, Omaha, NE)were maintained in DMEM supplemented with 20% FBS and100 �M each of penicillin and streptomycin in a humidifiedatmosphere of 5% CO2 at 37 °C. All the cells were tested anddetermined to be free of mycoplasma every month and prior tobeginning of any functional assay.

Reagents, Gene Constructs, and Antibodies—The followingreagents were used: Roswell Park Memorial Institute medium(RPMI 1640); Dulbecco’s Modified Eagle Medium (DMEM);fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville,GA); penicillin and streptomycin (Invitrogen, Carlsbad, CA);Adrenomedullin (Human)-ELISA Kit (Phoenix Pharmaceuti-cals, Inc. Burlingame, CA); MycoSensorPCR assay kit (Strat-agene, La Jolla, CA); FuGENE transfection reagent (Roche,Indianapolis, IN); a chromatin immunoprecipitation assay(ChIP) kit (Active Motif, Carlsbad, CA); Western blottingSuperSignal West Femto Maximum sensitivity substrate kit(Thermo Scientific, Logan, UT); immunohistochemical analy-sis reagent EZ-Dewax (Biogenex, Fremont, CA); backgroundsniper, polymer and probe (Biocare Medical, Concord, CA).The following antibodies were used: �-SMA (1:100, rabbit poly-clonal; S0010) (Epitomics, Burlingame, CA), ADM (1:1000, rab-bit polyclonal; ab69117), fibronectin (ab6328) and collagen I(ab88147) (1:100, mouse monoclonal) and SHH (1:1000; rabbitmonoclonal; ab53281) (Abcam, Cambridge, MA), mousemonoclonal biotinylated anti-�-actin (1:20,000; A3854; Sigma-Aldrich) and horseradish peroxidase (HRP)-labeled secondaryantibodies (1:2000; Santa Cruz Biotechnology).

Western Blotting Analysis—Western blotting was performedusing standard procedures as described earlier (36, 37). Briefly,cell lysates were resolved on 10% polyacrylamide gels and trans-ferred to PVDF membranes. Blots were subjected to a standardimmunodetection procedure using specific antibodies againstvarious proteins and visualized using SuperSignal West FemtoMaximum sensitivity substrate kit with a LAS-3000 image ana-lyzer (Fuji Photo Film Co., Tokyo, Japan).

RNA Isolation, cDNA Synthesis, and Quantitative Real Time-PCR (qRT-PCR)—Total RNA was extracted using TRIzol re-agent. Two microgram of total RNA was used for cDNAsynthesis using the High Capacity complementary DNAreverse transcription kit following manufacturer’s instruc-tions. Subsequently, quantitative RT-PCR was performed in96-well plates using cDNA as a template and SYBR Green Mas-

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ter Mix on an iCycler system (Bio-Rad) with specific primer pairsets SHH-Forward-5�-ACCGAGGGCTGGGACGAAGA-3�,SHH-Reverse- 5�-ATTTGGCCGCCACCGAGTT-3�, ADM-Forward-5�-CGGGATCCATGAAGCTG GTTTCCGTC-3�,ADM-Reverse-5�-CGGAA TTCCTAAAGAAAGTGGGG-AGC-3�, GAPDH-Forward- 5�-GGTGGTCTCCTCT GACT-TCAACA-3� and GAPDH-Reverse-5�-GTTGCTGTAGCCA-AATTCGTTGT-3�. The thermal conditions for real-time PCRassays were as follows: cycle 1: 95 °C for 10 min, cycle 2 (�40):95 °C for 10 s and 58 °C for 45 s.

Immunohistochemical and Histological Analyses—Immuno-histochemical analyses were performed as described earlier(38). All the antibodies were used at 1:100 dilutions except anti-body against fibronectin (1:50). Tumor tissue sections werestained with H&E and visualized under microscope (100� and400�), and photographed.

Enzyme-linked Immunosorbent Assay (ELISA)—Cells (1 �106/well) were seeded in 6-well plates in regular growthmedium. After 24 h, medium was replaced with low (2%)serum-containing medium and cells were further allowed togrow for next 48 h. Thereafter, culture supernatants were col-lected and centrifuged to clear the cellular debris. ADM andSHH levels were then detected using respective ELISA kit as permanufacturer’s instructions.

Chromatin Immunoprecipitation (ChIP) Assay—ChIP assaywas performed using a ChIP-IT enzymatic kit as previouslydescribed (27). Briefly, DNA-protein cross-linking was donewith paraformaldehyde (37%) followed by enzymatic DNAshearing. Sheared DNA was then subjected to immunoprecipi-tation using anti-MYB or normal rabbit IgG (as control). Sub-sequently, cross-linking was reversed, proteins digested withproteinase K, and DNA isolated. PCR was performed usingspecific primers sets (SHH-Chip-Forward-5�-CCCAACTCC-GATGTGTTCCG-3�, SHH-Chip-Reverse-5�-ATATAACCT-TGCCCGC CGC-3�, ADM-Chip-Forward-5�-CTCCGGC GT-ACTGTCTGAA-3� and ADM-Chip-Reverse-5�-CTTCCT-TTGGGGCTGGAGTTG-3�) flanking MYB-binding promoterregions and amplification products resolved on a 2.0% agarosegel and visualized using ethidium bromide staining. Input DNA(without immunoprecipitation) and normal IgG-precipitatedDNA were used as positive and negative controls, respectively.

Co-culture Assay—Pancreatic stellate cells (PSCs; 2 � 105/well) were seeded in the 6-well plates, and pancreatic cancercells (PCCs; 1 � 105/well) were seeded into insert chamber(1-�M pore size). Insert having PCCs was placed over the wellscontaining PSCs and PSCs/PCCs co-culture was allowed toestablish for next 24 h. Thereafter, medium (control or treat-ment) was replaced after every 48 h and number of viable cellswere counted at 144 h by trypan blue dye exclusion assay. Incontrol experiments, both well and insert chambers wereseeded with either PSCs or PCCs.

WST-1 Assays—Cells (3 � 103) were seeded in 96-well plates,and medium was replaced next day with control or treatmentmedium. Cells were allowed to grow for the next 6 days, andmedium replenished every 48 h. Subsequently, WST-1 assaywas performed as per the manufacturer’s instructions.

Statistical Analysis—All experiments were performed at leastthree times and numerical data expressed as mean � S.D. Wher-

ever appropriate, the data were also subjected to unpaired two-tailed Student’s t test, and p � 0.05 was considered as significant.

Author Contributions—A. B., A. P. S.: study design; A. B., S. K. S.,N. T., S. A.: acquisition of data; A. B., S. K. S., S. S., J. E. C., M. K.,A. P. S.: analysis and interpretation of data; A. B., S. K. S., S. S.,A. P. S.: manuscript preparation; A. P. S.: obtained funding.

Acknowledgment—We thank Dr. Aamir Ahmad (USAMCI) for care-ful reading of the manuscript.

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E. Carter, Moh'd Khushman and Ajay P. SinghArun Bhardwaj, Sanjeev K. Srivastava, Seema Singh, Nikhil Tyagi, Sumit Arora, James

Up-regulation and Cooperative Action of Sonic Hedgehog and AdrenomedullinMYB Promotes Desmoplasia in Pancreatic Cancer through Direct Transcriptional

doi: 10.1074/jbc.M116.732651 originally published online May 31, 20162016, 291:16263-16270.J. Biol. Chem. 

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