Membrane proximal ectodomain cleavage of MUC16 occurs in the acidifyingGolgi/post-Golgi compartments

Das, Srustidhar; Majhi, Prabin D.; Al-Mugotir, Mona H.; Rachagani, Satyanarayana; Sorgen, Paul; Batra, Surinder K.

doi:10.1038/srep09759

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Published: 05 June 2015

Membrane proximal ectodomain cleavage of MUC16 occurs in the acidifyingGolgi/post-Golgi compartments

Srustidhar Das¹,
Prabin D. Majhi¹,
Mona H. Al-Mugotir¹,
Satyanarayana Rachagani¹,
Paul Sorgen¹ &
…
Surinder K. Batra^1,2,3

Scientific Reports volume 5, Article number: 9759 (2015) Cite this article

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Abstract

MUC16, precursor of the most widely used ovarian cancer biomarker CA125, is upregulated in multiple malignancies and is associated with poor prognosis. While thepro-tumorigenic and metastatic roles of MUC16 are ascribed to the cell-associatedcarboxyl-terminal MUC16 (MUC16-Cter), the exact biochemical nature of MUC16 cleavagegenerating MUC16-Cter has remained unknown. Using different lengths of dual-epitope(N-terminal FLAG- and C-terminal HA-Tag) tagged C-terminal MUC16 fragments, wedemonstrate that MUC16 cleavage takes place in the juxta-membrane ectodomain stretchof twelve amino acids that generates a ~17 kDa cleaved product and isdistinct from the predicted sites. This was further corroborated by domain swappingexperiment. Further, the cleavage of MUC16 was found to take place in theGolgi/post-Golgi compartments and is dependent on the acidic pH in the secretorypathway. A similar pattern of ~17 kDa cleaved MUC16 was observed inmultiple cell types eliminating the possibility of cell type specific phenomenon.MUC16-Cter translocates to the nucleus in a cleavage dependent manner and binds tothe chromatin suggesting its involvement in regulation of gene expression. Takentogether, we demonstrate for the first time the oft-predicted cleavage of MUC16 thatis critical in designing successful therapeutic interventions based on MUC16.

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Introduction

Mucins are high molecular weight glycoproteins and are primarily expressed by thesecretory epithelial cells lining respiratory, gastrointestinal and reproductivetracts¹. Although mucins are thought to protect epithelialsurfaces from various physical insults, recent molecular studies have generated interestin their use as diagnostic and therapeutic targets^1,2,3 for theirrole in cancer. In particular, the transmembrane mucins have been implicated in variousoncogenic signaling pathways and their de novo expression in certain malignanciesrenders them very attractive targets^1,2,3. MUC16 (CA125), thebest-known biomarker for ovarian cancer⁴, is up regulated inmultiple malignancies and is strongly associated with poor prognosis^5,6,7. MUC16 is a type-I transmembrane protein with a heavily glycosylated N-terminalregion, a tandem repeat region comprising of approximately 60 repeats of~156 amino acids each, a transmembrane (TM) domain and a cytoplasmic taildomain (CTD) of 32 amino acids^8,9. MUC16 is predicted to harbor~56 SEA (Sperm protein, Enterokinase and Agrin) domains unlike other mucinssuch as MUC1, MUC12, MUC13 and MUC17 which possess only a single SEA module^9,10.

SEA domain(s) present in mucin and non-mucin proteins are shown to possessautoproteolytic activity^11,12,13. MUC16 is speculated to harbortwo proteolytic sites in the membrane proximal SEA domains i.e. 50 residues proximal tothe TM domain (site #1 at PLARRVDR) in the last (56^th)¹⁴ and the ‘DSVLV’ site (site #2) analogous to the MUC1‘GSVVV’ site in the penultimate (55^th) SEAdomains¹². However, neither has been experimentally validated.There have been studies addressing the functional significance of various lengths ofMUC16 carboxyl-terminal region (283 and 413 amino acids) in ovarian, breast and coloncancer cells, however, the exact biochemical nature of MUC16 cleavage was not addressedin these studies^15,16,17,18. Besides, while the autoproteolyticcleavage in the last and penultimate SEA domain has been hypothesized to be the majorproteolytic mechanism of MUC16 cleavage, proteases such as MMP-7, neutrophil elastase(NE) and bacterial metalloprotease (ZmpC) have been implicated in enhanced shedding ofMUC16 from the cell surface^19,20.

Given the importance of CA125 in ovarian cancer, antibodies such as Oregovomab andAbagovomab against CA125 have been used in clinical trials for ovarian cancer patientswithout positive outcomes^21,22,23. Since these antibodies bind tothe extracellular portion of MUC16 (i.e. CA125), the potential reasons for the failureof these antibodies are (i) binding of these antibodies to the circulating (shed) CA125,therefore, reducing the amount of antibodies available to target and kill the cancercells, (ii) the kinetics and dynamics of MUC16 cleavage/shedding from the tumor cells isnot well understood, therefore, the likelihood of shedding of cell-surface MUC16 wouldstill reduce the availability of these therapeutic antibodies to the cancer cells²¹. Taken together, an understanding of biochemical nature of MUC16cleavage and its potential regulators would be critical in devising successfultherapeutic strategy based on MUC16/CA125.

For the first time, we report here the experimental evidence for the oft-predictedcleavage of MUC16. This takes place in the juxta membrane ectodomain region and isdistinct from the predicted sites. The cleavage is independent of extracellularproteases (i.e. NE and MMP-7) and intracellular cues (i.e. phosphorylation). Further,the cellular location of cleavage is identified to be the Golgi/post-Golgi compartmentsand MUC16 cleavage is dependent on the acidic pH in the secretory pathway. MUC16-Ctertranslocates to the nucleus in a cleavage dependent manner and independent of theputative nuclear localization signal (NLS) and participates in the regulation of geneexpression²⁴. Altogether, the present study provides insightinto the cleavage of MUC16 that is critical towards understanding its functionalsignificance under physiological and pathological conditions and subsequent therapeutictargeting in multiple cancer types.

Results

Membrane proximal ectodomain cleavage of MUC16 is spatially distinct fromthe predicted cleavage sites

Cleavage of MUC16 has been proposed to take place in the DSVLV site ofpenultimate SEA domain, analogous to MUC1 cleavage site GSVVV and/or at 50residues proximal to transmembrane (TM) domain in the last SEA domain^12,14. However, neither has been experimentally validated.Due to lack of antibodies for the juxta-membrane region of MUC16, wedemonstrated cleavage of MUC16 using dual-epitope tagging (Fig.1a and 1b). We generated a mammalian expressionconstruct with last two SEA domains of MUC16 (321 amino acids fromthe C-terminal end, termed as F321HA) that included both the predicted cleavagesites and has N-terminal preprotrypsin leader peptide for appropriate membranetargeting. Further, multiple deletion constructs were generated from F321HA(Fig. 1a). Expression of different lengths ofdual-tagged MUC16 carboxyl-terminus (MUC16-Cter) in HEK293T, except the F53HAlacking any extracellular residues, resulted in a unique ~17 kDaproduct present in HA but not FLAG immunoblot (indicated by an arrow in Fig. 1b). This demonstrates that MUC16 undergoes cleavage inthe carboxyl-terminal region as the C-terminal (HA-tagged) product is physicallyseparated from the N-terminal portion (FLAG-tagged) resulting in a unique~17 kDa fragment. Our findings demonstrate that C-terminal 65residues of MUC16 (F65HA) is the minimal length that is capable of undergoingcleavage, which harbors only 12 extracellular amino acids in addition to thetransmembrane (TM) and cytoplasmic tail domain (CTD) and no SEA domain. However,this does not rule out further cleavage(s) upstream in the last and in thepenultimate SEA domains. To assess additional cleavage(s) of MUC16 in additionto the afore-mentioned cleavage, a triple epitope-tagged construct wasengineered wherein a FLAG-tag was introduced between the N-terminal HA-tag andC-terminal Myc-tag at 29^th membrane-proximal ectodomain residueinto the 321 amino acids fragment of MUC16 (Fig. 1c).Further upstream cleavage(s) in addition to the cleavage at the juxtamembraneectodomain would result in unequal sized HA- and FLAG-tagged products. However,the size of the FLAG-tagged product was same as the corresponding HA-taggedproduct, suggesting no additional cleavage further upstream (Fig.1d). For the first time, we present experimental evidencedemonstrating cleavage of MUC16 and that the site of cleavage is distinct fromthe predicted cleavage sites^12,14.

To further substantiate our findings, we swapped various domains (extracellulardomain (ECD), TM and CTD) of 150 amino acid fragment of another transmembranemucin MUC4 with that of 114 amino acid fragment of MUC16 (Fig.1e). MUC4 is a non-SEA domain mucin with a short CTD (20 amino acids)and F-M4-150HA does not undergo cleavage (Fig. 1f, lane 3)unlike that of 114 amino acid fragment of MUC16 (Fig. 1f,lane 2) and therefore was considered to be an ideal partner for swapping.Swapping the TM or CTD alone or together (TM-CTD) of MUC4 with that of MUC16 didnot prevent its cleavage (Fig. 1f, lanes 4, 5 and 7).However, replacing the extracellular domain (ECD) of MUC16 with MUC4 abrogatedits cleavage (Fig. 1f, lane 6) that was partially rescuedby inclusion of twelve (Fig. 1f, lane 8), but not six(Fig. 1f, lane 9) membrane-proximal residues of MUC16.These findings further corroborate membrane proximal ectodomain cleavage ofMUC16 and that the 12 extracellular residues are critical for this cleavage.

N-glycosylation and ubiquitylation of carboxy-terminus of MUC16 determineits size and stability

MUC16-Cter cleavage, studied with F114HA (a more detailed description includingthe amino acids composition of the 114 carboxyl-terminal residues of MUC16 isprovided in Supplementary Fig. 1) and other constructs,yielded a unique 17 kDa HA-only product but also showed a number ofproducts with both HA and FLAG-tags of molecular weight disproportionate to thepolypeptide length (Fig. 1b). This suggests that (i)MUC16-Cter undergoes partial cleavage, leaving much of the protein uncleavedwhich undergoes various post-translational modifications and/or (ii) both endsof the cleaved protein remain associated, in specific (heterodimeric) ornon-specific interactions (aggregation). Tunicamycin or MG132 treatment ofMiaPaCa-2 and T3M4 pancreatic cancer cells stably transfected with CMV9-F114HAresulted significant abrogation of higher molecular weight forms and increasedaccumulation of MUC16-Cter, respectively (Fig. 2a)suggesting MUC16-Cter undergoes N-glycosylation and ubiquitylation. To assessthe global contribution of N-glycosylation, Chinese Hamster Ovary (CHO) cellswith intact (Pro⁻⁵) and defective N-glycosylation(Lec1 and Lec8)²⁵ were transfected with different lengthsof MUC16-Cter, which affected both the size and stability of the proteins (Fig. 2b). Further, mutation of three N-glycosylation sites(N-X-S/T) (Supplementary Fig. 1) in F114HA to Gln (Q)corroborated the above findings (Fig. 2c). To investigatewhether MUC16-Cter undergoes ubiquitylation, in vivo ubiquitylation assaywas performed using the wild type, Lys and Cys mutants. In vivoubiquitylation of wild type, Lys (2K → A) and Cys (3C → A)mutants of MUC16-Cter (F114HA) showed that the Lys in the CTD (Supplementary Fig. 1) of MUC16 undergoes polyubiquitylation (Fig. 2d) and therefore proteasomal degradation (Fig. 2a). Similar ubiquitylation analysis using individualLys mutations demonstrated that Lys90 is preferentially ubiquitylated than Lys89(Fig. 2e). Taken together, while both N-glycosylationand ubiquitylation can influence the function of MUC16-cter by regulating itsstability, the exact function(s) of site specific N-glycosylation andubiquitylation remains to be elucidated.

MUC16 cleavage is unaffected by proteases involved in regulatedintramembrane proteolysis (RIP), neutrophil elastase or MMP-7 and is independentof intracellular cue(s)

Having demonstrated no upstream cleavage(s) in the last and penultimate SEAdomains, we next explored the possibility of downstream cleavage site(s).Specifically, we investigated the regulated intramembrane proteolysis (RIP) byγ-secretase, since (i) exclusion of the extracellular12 amino acids in F53HA that does not undergo cleavage, would haveabrogated the cleavage by priming proteases (such as TACE for Notch1²⁶ and α-secretase for APP²⁷)and (ii) γ-secretase inhibitor treatment in HCLE and HCjE cellsreduced MUC16 protein without affecting mRNA²⁸. While thereduced MUC16 protein is attributed to Notch signaling²⁸, adirect effect of γ-secretase on MUC16 cannot be ruled out. Using aquantitative luciferase assay based on the GAL4-VP16 system²⁹, we showed that MUC16 is not a target of γ-secretase (Fig. 3a) where the amyloid precursor protein(APP-C99-GAL4-VP16) was used as a positive control. Althoughγ-secretase³⁰ mediated RIP is the mostwidely studied regulated intramembrane proteolysis, other proteases such assite-2 proteases^30,31 and rhomboid proteases³⁰ have been shown to belong to the same clan. However,swapping the TM domain alone or TM-CTD together of MUC4 with MUC16 did notabrogate the cleavage of MUC16 ruling out the involvement of any form of RIP(Fig. 1f, lanes 5 and 7).

Previous studies have reported that Ser/Thr phosphorylation in the CTD of MUC16(Supplementary Fig. 1) could be a trigger forproteolytic cleavage of MUC16 in response to EGF treatment^32,33; however, mutating Ser106Ala or Thr84/85Ala did not affect the cleavageof MUC16-Cter (Fig. 3b, lanes 6 and 7). Further, aminoacids capable of any post-translational modification(s) were mutated to Alawithout any effect on cleavage (Fig. 3b right panel,see cleaved fraction calculation in Methods) and replacement of theentire CTD of MUC4 with that of MUC16 did not abrogate cleavage (Fig. 1f, lane 4). These results indicate no requirement ofcytoplasmic cues for MUC16 cleavage. However, mutating Tyr94/103/104Ala(F114HA3Y → A) (Supplementary Fig. 1) resulted inan increase in both the unique HA-tagged and common FLAG and HA-tagged products(Fig. 3b, lane 5). Although it appears that Tyrmutations lead to increased accumulation of the cleaved MUC16, the ratio ofcleaved (17 kDa HA-tagged product) to the total HA-tagged fraction(17 kDa + higher molecular weight HA-tagged products) remainsunchanged (see cleaved fraction calculation in Methods). This suggeststhat Tyr phosphorylation leads to increased degradation (rapid turnover) ofMUC16. A recent study¹⁷ has demonstrated that Tyr22142(same as Tyr104 in our study) of MUC16 is phosphorylated by c-Src.

Without intracellular cues for MUC16 cleavage, we examined the involvement ofproteases such as NE and MMP-7 as their treatment resulted in enhanced sheddingof MUC16 in HCLE cells¹⁹. We analyzed many types ofnon-neutrophil cell lines for NE production, but none expressed NE (Fig. 3c). However, MUC16 cleavage was observed in all thecell types (Supplementary Fig. 2), therefore, ruling out theinvolvement of NE. In addition, the observation of a similar cleavage pattern inall the cell lines examined showed similar processing of MUC16 irrespective ofcell types used, eliminating the possibility of cell type specific effect (Supplementary Fig. 2). This suggests that while MUC16cleavage takes place in all the cell types, whether it will have similarphenotypes and mode of action need to be addressed in future studies. AlthoughMMP-7 was expressed by all the cells (Fig. 3c), cleavageof MUC16 occurs in Mmp7^−/− skin fibroblasts(Fig. 3d and 3e), ruling out its involvement in MUC16cleavage. Taken together, these results indicate that MUC16 is neither a targetof previously reported extracellular proteases such as NE and MMP-7 norintramembrane proteolysis and its cleavage is independent of post-translationalmodifications in the CTD.

Cleavage of MUC16 takes place in the acidic pH of Golgi/post-Golgicompartments

Next, to find out the exact cellular location for cleavage, we used brefeldin-A(BFA), which induces fusion of Golgi to the endoplasmic reticulum (ER) (Fig. 4a), resulting in redistribution of Golgi proteins intothe ER³⁴ and a rapid and reversible block in the ER-Golgitrafficking that prevents further secretion³⁵. Pretreatmentof HeLa cells with BFA resulted in abrogation of MUC16-Cter cleavage (Fig. 4b, lanes 2–3) that was rescued by BFAremoval (Fig. 4b, lanes 4–6). One majordistinction between the ER and the Golgi/post-Golgi compartments is theexistence of a pH gradient with values decreasing towards the secretorydestination from the ER (pH ~ 7.1), cis-medial-trans Golgi (pH ~7.0–6.0) and endosomes (pH ~ 5.0)³⁶. If MUC16cleavage was induced at the acidic pH of the Golgi/post-Golgi compartments, wereasoned that alkalinization of the Golgi/post-Golgi compartments could abrogatecleavage. Towards this, we used two pH-disrupting agents: NH₄Cl, aweak base, which neutralizes the acidic pH by releasing ammonia into the cell,and bafilomycin-A1, an inhibitor of theH⁺-K⁺-ATPase pumps present in both secretory andendocytic pathways³⁷. HeLa cells pretreated with eitherNH₄Cl or bafilomycin-A1 demonstrated that neutralizing the pH ofthe Golgi indeed significantly abrogated MUC16-Cter cleavage (Fig. 4c). To rule out degradation, we showed that MG132 treatment ofBFA treated cells did not lead to accumulation of the cleaved fragment (Fig. 4d, lanes 2 and 5). Taken together, these resultsindicate that cleavage of MUC16 takes place in the acidic pH of theGolgi/post-Golgi compartments.

Cleavage of MUC16 is not dictated by its primary amino acidsequence

As shown earlier, 12 membrane proximal ectodomain residues appear to be criticalfor MUC16 cleavage (Fig. 1b and 1f lane 8). In an effortto identify the exact site of cleavage, we performed alanine scan mutagenesis ofthe 12 residues, however, to our surprise it did not affect MUC16 cleavage(Fig. 5a). Further, complete deletion of the 12 aminoacids from F114HA only partially abrogated MUC16 cleavage (Fig.5b, lanes 1 and 2) and insertion of the 12 amino acids into MUC4-Cter(F-M4-150-HA) at the junction of TM and ECD resulted in its partial cleavage(Fig. 5c) that otherwise does not undergo cleavage.Our findings suggest that the cleavage of MUC16 is not entirely dictated by itsprimary amino acid sequence.

Nuclear localization of MUC16-Cter is independent of its putative nuclearlocalization signal

MUC16-Cter is predicted to have a nuclear localization signal (NLS i.e. RRRKKE)without DNA-binding domain and was detected in both soluble nuclear extract(SNE) and the chromatin bound (CB) fraction (Fig. 6a and6b). However, mutation of the putative NLS (RRRKKE →AAAAAA; NLS → Ala) did not abrogate its nuclear localization (Fig. 6a), indicating involvement of a non-classical nuclearimport pathway. The MUC1-Cter, despite having one RRK motif in the CTD,translocates into the nucleus by interacting with Nup62 independent of the RRKmotif³⁸. A prerequisite for this nuclear import isMUC1-Cter oligomerization³⁸ mediated by disulfide bridges,the inhibition of which prevents Nup62 binding. To gain insight into theinteraction of MUC16-Cter with itself, we investigated possiblecysteine-mediated oligomerisation through expression of a pair of dual taggedMUC16-Cter constructs. Co-expression of FA114HA (N-ter FLAG and C-ter HA-tag)with V5-114MyC (N-ter V5-tag and C-ter Myc-tag) and co-immunoprecipitationshowed self-interaction of MUC16-Cter. The results indicate possibleheterodimerization between the cleaved fragments (white arrow head) as well asbetween cleaved and uncleaved MUC16-Cter fragments (yellow arrow heards) (Fig. 6c). Mutation of cysteine residues (C76,79A) did notaffect the pattern or extent of MUC16-Cter self-interaction, suggesting strongnon-covalent association resistant to denaturing conditions (Fig.6c). Next, to understand if the cleavage of MUC16-Cter has anyinfluence on nuclear translocation, chimeric constructs with C-terminalGAL4-VP16 (GV)³⁹ were generated for MUC16-Cter(M16-114-GV), MUC4-Cter (M4-150-GV) and MUC4-ECD and MUC16-TMCT chimera(M4-ECD-M16TMCT-GV). There were significant reductions in the luciferaseactivities of M4-150-GV and M4-ECD-M16TMCT-GV, which unlike MUC16-Cter do notundergo cleavage (Fig. 6d), demonstrating the dependenceof nuclear translocation on the cleavage.

Discussion

CA125, since its discovery in 1981⁴⁰, is used as the goldstandard biomarker for ovarian cancer. Its molecular identity was revealed to be atransmembrane mucin, MUC16 in 2001^14,41. Recent studies havefurther underscored its importance; owing to its de novo and/or increasedexpression in cancer^5,6,7 as well as being one of the topthree frequently mutated genes across various cancer types⁴².Given the importance of oncogenic signaling mediated by mucin cytoplasmic tailfollowing cleavage², we investigated the speculations aboutMUC16 cleavage^12,14. Here, we demonstrate for the first timethat the 12 extracellular amino acids proximal to the TM domain are sufficient forMUC16 cleavage opposed to the proposed 50 aa in the last SEA domain (site#1, PLARRVDR)¹⁴ and a second cleavage (site # 2) at‘DSVLV’ site in the penultimate SEA domain¹² analogous to ‘GSVVV’ of MUC1. Although we have notbeen able to demonstrate cleavage of endogenous MUC16 due to commercialunavailability of CTD specific antibodies, a recent study published by Davies etal.⁴³, using an in-house antibody raised against theMUC16 CTD, demonstrates the existence of a ~17 kDa cleaved product inNHBE cells. This supports our findings in an overexpression system. Although wedemonstrated that the membrane proximal 12 amino acids were sufficientfor MUC16 cleavage, deletion of the same only partially abrogated its cleavagesuggesting this stretch of 12 amino acids is not an absolute necessityfor its cleavage. The possible reasons for this could be: (i) cleavage of MUC16 isnot dictated by its primary amino acid sequence, instead by a change in itsstructure as it encounters the acidic pH of the secretory pathway, or (ii) existenceof closely apposed multiple cleavage sites that are independent of each other.Therefore, our future work will be directed towards testing the above-mentionedhypotheses of MUC16 cleavage.

The protein backbone of MUC16 is ~22,152 amino acids, the largest amongall the known mucins and corresponds to ~2.5 MDa unglycosylated and20 MDa glycosylated mass⁸. While CA125 is consideredto be the extracellular shed portion of MUC16 following cleavage, it is hypothesizedto be a discontinuous repetitive epitope distributed across the tandem repeat regionof MUC16²¹. Since the antibodies against CA125 detect a proteinof approximately 200–250 kDa (i.e.0.2–0.25 MDa), much smaller than the N-ter extracellularfragment of MUC16, our findings of the most distal cleavage of MUC16 at the juxtamembrane ectodomain region do not necessarily generate CA125. Therefore, while thecleavage reported in here provides an explanation for the release of MUC16 from thecell surface, the mechanism(s) of CA125 generation still remains to beelucidated.

Since cleavage of MUC16 takes place in the membrane proximal ectodomain enroute to the plasma membrane, use of antibodies targeting extracellulartandem repeat regions of MUC16 is not the most effective means of targeting cancercells and is considered to be one of the major factors for the failures in clinicaltrials using this approach. In a recent study, Dharma Rao et al.⁴⁴ have generated several monoclonal antibodies targeting themembrane proximal ectodomain and the CTD of MUC16. These have been shown to beeffective in multiple applications and therefore are expected to be useful indiagnostics and therapeutics. However, the antibodies for the membrane proximalregion are generated assuming cleavage of MUC16 at 50 residues upstream of the TMdomain (site #1, PLARRVDR) and therefore does not bind to amino acids in the F65HAfragment that we demonstrate here to be the minimum length of MUC16carboxyl-terminal fragment that undergoes cleavage. Therefore in addition to theabove-mentioned antibodies, additional antibodies towards carboxyl-terminus MUC16including the membrane proximal 12 residues will be critical for diagnostic andtherapeutic targeting using MUC16.

We further demonstrated that the molecular weight disproportionate to the differentlengths of polypeptides expressed is due to post-translational modifications (PTMs)such as N-glycosylation, ubiquitylation and/or self di-/oligomerization. These PTMsare probably critical for its subcellular localizations as well as biologicalfunctions as they regulate the stability of MUC16-Cter. Specifically, mutations ofthe N-glycosylation site(s) such as Asn30Gln and Asn24,30Gln abrogated the highermolecular weight glycoforms of F114HA. In addition, the intensity of17 kDa cleaved MUC16 was reduced in the N-glycosylation mutantssuggesting cleavage of MUC16 to be influenced at least in part by N-glycosylation.However, F65HA with no N-glycosylation sites was cleaved as efficiently as otherlonger fragments with different numbers of N-glycosylation sites. Therefore, whileit appears that N-glycosylation influences MUC16 cleavage, further studies would berequired to either implicate or rule out the involvement of N-glycosylation in MUC16cleavage.

The juxtamembrane stretch of positively charged Lys/Arg residues (RRRKK), though wasconsidered to be the putative nuclear localization signal, mutation of this stretchto Ala did not abrogate its ability to undergo nuclear localization, suggestinginvolvement of non-canonical nuclear transport. One possibility could be thedi/oligomerization induced importin-β mediated nuclear transport as hasbeen shown for MUC1-Cter³⁸. However, the oligomerization ofMUC1-Cter is mediated by the disulfide linkages mediated by Cys residues, which isnot true for MUC16-Cter. Therefore, future studies should be directed towardsunderstanding the mechanistic basis of di/oligomerization as well as nucleartranslocation of MUC16-Cter. This suggests that MUC16-Cter could have regulatoryeffect on the gene expression possibly mediated by interaction with othertranscription factor(s) and regulatory protein(s) in the nucleus. Indeed, wedemonstrated that expression of MUC16-Cter in MiaPaCa-2 pancreatic cancer cellsresulted in up regulation of LMO2 and NANOG, implicated in inducingstem-cell like features during carcinogenesis, in a JAK2 dependent manner²⁴. Further, our demonstration of the dependence of increasednuclear activity using GAL4-VP16 system on the ability of MUC16-Cter to undergocleavage suggests that understanding the exact mechanism of cleavage would enable usto design specific therapeutic intervention(s) targeted to prevent cleavage ofMUC16.

Understanding cleavage and post-cleavage events of MUC16 would aid in dissecting itsrole in multiple malignancies and its de novo expression in PC makes it asuitable candidate to be exploited for targeted therapy. Preventing cleavage ofMUC16 would serve two important purposes such as (i) reduced nuclear translocation,and (ii) increased cell surface representation of MUC16 that will enhance theefficacy of the CA125 antibody based drugs such as Oregovomab and Abagovomab. Here,we showed that (i) increased nuclear translocation of the cleaved fragment isdependent of MUC16 cleavage and (ii) treatment of BFA results in abrogation ofMUC16 cleavage. Although use of drugs that perturbs the secretory pathway raises theobvious concern of affecting the functions of normal cells, studies havedemonstrated that normal peripheral blood mononuclear cells, fibroblasts and retinalpigment epithelial cells are much less sensitive to BFA treatment compared to themalignant cells, possibly due to the increased reliance of the tumor cells on thesecretory pathway compared to the normal cells^35,45. Breflate(a prodrug form of brefeldin-A, NSC656202) can be used to prevent cleavage of MUC16and its associated tumorigenic functions and therefore can be viewed as aninteresting therapeutic avenue³⁵.

Methods

Cell culture and transfections

HEK293T, HeLa, MiaPaCa-2, T3M4, MCF7 and SKOV3 cells were grown in DMEMsupplemented with 10% heat-inactivated FBS (Sigma) and 100 U/mlpenicillin, 100 μg/ml streptomycin (penstrep). HPDE wascultured in keratinocyte serum free medium supplemented with EGF, bovinepituitary extract and penstrep. MCF10-A cells were cultured in DMEM/F12 mediumsupplemented with 5% heat-inactivated FBS, EGF (20 μg/ml),hydrocortisone (0.5 μg/ml), cholera toxin(0.1 μg/ml), insulin (10 μg/ml) andpenstrep. Parental (PRO⁻⁵) and N-glycosylationdeficient (Lec-1 and Lec-8) CHO and U-937 cells (suspension culture) cells weregrown in RPMI medium supplemented with 5% heat-inactivated FBS and penstrep.Transient transfections of HEK293T cells were performed using PEI(polyethyleneimine) at a ratio of 5 ug PEI/ug of DNA. MiaPaCa-2,T3M4, HPDE, MCF10-A, MCF7, SKOV3, HeLa and CHO cells were transfected usingLipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions. MiaPaCa-2 and T3M4 cells were selected for G418(400–600 μg/ml) resistance to generate thestable cells. These stable cells were maintained in400 μg/ml G418 only except during the experimentalprocedure.

Plasmids and cloning strategy

Standard PCR and molecular cloning techniques were used to make constructs. Forexpression in the mammalian system, p3X-FLAG-CMV9 (Sigma) and pSecTag2C(Invitrogen) plasmids were used to make various constructs. DNA fragmentsencoding the carboxyl-terminal region of MUC16 (321 amino acids), MUC4 (150amino acids), was amplified by RT-PCR and was cloned into the respectiveexpression vectors. These constructs were further manipulated to generatevarious point mutations (by site-directed mutagenesis), deletions and domainswapping variants and are listed in Supplementary Table 1.Gal4-VP16 was PCR-amplified from pSG5-SP-C99-Gal4-VP16 plasmid and clonedC-terminal to MUC16-114, MUC4-150 and MUC4-ECD-MUC16TMCT in the p3X-FLAG-CMV9vector.

Establishment of skin fibroblasts from adult WT,Mmp7^−/− andMmp2^−/− mice

Fibroblasts were prepared from the skin of wild type (C57BL/6),Mmp2^−/− andMmp7^−/− KO mice as describedpreviously⁴⁶. Within 3–6 passages thesefibroblasts were used for the purpose of transfection and RNA extraction. TheInstitutional Animal Care and User Committee at the University of NebraskaMedical Center approved all animal work.

Immunoprecipitation and immunoblotting

Cells were lysed in IP buffer (50 mM Tris-HCl pH-7.4,300 mM NaCl, 5 mM EDTA, 1% NP-40) containing completeprotease inhibitor cocktail (Roche), 2 mMNa₃VO₄, 10 mM NaF and 1 mM PMSFon ice for 30 minutes. Cell lysates were clarified by centrifugationand were immunoprecipitaed with indicated antibodies overnight at4°C. Protein complexes were isolated by incubation with Protein-A,Protein-G or Protein-A/G Agarose beads (Santa Cruz Biotechnology) for2–4 h. Immunoprecipitates were washed 3–5 timeswith IP buffer, boiled with SDS sample buffer and analysed by immunoblotting asdescribed below using indicated antibodies. Standard methods were used forimmunoblotting. Cells were lysed with RIPA buffer (50 mM Tris-HClpH-7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1%SDS) supplemented with complete protease inhibitor mixture (Roche),2 mM Na₃VO₄, 10 mM NaF and1 mM PMSF on ice. Cell lysates were cleared by centrifugation andquantified using the bicinchoninic acid method. Proteins(10–40 μg) were separated bySDS–PAGE under reducing conditions and blotted onto a polyvinylidenedifluoride membrane (Millipore). Membranes were probed with specific antibodies.Blots were washed and probed with respective secondary peroxidase-conjugatedantibodies and the bands visualized by chemiluminescence (Thermo Scientific).The following antibodies were used: mouse monoclonal antibodies for FLAG-Tag(1:3000; M2), β-Actin (1:5000) from Sigma, Myc-tag (1:2000) from Cellsignaling, EGFR (1:1000; Santa Cruz Biotechnology), rabbit monoclonal antibodiesfor HA-Tag (1:2000), GAPDH (1:1000), SP1 (1:1000) from Cell Signaling, andrabbit polyclonal antibody for Histone H3 (1:1000; Abcam).

In vivo ubiquitylation assay

HEK293T cells coexpressing HA-tagged wild type (F114HA) or lysine (F114HA-2K→ A), cysteine (F114HA-3C → A), lysine and cysteine(F114HA-2K → A/3C → A), Lys89Ala (F114HA K89A), Lys90Ala(F114HA K90A) mutants of MUC16-Cter and Myc-tagged Ubiquitin were lysed with IPbuffer (50 mM Tris-HCl pH-7.4, 300 mM NaCl,5 mM EDTA, 1% NP-40) supplemented with complete protease inhibitormixture (Roche), 2 mM Na₃VO₄, 10 mMNaF, 1 mM PMSF and placed on ice for 20 min. Cell lysateswere cleared by centrifugation and immunoprecipitated using indicated antibodiesfor 2 h to overnight at 4°C. Protein complexes werecollected by incubation for 2–4 h with Protein-A,Protein-G Agarose beads (Santa Cruz Biotechnology). Immunoprecipitates werewashed 3–5 times with IP buffer, boiled in SDS sample buffer andanalyzed by immunoblotting with appropriate antibodies.

Subcellular fractionation

Subcellular fractionations were carried out using subcellular proteinfractionation kits (Thermo Scientific and G-Biosciences) according to themanufacturers' instructions. Fraction purity was determined bywestern blotting, using the following antibodies: GAPDH for cytoplasmic, EGFRfor membrane, SP1 for total and soluble nuclear and Histone-3 forchromatin-bound fractions.

Luciferase assay

0HEK293T cells were transfected with Renilla Luciferase plasmid(25 ng), pFR-Luciferase plasmid (500 ng) and the inducerplasmids (1000 ng) at a ratio of 1:20:40 respectively. Inducerplasmids included pCMV9-M16-114-GAL4-VP16, pCMV9-M4-150-GAL4-VP16,pCMV9-M4ECD-M16TMCT-GAL4-VP16, pSG5-APP-C99-GAL4-VP16, pSG5-GAL4-VP16 or pCMV9empty vector. Luciferase readings were measured 24 h followingtransfection according to manufacturers' instructions (Promega) withthe exception of Υ-secretase mediated RIP assay. For this,24 h after transfection cells were treated with or without inhibitorX (25 nM Inh X; Calbiochem) for a period of 16 h beforeluciferase readings were measured.

Immunofluorescence microscopy

HeLa cells were grown on coverslips for twenty-four hours and were washed twicewith PBS and fixed with 4% paraformaldehyde in PBS (pH-7.4) for10 min. After washing with PBS, cells were quenched with30 mM glycine. Cells were then permeabilized with 0.1% Triton-X-100for 10 min and blocked with 10% normal goat serum (NGS) in PBS for1 h. Cells were incubated with appropriate antibodies (anti-Calnexin;1:200 from SCB, anti-Giantin; 1:1000 from Abcam) for 1 h in PBScontaining 2% NGS. The cells were washed three times with PBST and incubatedwith Alexa-Fluor 488-conjugated donkey anti-mouse and Alexa-Fluor 568-conjugateddonkey anti-rabbit (Life Technologies) antibodies for 30 min. Thecells were washed three times with PBST and mounted in Vectashield with DAPI(Vector Laboratories).

RNA isolation, reverse transcription and PCR analysis

RNA was extracted from the cells using the RNeasy mini kit (Qiagen) according tothe manufacturer's instructions. cDNA was synthesized from total RNAusing oligo-dT or random hexamers using SuperScript reverse transcriptase II(Life Technologies) kit. For analysis of neutrophil elastase (ELANE),MMP7, Mmp2 and Mmp7, gene specific primers weredesigned using NCBI primer designing tool and endpoint PCR was carried out for30 cycles with a melting temperature of 58°C.β-ACTIN and Gapdh were used as loadingcontrols.

Primers used are MMP-7 FP: 5′- CAGGAAACACGCTGGCTCAT-3′ RP:5′-AGACTGCTACCATCCGTCCA-3′, ELANE FP:5′-CATATAGATCTCATCTGGGCATCC-3′ RP:5′-TGCCAGATGCTGGAGAGTGT-3′, Mmp-7 FP:5′-TGGAGACAGCTTCCCCTTTG-3′ RP: 5′-TGGAGACAGCTTCCCCTTTG-3′, Mmp-2 FP: 5′-TCCCCCGATGCTGATACTGA-3′ Mmp-2 RP: 5′-TCCCCCGATGCTGATACTGA-3′, β-ACTIN FP:5′-TGGACATCCGCAAAGACCTG-3′ RP:5′-TGGACATCCGCAAAGACCTG-3′, Gapdh FP:5′-GCTCACTGGCATGGCCTTCCGTG-3′ RP:5′-TGGAAGAGTGGGAGTTGCTGTTGA-3′.

Cleaved fraction calculation

Since MUC16-Cter cleavage does not proceed to completion (Fig.1b and 3b) and the cleavage takes place in theGolgi/post-Golgi compartments (Fig. 4b), measuring theFLAG-tagged product in the culture medium (assuming it is released) may not be atrue measure of cleavage. To estimate the cleavage efficiency, we firstnormalized the bottom (<17 kDa C-ter HA-tagged productthat is physically separated from the N-ter) and top (higher molecular weight)HA-tagged products with β-actin separately. A ratio of the normalizedbottom to the total (bottom + top) HA-tagged fraction was calculated and wasconsidered to be a better measure of cleavage efficiency, assuming the topHA-tagged to be either uncleaved and/or heterodimeric and the bottom HA-taggedto be the actual cleaved fraction (Fig. 3b and 5b, right panel). We employed this quantification todemonstrate no influence of the cytoplasmic tail amino acid mutations onMUC16-Cter cleavage (Fig. 3b, right panel). Though themutations such as F114HA Tyr94/103/104Ala (3Y → A) appear to have anincreased amount of 17 kDa cleaved fragment, the normalized cleavedfraction is unaltered compared to the WT (Fig. 5b, rightpanel), indicating these mutations may affect stability but not cleavage ofMUC16-Cter.

References

Hollingsworth, M. A. & Swanson, B. J. Mucins in cancer: protection and control of the cell surface. Nat. Rev. Cancer 4, 45–60 (2004).
CAS PubMed Google Scholar
Kufe, D. W. Mucins in cancer: function, prognosis and therapy. Nat. Rev. Cancer 9, 874–885 (2009).
CAS PubMed PubMed Central Google Scholar
Senapati, S., Das, S. & Batra, S. K. Mucin-interacting proteins: from function to therapeutics. Trends Biochem. Sci. 35, 236–245 (2010).
CAS PubMed Google Scholar
Mai, P. L., Wentzensen, N. & Greene, M. H. Challenges related to developing serum-based biomarkers for early ovarian cancer detection. Cancer. Prev. Res. (Phila) 4, 303–306 (2011).
Google Scholar
Einama, T. et al. Co-expression of mesothelin and CA125 correlates with unfavorable patient outcome in pancreatic ductal adenocarcinoma. Pancreas 40, 1276–1282 (2011).
CAS PubMed Google Scholar
Streppel, M. M. et al. Mucin 16 (cancer antigen 125) expression in human tissues and cell lines and correlation with clinical outcome in adenocarcinomas of the pancreas, esophagus, stomach and colon. Hum. Pathol. 43, 1755–1763 (2012).
CAS PubMed PubMed Central Google Scholar
Shimizu, A. et al. Coexpression of MUC16 and mesothelin is related to the invasion process in pancreatic ductal adenocarcinoma. Cancer. Sci. 103, 739–746 (2012).
CAS PubMed Google Scholar
Hattrup, C. L. & Gendler, S. J. Structure and function of the cell surface (tethered) mucins. Annu. Rev. Physiol. 70, 431–457 (2008).
CAS PubMed Google Scholar
Govindarajan, B. & Gipson, I. K. Membrane-tethered mucins have multiple functions on the ocular surface. Exp. Eye Res. 90, 655–663 (2010).
CAS PubMed PubMed Central Google Scholar
Duraisamy, S., Ramasamy, S., Kharbanda, S. & Kufe, D. Distinct evolution of the human carcinoma-associated transmembrane mucins, MUC1, MUC4 AND MUC16. Gene 373, 28–34 (2006).
CAS PubMed Google Scholar
Wreschner, D. H. et al. Generation of ligand-receptor alliances by “SEA” module-mediated cleavage of membrane-associated mucin proteins. Protein Sci. 11, 698–706 (2002).
CAS PubMed PubMed Central Google Scholar
Macao, B., Johansson, D. G., Hansson, G. C. & Hard, T. Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin. Nat. Struct. Mol. Biol. 13, 71–76 (2006).
CAS PubMed Google Scholar
Akhavan, A., Crivelli, S. N., Singh, M., Lingappa, V. R. & Muschler, J. L. SEA domain proteolysis determines the functional composition of dystroglycan. FASEB J. 22, 612–621 (2008).
CAS PubMed Google Scholar
O'Brien, T. J. et al. The CA 125 gene: an extracellular superstructure dominated by repeat sequences. Tumour Biol. 22, 348–366 (2001).
CAS PubMed Google Scholar
Theriault, C. et al. MUC16 (CA125) regulates epithelial ovarian cancer cell growth, tumorigenesis and metastasis. Gynecol. Oncol. 121, 434–443 (2011).
CAS PubMed Google Scholar
Boivin, M., Lane, D., Piche, A. & Rancourt, C. CA125 (MUC16) tumor antigen selectively modulates the sensitivity of ovarian cancer cells to genotoxic drug-induced apoptosis. Gynecol. Oncol. 115, 407–413 (2009).
CAS PubMed Google Scholar
Akita, K. et al. CA125/MUC16 interacts with Src family kinases and over-expression of its C-terminal fragment in human epithelial cancer cells reduces cell-cell adhesion. Eur. J. Cell Biol. 92, 257–263 (2013).
CAS PubMed Google Scholar
Matte, I., Lane, D., Boivin, M., Rancourt, C. & Piche, A. MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression. BMC Cancer 14, 234-2407-14-234 (2014).
Blalock, T. D., Spurr-Michaud, S. J., Tisdale, A. S. & Gipson, I. K. Release of membrane-associated mucins from ocular surface epithelia. Invest. Ophthalmol. Vis. Sci. 49, 1864–1871 (2008).
PubMed PubMed Central Google Scholar
Govindarajan, B. et al. A metalloproteinase secreted by Streptococcus pneumoniae removes membrane mucin MUC16 from the epithelial glycocalyx barrier. PLoS One 7, e32418 (2012).
ADS CAS Google Scholar
Felder, M. et al. MUC16 (CA125): tumor biomarker to cancer therapy, a work in progress. Mol. Cancer 13, 129-4598-13-129 (2014).
Berek, J. S. et al. Randomized, placebo-controlled study of oregovomab for consolidation of clinical remission in patients with advanced ovarian cancer. J. Clin. Oncol. 22, 3507–3516 (2004).
ADS CAS PubMed Google Scholar
Sabbatini, P. et al. Abagovomab as maintenance therapy in patients with epithelial ovarian cancer: a phase III trial of the AGO OVAR, COGI, GINECO and GEICO--the MIMOSA study. J. Clin. Oncol. 31, 1554–1561 (2013).
CAS PubMed PubMed Central Google Scholar
Das, S. et al. Carboxyl-terminal domain of MUC16 imparts tumorigenic and metastatic functions through nuclear translocation of JAK2 to pancreatic cancer cells. Oncotarget (2015).
Patnaik, S. K. & Stanley, P. Lectin-resistant CHO glycosylation mutants. Methods Enzymol. 416, 159–182 (2006).
CAS Google Scholar
Mumm, J. S. et al. A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1. Mol. Cell 5, 197–206 (2000).
CAS PubMed Google Scholar
Sisodia, S. S. & St George-Hyslop, P. H. gamma-Secretase, Notch, Abeta and Alzheimer's disease: where do the presenilins fit in? Nat. Rev. Neurosci. 3, 281–290 (2002).
CAS PubMed Google Scholar
Xiong, L., Woodward, A. M. & Argueso, P. Notch signaling modulates MUC16 biosynthesis in an in vitro model of human corneal and conjunctival epithelial cell differentiation. Invest. Ophthalmol. Vis. Sci. 52, 5641–5646 (2011).
CAS PubMed PubMed Central Google Scholar
Tousseyn, T. et al. ADAM10, the rate-limiting protease of regulated intramembrane proteolysis of Notch and other proteins, is processed by ADAMS-9, ADAMS-15 and the gamma-secretase. J. Biol. Chem. 284, 11738–11747 (2009).
CAS PubMed PubMed Central Google Scholar
Urban, S. Making the cut: central roles of intramembrane proteolysis in pathogenic microorganisms. Nat. Rev. Microbiol. 7, 411–423 (2009).
CAS PubMed PubMed Central Google Scholar
Rawson, R. B. et al. Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs. Mol. Cell 1, 47–57 (1997).
CAS PubMed Google Scholar
Fendrick, J. L. et al. CA125 phosphorylation is associated with its secretion from the WISH human amnion cell line. Tumour Biol. 18, 278–289 (1997).
CAS PubMed Google Scholar
Konishi, I., Fendrick, J. L., Parmley, T. H., Quirk, J. G., Jr & O'Brien, T. J. Epidermal growth factor enhances secretion of the ovarian tumor-associated cancer antigen CA125 from the human amnion WISH cell line. J. Soc. Gynecol. Investig. 1, 89–96 (1994).
CAS PubMed Google Scholar
Colanzi, A. et al. Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS. Proc. Natl. Acad. Sci. U. S. A. 110, 9794–9799 (2013).
ADS CAS PubMed PubMed Central Google Scholar
Carew, J. S. Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL. Blood 107, 222; 222–231; 31 (2006).
Nakamura, N., Tanaka, S., Teko, Y., Mitsui, K. & Kanazawa, H. Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation. J. Biol. Chem. 280, 1561–1572 (2005).
CAS PubMed Google Scholar
Axelsson, M. A. et al. Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins. Glycobiology 11, 633–644 (2001).
CAS PubMed Google Scholar
Leng, Y. et al. Nuclear import of the MUC1-C oncoprotein is mediated by nucleoporin Nup62. J. Biol. Chem. 282, 19321–19330 (2007).
CAS PubMed Google Scholar
Chapin, H. C., Rajendran, V., Capasso, A. & Caplan, M. J. Detecting the surface localization and cytoplasmic cleavage of membrane-bound proteins. Methods Cell Biol. 94, 223–239 (2009).
CAS PubMed PubMed Central Google Scholar
Bast, R. C., Jr et al. Reactivity of a monoclonal antibody with human ovarian carcinoma. J. Clin. Invest. 68, 1331–1337 (1981).
PubMed PubMed Central Google Scholar
O'Brien, T. J., Beard, J. B., Underwood, L. J. & Shigemasa, K. The CA 125 gene: a newly discovered extension of the glycosylated N-terminal domain doubles the size of this extracellular superstructure. Tumour Biol. 23, 154–169 (2002).
CAS PubMed Google Scholar
Kim, N., Hong, Y., Kwon, D. & Yoon, S. Somatic mutaome profile in human cancer tissues. Genomics Inform. 11, 239–244 (2013).
Google Scholar
Davies, J. R., Kirkham, S., Svitacheva, N., Thornton, D. J. & Carlstedt, I. MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells. Int. J. Biochem. Cell Biol. 39, 1943–1954 (2007).
CAS PubMed Google Scholar
Dharma Rao, T. et al. Novel monoclonal antibodies against the proximal (carboxy-terminal) portions of MUC16. Appl. Immunohistochem. Mol. Morphol. 18, 462–472 (2010).
CAS PubMed Google Scholar
Larsson, D. E. et al. Identification and evaluation of potential anti-cancer drugs on human neuroendocrine tumor cell lines. Anticancer Res. 26, 4125–4129 (2006).
CAS PubMed Google Scholar
Seluanov, A., Vaidya, A. & Gorbunova, V. Establishing primary adult fibroblast cultures from rodents. J. Vis. Exp. 44, e2033 (2010).
Google Scholar

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Acknowledgements

We would like to thank Dr. William Chaney (UNMC) for the CHO cells and Dr. JoyceSolheim for U937 cells, Dr. B. Timothy Baxter (UNMC) forMmp2^−/− andMmp7^−/− KO mice, Prof. Bart De Strooper(VIB, Belgium) for pSG5-SP-VP16-Gal4 and pSG5-SP-C99-Gal4-VP16 plasmids, Dr. YapingTu (Creighton University, Nebraska) for pFR-Luciferase plasmid, Prof. SpyrosArtavanis-Tsakonas, Dr. Youping Sun (Harvard University, Boston) for Myc-Ubiquitinconstructs. We would like to thank UNMC Cell Analysis Facility and the UNMC DNASequencing Core Facility. We would like to thank Dr. Joyce Solheim for editing themanuscript, Dr. Ying Yan and Poomy Pandey (UNMC) for sharing reagents and usefuldiscussion and Kavita Mallya (UNMC) for technical support. This work was supportedby grants from the Department of Defense (BC101014) and NIH (U54 CA163120, UO1CA111294 and P50 CA127297).

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Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, 68198, USA
Srustidhar Das, Prabin D. Majhi, Mona H. Al-Mugotir, Satyanarayana Rachagani, Paul Sorgen & Surinder K. Batra
Department of Pathology, University of Nebraska Medical Center, Omaha, NE, 68198, USA
Surinder K. Batra
Buffett Cancer Center, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, 68198, USA
Surinder K. Batra

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Srustidhar Das
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Prabin D. Majhi
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Mona H. Al-Mugotir
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Satyanarayana Rachagani
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S.D., P.D.M. and S.K.B. designed the experiments. S.D., P.D.M., S.R. and M.H.A.performed the experiments, P.S. provided experimental materials, S.D., P.D.M.,M.H.A., P.S. and S.K.B. analyzed the data, S.D., P.D.M. and S.K.B. wrote themanuscript.

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Das, S., Majhi, P., Al-Mugotir, M. et al. Membrane proximal ectodomain cleavage of MUC16 occurs in the acidifyingGolgi/post-Golgi compartments. Sci Rep 5, 9759 (2015). https://doi.org/10.1038/srep09759

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Received: 08 January 2015
Accepted: 13 March 2015
Published: 05 June 2015
DOI: https://doi.org/10.1038/srep09759

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