Mucin 1 (MUC1) is a novel partner for MAL2 in breast carcinoma cells
© Fanayan et al; licensee BioMed Central Ltd. 2009
Received: 21 August 2008
Accepted: 28 January 2009
Published: 28 January 2009
The MAL2 gene, encoding a four-transmembrane protein of the MAL family, is amplified and overexpressed in breast and other cancers, yet the significance of this is unknown. MAL-like proteins have trafficking functions, but their molecular roles are largely obscure, partly due to a lack of known binding partners.
Yeast two-hybrid screening of a breast carcinoma cDNA expression library was performed using a full-length MAL2 bait, and subsequent deletion mapping experiments were performed. MAL2 interactions were confirmed by co-immunoprecipitation analyses and confocal microscopy was employed to compare protein sub-cellular distributions. Sucrose density gradient centrifugation of membranes extracted in cold Triton X-100 was employed to compare protein distributions between Triton X-100-soluble and -insoluble fractions.
The tumor-associated protein mucin 1 (MUC1) was identified as a potential MAL2 partner, with MAL2/MUC1 interactions being confirmed in myc-tagged MAL2-expressing MCF-10A cells using co-immunoprecipitation assays. Deletion mapping experiments demonstrated a requirement for the first MAL2 transmembrane domain for MUC1 binding, whereas the MAL2 N-terminal domain was required to bind D52-like proteins. Confocal microscopy identified cytoplasmic co-localisation of MUC1 and MAL2 in breast cell lines, and centrifugation of cell lysates to equilibrium in sucrose density gradients demonstrated that MAL2 and MUC1 proteins were co-distributed between Triton X-100-soluble and -insoluble fractions. However co-immunoprecipitation analyses detected MAL2/MUC1 interactions in Triton X-100-soluble fractions only. Myc-MAL2 expression in MCF-10A cells was associated with both increased MUC1 detection within Triton X-100-soluble and -insoluble fractions, and increased MUC1 detection at the cell surface.
These results identify MUC1 as a novel MAL2 partner, and suggest a role for MAL2 in regulating MUC1 expression and/or localisation.
Human MAL2, a 19 kDa protein with four transmembrane (TM) domains [1, 2] is a member of the MAL protein family. The founding member MAL  resides in lipid rafts [4, 5] and is required in apical vesicle transport [6–9]. The MAL family also includes less characterised members, including BENE, which is also a raft-associated integral membrane protein , plasmolipin, a 20 kDa proteolipid expressed in compact myelin and epithelial cells  and chemokine-like factor superfamily 8 (CKLFSF8), a novel regulator of EGF-induced signalling . MAL2 was identified as a partner for tumor protein D52-like proteins through yeast two-hybrid (Y2H) expression screening of a human breast carcinoma library . The MAL2 protein is now known to be expressed in many epithelial cell types, as well as peripheral neurons, mast cells and dendritic cells . In HepG2 hepatoma cells, MAL2 resides exclusively within lipid rafts, and represents an essential component for indirect basolateral-to-apical transcytosis , where it shows a highly dynamic subcellular localisation . MAL2 has also been reported to be distributed in both lipid raft and non-raft fractions in primary thyrocytes  and PC-3 prostate carcinoma cells , predicting additional, uncharacterised cellular functions for MAL2 outside lipid rafts.
The initial identification of MAL2 suggested its overexpression in breast cancer , which is supported by the MAL2 gene being found at chromosome 8q24, which is frequently gained in breast and other cancers . Several studies have now identified MAL2 amplification and/or overexpression in breast cancer [18–22]. Overexpression of MAL2 has also been reported in other cancers, including primary ovarian carcinoma [23, 24] and ascites , and pancreatic carcinoma , where MAL2 has since been employed as a discriminator of pancreatic carcinoma versus chronic pancreatitis . Expression profiling has also indicated MAL2 overexpression in malignant pleural mesothelioma of the epithelial type , and in head and neck squamous cell carcinoma . Immunohistochemical analyses first revealed differential MAL2 expression in renal carcinomas , with this being recently confirmed in chromophobe renal cell carcinoma versus oncocytoma .
Despite numerous reports of MAL2 overexpression in breast cancer, little is known about how increased MAL2 expression may provide an advantage to cancer cells. While MAL2 cellular localisation and function have been explored in previous studies [2, 14, 15], the only known MAL2 partners are members of the D52-like protein family . Interactions between MAL2 and both D52 and D53 have since been identified in a large scale Y2H analysis , which supports further use of the Y2H system to analyse MAL2 function. We therefore carried out a Y2H screening of a breast carcinoma cDNA expression library [1, 32] to identify novel MAL2 binding partners. One protein thus identified was mucin 1 (MUC1), a transmembrane protein expressed on the apical surface of epithelial cells  and overexpressed in multiple cancers , in part through MUC1 gene amplification . Like other mucins, MUC1 protects and lubricates normal glandular epithelia, whereas MUC1 overexpression in cancer alters many cellular properties, including intercellular adhesion and immune recognition [33, 34]. As MUC1 therefore represented a candidate MAL2 partner of particular interest, subsequent experiments were performed to confirm MAL2/MUC1 interactions, and examine their significance in breast epithelial and cancer cells. As we will describe, this work identifies a MAL2 as a cytoplasmic MUC1 partner which binds MUC1 in non-lipid raft fractions, and may regulate MUC1 expression and/or subcellular distribution.
Deleted MAL2 yeast two-hybrid constructs, primer sequences and MAL2 regions deleted
PCR Primer Sequences (5'-3')
MAL2 region deleted
Yeast two-hybrid system and screening
Yeast cultures of the Saccharomyces cerevisiae Hf7c strain were grown at 30°C in standard liquid or solid media, based upon either rich YPD media (2% bacto-peptone, 1% yeast extract, 2% dextrose), or minimal SD medium (0.67% yeast nitrogen base without amino acids, 2% dextrose, with appropriate amino acid supplements) for expression library screening and direct interaction testing. For cDNA library expression screening, bait (pAS2-1MAL2) and human breast carcinoma pAD-GAL4 library plasmids were transfected simultaneously into Hf7c cells. Subsequent screening and the recovery of plasmid DNA from yeast cells were carried out as described . For the direct testing of interactions, paired baits (pAS2-1 constructs) and preys (pACT2 or pAD-GAL4 constructs) were transfected into Hf7c cells as described . Interactions between baits and preys were assessed by qualitatively determining HIS3 reporter gene activity .
The BC2 (an anti-MUC1 VNTR epitope mouse IgG1 monoclonal) and FITC-BC2 antibodies have been previously described . Rabbit polyclonal c-Myc (A-14) and CAV1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and BD Biosciences (BD Biosciences, San Jose, CA, USA), respectively. Affinity-purified D52 rabbit polyclonal antibody has been described previously . Peroxidase-conjugated donkey anti-rabbit and anti-mouse, FITC-conjugated donkey anti-mouse and CY3-conjugated donkey anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch, Inc (West Grove, PA, USA).
Preparation and affinity purification of polyclonal MAL2 antisera
For the production of sheep antisera, one sheep was injected subcutaneously with 2 mg coupled MAL2 C165-P176 peptide antigen on 2 occasions, spaced by 3 weeks. For the production of rabbit antisera, two rabbits were injected subcutaneously with 0.5 mg coupled N13-V24 and C165-P176 MAL2 peptide antigens on 2 occasions, spaced by 2 weeks. Antisera were affinity-purified using relevant MAL2 peptides as previously described .
Human breast cell lines
The MDA-MB-435 (a kind gift from Dr Janet Price, MD Anderson Cancer Centre, Houston, TX), MDA-MB-453, SK-BR-3 and MCF-7 breast cancer cells were cultured as described in the American Type Culture Collection Catalogue. MCF-10A cells are described in the American Type Culture Collection Catalogue and were cultured as described .
Derivation of stably-transfected cell lines
The Myc-tagged MAL2 expression construct was stably transfected into MCF-10A cells. Cells were seeded at approximately 60% confluence in 100 mm dishes and transfected 18 h later with 20 μg plasmid DNA using LipofectAMINE 2000 Reagent (Life Technologies, Inc., Gaithersburg, MD, USA), according to the manufacturer's instructions. After 24 h, G418 was added to a concentration of 1 mg/ml. Media were replenished every 2–3 days and a G418-resistant mixed population was selected 14 days post-transfection. MDA-MB-435 and MDA-MB-453 breast cancer cells were transfected by electroporation with a MUC1 cDNA containing 22 VNTR repeats in the pcDNA3 vector (Invitrogen) or the vector alone. Stable G418 resistant clones were isolated and MUC1 expression determined by flow cytometry.
Preparation of total protein extracts and Western blot analyses
Cells were harvested in cold PBS, pelleted and washed twice in cold PBS. Total cell protein extracts were prepared by resuspending the pellet in SDS extraction buffer (125 mM Tris-HCl pH 6.8, 3% SDS, 5% 2-mercaptoethanol, 1 mM PMSF, and protease inhibitors [Roche, Basel, Switzerland]), which were then briefly sonicated. Samples were resolved using SDS-PAGE on 12.5% polyacrylamide gels, and electrotransferred to nitrocellulose filters (Millipore, Billerica, MA, USA). Protein loading was analysed using Ponceau S staining, and filters were blocked overnight at 4°C in 5% skim milk powder in TBS. Membranes were washed twice with TBS and incubated with either affinity-purified rabbit polyclonal MAL2 antisera (1/100), affinity-purified rabbit polyclonal D52 antisera (1/100), BC2 (1/100) or CAV1 antibody (1/2000) in 0.1% Tween 20 in TBS, for 2 h. Membranes were washed 3 times in 0.1% Tween 20 in TBS, and then incubated with horseradish peroxidase-conjugated donkey anti-rabbit or donkey anti-mouse secondary antibody (Jackson ImmunoResearch, Inc) (1/5000) for 2 h. Blots were washed 4 times with 0.1% Tween 20 in TBS, followed by 2 washes in TBS and antigen-antibody complexes were visualised by Western lightning chemiluminescent reagent (Perkin Elmer, Waltham, Massachusetts, USA).
MCF-10A cells were grown to 80% confluence in 100 mm dishes and washed with cold PBS. For each co-immunoprecipitation, proteins were extracted by scraping cells from 6 dishes into 0.15 ml lysis buffer per dish. For co-immunoprecipitation of MAL2 and D52 proteins, 10 mM Tris with 1 mg/ml Saponin (Sigma-Aldrich, St. Louis, MO, USA) was employed as a lysis buffer . For co-immunoprecipitation of MAL2 and MUC1 proteins, SDS lysis buffer (125 mM Tris-HCl pH 6.8, 3% SDS, 5% 2-mercaptoethanol, 1 mM PMSF, and protease inhibitors (Roche) was employed. Protein A-Sepharose beads (Sigma-Aldrich) plus rabbit MAL2 antisera (1/50), either alone or with 1 μg/ml synthetic peptides N13-V24 and C165-P176, or protein G-agarose beads (Sigma-Aldrich) plus BC2 (1/50) were added to cell lysates and incubated on a rotary mixer at 4°C for 16 h. Beads were washed 4 times with 1 ml lysis buffer, followed by a final wash with PBS. Eluted proteins (15 μl) were separated using SDS-PAGE on 12.5% polyacrylamide gels and electrotransferred to nitrocellulose filters (Millipore) for Western blot analyses, as above.
Immunofluorescent labelling of breast cell lines
Cell lines were cultured to near confluence, and harvested by trypsinisation. Cells were diluted 3- to 10-fold and cultured overnight on glass coverslips. For immunofluorescent staining, cells were washed twice with PBS, and fixed in 4% paraformaldehyde supplemented with 0.1% saponin for 20 min at RT. Cells were washed twice with PBS, and incubated at RT for 2 h with affinity-purified rabbit MAL2 (1/50), D52 (1/100) antisera or mouse BC2-FITC (40 μg/ml), in 0.1% bovine serum albumin (BSA) in PBS. Primary antibody was omitted in control incubations. Cells were washed twice with PBS and incubated with a CY3-conjugated donkey anti-rabbit secondary antibody (1/500) (Jackson ImmunoResearch) in 0.1% BSA in PBS, for 1 h in the dark. Cells were washed again and DNA was counterstained with 10 nM DAPI (Sigma-Aldrich). Following 2 washes in PBS, cells were mounted in DAPCO (Sigma-Aldrich) prepared according to the manufacturer's instructions. Images were taken using a TCS SP2 Laser Scanning Confocal microscope (Leica Technologies, Wetzlar, Germany), using a 63× oil-immersion objective and a 4× zoom factor.
Membrane fractionation analyses
Triton X-100 soluble and insoluble fractions were prepared essentially as described . Cells grown in 4 × 100 mm dishes were treated with 20 mM methyl β-cyclodextrin (MβCD) at 37°C for 30 min. Cells were then rinsed with PBS and lysed for 20 min in 1 ml 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 at 4°C. The lysate was brought to 40% sucrose in a final volume of 4 ml, placed at the bottom of a 12 ml tube, and then layered with 6 ml 30% sucrose followed by 2 ml 5% sucrose, made in the same buffer without Triton X-100. Gradients were centrifuged for 22 h at 36,000 rpm at 4°C in a Beckman SW41 rotor. Fractions of 1 ml were harvested from the top of the tube and aliquots were subjected to Western blot analyses.
Yeast two-hybrid screening identifies MUC1 as a putative MAL2 partner
Derivation of polyclonal antisera which specifically recognise MAL2 protein
Co-immunoprecipitation of MUC1 and MAL2 from MCF-10A/Myc-MAL2 cells
Mapping the MAL2 region required for MUC1 binding
MAL2 and MUC1 co-localise in the cytoplasm of breast carcinoma cells
MAL2 and MUC1 are present in lipid raft fractions in human breast carcinoma cells
Increased MAL2 expression leads to increased cell surface expression of MUC1
MAL2 was first identified through its expression in breast carcinoma and interactions with D52-like proteins within the Y2H system . We therefore undertook a Y2H screen to identify MAL2 binding partners expressed in human breast cancer tissue, which identified a number of novel putative binding proteins, including MUC1. MUC1 represented a candidate partner of particular interest, given the fact that it is overexpressed in cancer types where MAL2 overexpression has also been reported, and as MUC1 overexpression contributes to cancer progression through a number of mechanisms [43, 44]. Interactions between human MUC1 and MAL2 proteins are also broadly consistent with the previously reported interactions between the yeast signalling mucin Msb2 and the tetraspanin protein Sho1 . Subsequent results collectively identify MAL2 as a novel cytoplasmic MUC1 partner, and a possible regulator of MUC1 expression and/or subcellular distribution. It is striking to note that MAL2, a chromosome 8q24 amplification target, has now been shown to bind MUC1 and D52, both of which are amplified and/or overexpressed in breast and other cancers [21, 22, 34, 35, 46] strongly suggesting that these proteins have co-operating functions in cancer cells.
While a recent study by Kinlough et al.  indicated that palmitoylation is the dominant feature modulating MUC1 recycling to the plasma membrane, the mechanisms by which MUC1 is targeted and maintained at the plasma membrane are not fully understood. Since ectopic Myc-MAL2 expression in MCF-10A cells was associated with increased MUC1 detection at the cell periphery in the present study, MAL2 may play a direct or indirect role in MUC1 targeting. Interestingly, MAL2 incompletely co-localised with peripheral MUC1 in MCF-10A/Myc-MAL2 cells, which supports previous findings that altering MAL2 expression can alter cargo accumulation distant to the predominant site of MAL2 expression [2, 14]. However, ectopic Myc-MAL2 expression also produced an apparent expansion of cell surface domains in MCF-10A cells, which may also contribute to increased MUC1 detection at the cell periphery. Similar observations have been made in a previous study where MAL overexpression altered the morphology of MDCK cells, by seemingly expanding apical cell surface domains through increased apical delivery . Finally, we also noted that ectopic MAL2 expression produced an increase in MUC1 detection across Triton X-100-soluble and -insoluble protein fractions in MCF-10A/Myc-MAL2 cells, indicating that Myc-MAL2 may positively regulate MUC1 expression. This may also lead to increased peripheral MUC1 detection. While it remains to be determined whether MAL2 can alter MUC1 distribution in other cell types, the broad expression of MAL2 within epithelia  is consistent with MAL2 contributing to MUC1 targetting under physiological conditions. It will also be of interest to examine whether MAL2 can similarly regulate MUC1 secretion.
If MAL2 similarly regulates MUC1 expression and/or distribution in cancer cells, increased MAL2 levels could alter cancer cell biology in several ways. Increased MUC1 localisation at the cell surface could reduce intercellular adhesion and promote invasiveness , and increased MAL2 expression would thus be expected to have adverse significance in cancer cells. Accordingly, MAL2 has been included within a gene signature of poor prognosis in breast cancer , and MAL2 overexpression was associated with resistance to doxorubicin therapy in breast cancer patients . MAL2 overexpression might also reduce cytoplasmic MUC1 accumulation, which has been indicated to be an adverse finding in breast and ovarian carcinomas [50–52], tumor types which also overexpress MAL2 [20, 24, 25]. Interestingly, suppression of MUC1 expression in a pancreatic cancer cell line reduced these cells' metastatic potential, and was accompanied by reduced MAL2 levels . Further direct analyses are therefore required to determine whether MAL2 and MUC1 levels are positively correlated in cancer types commonly expressing these proteins, and whether MAL2 expression is significantly associated with cell surface expression of MUC1 in cancer cells.
The present study also provides the first report that MUC1 localises within lipid raft fractions in breast carcinoma cells, and raises the possibility that some of MUC1's signalling functions [43, 44] may occur within lipid rafts. Previous studies reported that MUC1 in T-lymphocyte cell lines was insoluble in cold Triton X-100 and associated with low density membrane fractions [54, 55], yet Kinlough et al.  showed MUC1 from MDCK cells was fully soluble in Triton X-100. The reported differences in MUC1 solubility may be due to cell specific differences in membrane composition and their selectivity for detergents, as reported by Schuck et al. , or alternatively MUC1 may not reside in lipid rafts in all cell types. We demonstrated associations between MUC1 and MAL2 in Triton X-100 soluble fractions, in agreement with MUC1 and MAL2 being predominantly detected in non-raft fractions in all cell lines analysed, which was also noted for the MAL2 partner D52. While we were unable to determine whether MAL2 and MUC1 associate within lipid rafts, our results indicate that MAL2 associates with MUC1 and potentially other proteins outside lipid raft membrane microdomains, as indicated for other tetraspanins , and highlights the fact that MAL2 may have independent functions within and outside lipid rafts.
Our analysis of MAL2 partners has also indicated that MAL2 may represent a multifunctional transmembrane adaptor protein, capable of binding more than one partner simultaneously. We noted that both MAL2 and D52 co-immunoprecipitated with MUC1 in the present study, despite the fact that direct interactions between MUC1 and D52 were not detected in the Y2H system. Independent interaction domains for MAL2/MUC1 and MAL2/D52 binding were also indicated by the MAL2 N-terminal domain being required to bind D52-like proteins, yet the first TM domain was required for binding MUC1. Correspondingly, D52-like proteins did not bind MAL, whose N-terminal sequence is poorly conserved with respect to that of MAL2 , yet MUC1 bound both MAL and MAL2 in the Y2H system. These results therefore predict shared and isoform-specific functions for MAL-like proteins, through shared and discrete binding partners.
This study has identified MAL2 as a novel cytoplasmic MUC1 partner and a potential regulator of MUC1 subcellular distribution. MAL2/MUC1 interactions were detected in Triton X-100-soluble membrane fractions, indicating that MAL2 has specific functions within non-raft membrane compartments in breast cancer cells. Since MAL2 is known to be amplified and overexpressed in breast and other cancers, it is striking that MAL2 has now been shown to bind two proteins, MUC1 and D52, which are also amplified and/or overexpressed. Based on these results, and a previous study highlighting the co-amplification of MAL2 and D52 genes in breast cancer , it will be interesting to examine whether MAL2 and MUC1 are commonly amplified and overexpressed in breast cancer, and whether MAL2 expression is significantly associated with cell surface expression of MUC1.
epidermal growth factor
chemokine-like factor superfamily8
colony forming unit
Mal T-cell differentiation protein
polymerase chain reaction
bovine serum albumin
We wish to thank Professor Peter Gunning (CHW) for his ongoing support, and Ms Roumayne Schepers (CHW, UU) for excellent technical assistance. This work was supported by a National Health and Medical Research Council of Australia Peter Doherty Fellowship (to SF), a Cancer Institute NSW Fellow (to JAB), funding from the Dr. Saal van Zwanenberg Foundation and the KWF Kankerbestrijding (to APA), donations to the Oncology Department of the Children's Hospital at Westmead, and the Oncology Children's Foundation. MAA is the recipient of grants BFU2006-01925 and GEN2003-20662-C07-02 from the Spanish Ministry of Education and Science.
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