Cells were treated with a biotinylation reagent to label surface proteins and then collected, lysed and labeled proteins were purified using neutravidin agarose resin. in HER2-amplified breast tumors and inhibiting HER2 activity in tumor cells resulted in a decreased MUCL1 expression. In-depth investigation demonstrated that phosphoinositide3-kinase/Akt pathway, but not Ras/MEK pathway, controls MUCL1 expression downstream of HER2. Phenotypic assays revealed a strong dependence of HER2-positive cells on MUCL1 for cell proliferation. We further identified the mechanism by which MUCL1 regulates cell growth. Knockdown of MUCL1 induced a G1/S phase arrest concomitant with decreased cyclin D and increased p21 and p27 levels. Finally, we investigated the impact of MUCL1 loss on kinase signaling pathways in N-Desethyl Sunitinib breast cancer cells through phospho-kinase array profiling. MUCL1 silencing abrogated phospho-focal adhesion kinase (FAK), Jun NH2-terminal kinase (JNK) and c-Jun signals, but not extracellular signal-regulated kinase or Akt pathway activities, thereby pointing to FAK/JNK pathway as the downstream effector of MUCL1 signaling. We are the first to identify an important role for MUCL1 in the proliferation of breast cancer cells, probably mediated via the FAK/JNK signaling pathway. Taken together, these data suggest a potential utility for therapeutic targeting of this protein in breast cancer. Introduction Mucin-like 1 (transcript. Early studies demonstrated by reverse transcriptionCPCR analysis that 90% of breast cancer cell lines express transcript as a biomarker for disease progression and metastasis in breast cancer patients.7, 8, 9, 10 Its limited N-Desethyl Sunitinib normal tissue expression also renders MUCL1 an attractive tumor-associated antigen for targeted therapy of breast cancers. Despite our understanding of the expression of MUCL1 in breast cancer, the cellular localization of the MUCL1 protein has remained largely unstudied, which will have a major impact on drug developmentability. Although most mucins are secreted, several members of this protein family such as MUC1 and MUC4 are tethered to the plasma membrane with a hydrophobic membrane-spanning domain. MUCL1 was detected while assessing expression of tumor-derived cDNA fragments on yeast surface by screening with breast cancer patient sera, suggesting that it is membrane bound.11 Protein sequence analysis software yielded an ambiguous prediction that MUCL1 contains an N-terminal peptide signal sequence for targeting to the endoplasmic reticulum/Golgi secretory pathway, which could also double as a weak transmembrane domain (Figure 1). Whether the protein is secreted or tethered to the plasma membrane remains unknown. Early studies reported a secreted form of the protein in engineered NIH293 cells,1 but this was done in an artificial ectopic overexpression system and has not yet been verified in breast cancer cells. In addition to our lack of understanding of MUCL1 localization, a MUCL1 cellular function has not yet been characterized. Here we describe our efforts to fully define the cellular localization of MUCL1 and discover the biological function and signaling network of MUCL1 in breast cancer. Open in a separate window Figure 1 A schematic of the MUCL1 amino acid sequence is presented. A hydrophobic signal peptide is present at residues 1C20 and a triple serine- and threonine-rich tandem repeat is present at residues 46C69. The antibody used for the current studies was generated against amino acids 19C53. Results MUCL1 characterization in breast cancer Earlier characterizations of expression examined a limited number of breast cancer and normal tissue samples. To build on these studies, we assessed N-Desethyl Sunitinib the levels of expression across 48 normal tissue types using a cDNA array. The highest expression was found in the mammary gland, verifying the previously reported findings (Figure 2a). Significant mRNA expression was also detected in the skin but at a level three times lower than in the mammary gland. All other normal cells either exhibited undetectable RNA in over 1000 malignancy cell lines representing 37 malignancy types in the Broad-Novartis Malignancy Cell Collection Encyclopedia. As expected, the highest level of manifestation was observed in breast tumor cell lines (Supplementary Number S1b). Correspondingly, when we examined the manifestation of across a panel of human being tumor samples using Oncomine Power Tools, breast cancer displayed the highest manifestation level of all cancers surveyed (Number Rabbit Polyclonal to SLC5A2 2b). Further highlighting its restricted manifestation, breast cells exhibited the highest gene manifestation among all the normal tissues included in the Oncomine analysis. Collectively, these multipronged genomic analyses suggest a restricted manifestation profile of is definitely highly indicated in normal breast cells and breast.
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