ΔNp63, also known as p40, regulates stemness of normal mammary gland epithelium and provides stem cell characteristics in basal and HER2-driven murine breast cancer models. Whilst ΔNp63/p40 is a characteristic feature of normal basal cells and basal-type triple-negative breast cancer, some receptor-positive breast cancers express ΔNp63/p40 and its overexpression imparts cancer stem cell-like properties in ER+ cell lines. However, the incidence of ER+ and HER2+ tumours that express ΔNp63/p40 is unclear and the phenotype of ΔNp63/p40+ cells in these tumours remains uncertain. Using immunohistochemistry with p63 isoform-specific antibodies, we identified a ΔNp63/p40+ tumour cell subpopulation in 100 of 173 (58%) non-triple negative breast cancers and the presence of this population associated with improved survival in patients with ER- /HER2+ tumours (p = 0.006). Furthermore, 41% of ER+ /PR+ and/or HER2+ locally metastatic breast cancers expressed ΔNp63/p40, and these cells commonly accounted for <1% of the metastatic tumour cell population that localised to the tumour/stroma interface, exhibited an undifferentiated phenotype and were CD44+ /ALDH- . In vitro studies revealed that MCF7 and T47D (ER+ ) and BT-474 (HER2+ ) breast cancer cell lines similarly contained a small subpopulation of ΔNp63/p40+ cells that increased in mammospheres. In vivo, MCF7 xenografts contained ΔNp63/p40+ cells with a similar phenotype to primary ER+ cancers. Consistent with tumour samples, these cells also showed a distinct location at the tumour/stroma interface, suggesting a role for paracrine factors in the induction or maintenance of ΔNp63/p40. Thus, ΔNp63/p40 is commonly present in a small population of tumour cells with a distinct phenotype and location in ER+ and/or HER2+ human breast cancers.

• Keywords
• CD44, HER2, aldehyde dehydrogenase, breast, cancer stem cells, oestrogen receptor, p40, p63, ΔNp63,
• MeSH
• Phenotype MeSH
• Heterografts MeSH
• Humans MeSH
• Mice MeSH
• Biomarkers, Tumor analysis   metabolism   MeSH
• Neoplastic Stem Cells metabolism   pathology   MeSH
• Tumor Suppressor Proteins metabolism   MeSH
• Breast Neoplasms genetics   metabolism   pathology   MeSH
• Receptor, ErbB-2 genetics   metabolism   MeSH
• Receptors, Estrogen genetics   metabolism   MeSH
• Transcription Factors metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ERBB2 protein, human MeSH Browser
• Biomarkers, Tumor MeSH
• Tumor Suppressor Proteins MeSH
• Receptor, ErbB-2 MeSH
• Receptors, Estrogen MeSH
• TP63 protein, human MeSH Browser
• Transcription Factors MeSH

Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53-independent MDM2-drug responsive-binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin-3 responsive MDM2-binding proteins that are perturbed independent of cell density using SWATH-MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin-3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin-3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin-3 treated cells. Mitotracker confirmed that Nutlin-3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin-3 treatment. Proximity ligation identified rearrangements of cellular protein-protein complexes in situ. In response to Nutlin-3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein-protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein-protein complexes in drug-treated cells.

• Keywords
• Cell biology, MDM2, Mitochondria, Nutlin-3, P53, SWATH-MS,
• MeSH
• Dihydrolipoamide Dehydrogenase metabolism   MeSH
• HCT116 Cells MeSH
• Imidazoles pharmacology   MeSH
• Humans MeSH
• Protein Interaction Maps drug effects   MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• Mitochondria drug effects   metabolism   MeSH
• Piperazines pharmacology   MeSH
• Proto-Oncogene Proteins c-mdm2 metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Dihydrolipoamide Dehydrogenase MeSH
• Imidazoles MeSH
• nutlin 3 MeSH Browser
• Piperazines MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH

The tumor suppressor p53 plays a key role in malignant transformation and tumor development. However, the frequency of p53 mutations within individual types of cancer is different, suggesting the existence of other mechanisms attenuating p53 tumor suppressor activity. Changes in upstream regulators of p53 such as MDM2 amplification and overexpression, expression of viral oncoproteins, estrogen receptor signaling, or changes in p53 transcriptional target genes were previously described in wild-type p53 tumors. We identified a novel pathway responsible for attenuation of p53 activity in human cancers. We demonstrate that AGR2, which is overexpressed in a variety of human cancers and provides a poor prognosis, up-regulates DUSP10 which subsequently inhibits p38 MAPK and prevents p53 activation by phosphorylation. Analysis of human breast cancers reveals that AGR2 specifically provides a poor prognosis in ER+ breast cancers with wild-type p53 but not ER- or mutant p53 breast cancers, and analysis of independent data sets show that DUSP10 levels also have prognostic significance in this specific sub-group of patients. These data not only reveal a novel pro-oncogenic signaling pathway mediating resistance to DNA damaging agents in human tumors, but also has implications for designing alternative strategies for modulation of wild-type p53 activity in cancer therapy.

• Keywords
• AGR2, Breast cancer, DUSP10, Drug resistance, p38 MAPK, p53,
• MeSH
• Enzyme Activation drug effects   MeSH
• Drug Resistance, Neoplasm MeSH
• Adult MeSH
• Mitogen-Activated Protein Kinase Phosphatases metabolism   MeSH
• Dual-Specificity Phosphatases metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• p38 Mitogen-Activated Protein Kinases metabolism   MeSH
• Mucoproteins MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• Breast Neoplasms drug therapy   metabolism   pathology   MeSH
• Neoplasms drug therapy   metabolism   pathology   MeSH
• Oncogene Proteins MeSH
• Proteins metabolism   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Breast drug effects   metabolism   pathology   MeSH
• Aged MeSH
• Signal Transduction * drug effects   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• AGR2 protein, human MeSH Browser
• DUSP10 protein, human MeSH Browser
• Mitogen-Activated Protein Kinase Phosphatases MeSH
• Dual-Specificity Phosphatases MeSH
• p38 Mitogen-Activated Protein Kinases MeSH
• Mucoproteins MeSH
• Tumor Suppressor Protein p53 MeSH
• Oncogene Proteins MeSH
• Proteins MeSH
• Antineoplastic Agents MeSH