Triple negative breast cancer (TNBC) is a group of cancers whose heterogeneity and shortage of effective drug therapies has prompted efforts to divide these cancers into molecular subtypes. Our ...computational platform, entitled GenEx-TNBC, applies concepts in systems biology and polypharmacology to prioritize thousands of approved and experimental drugs for therapeutic potential against each molecular subtype of TNBC. Using patient-based and cell line-based gene expression data, we constructed networks to describe the biological perturbation associated with each TNBC subtype at multiple levels of biological action. These networks were analyzed for statistical coincidence with drug action networks stemming from known drug-protein targets, while accounting for the direction of disease modulation for coinciding entities. GenEx-TNBC successfully designated drugs, and drug classes, that were previously shown to be broadly effective or subtype-specific against TNBC, as well as novel agents. We further performed biological validation of the platform by testing the relative sensitivities of three cell lines, representing three distinct TNBC subtypes, to several small molecules according to the degree of predicted biological coincidence with each subtype. GenEx-TNBC is the first computational platform to associate drugs to diseases based on inverse relationships with multi-scale disease mechanisms mapped from global gene expression of a disease. This method may be useful for directing current efforts in preclinical drug development surrounding TNBC, and may offer insights into the targetable mechanisms of each TNBC subtype.
In several cancers, including breast cancer, loss of E-cadherin expression is correlated with a loss of the epithelial phenotype and with a gain of invasiveness. Cells that have lost E-cadherin ...expression are either poorly invasive with a rounded phenotype, or highly invasive, with a mesenchymal phenotype. Most cells lacking E-cadherin still retain weak calcium-dependent adhesion, indicating the presence of another cadherin family member. We have now examined the expression of the mesenchymal cadherin, cadherin-11, in breast cancer cell lines. Cadherin-11 mRNA and protein, as well as a variant form, are expressed in the most invasive cell lines but not in any of the noninvasive cell lines. Cadherin-11 is localized to a detergent-soluble pool and is associated with both alpha- and beta-catenin. Immunocytochemistry shows that cadherin-11 is localized to the cell membrane at sites of cell-cell contact as well as at lamellipodia-like projections, which do not interact with other cells. These results suggest that cadherin-11 expression may be well correlated with the invasive phenotype in cancer cells and may serve as a molecular marker for the more aggressive, invasive subset of tumors. Cadherin-11 may mediate the interaction between malignant tumor cells and other cell types that normally express cadherin-11, such as stromal cells or osteoblasts or perhaps even with the surrounding extracellular matrix, thus facilitating tumor cell invasion and metastasis.
Vitamin D is implicated in the etiology of cancers of the gastrointestinal tract, usually characterized by alteration in the APC/β-catenin/TCF tumor suppressor pathway. The vitamin D receptor (VDR) ...is also implicated in cardiovascular and skin diseases as well as in immunity. Activated VDR can indirectly alter β-catenin nuclear localization and directly suppress β-catenin/TCF mediated transcriptional activity. We treated VDR null mice with the carcinogen azoxymethane (AOM) and generated mice bearing a mutated APC (hypomorph) on a VDR null background (Apc1638N/+Vdr-/-). VDR null mice do not develop GI or extra-colonic tumors but loss of VDR decreased intestinal tumor latency and increased progression to adenocarcinoma in both models. AOM treatment of VDR null mice also caused squamous cell carcinoma of the anus. Although levels and distribution of total or activated β-catenin in the epithelial component of tumors were unaffected by loss of VDR, β-catenin dependent cyclin D1 expression was affected suggesting a direct VDR effect on β-catenin co-activator activity. Extra-colonic mucosa manifestations in Apc1638N/+Vdr-/- animals included increased nuclear β-catenin in submucosal stromal cells, spleno- and cardiomegaly and large epidermoid cysts characteristic of the FAP variant, Gardner's syndrome. Consistent with this, SNPs in the VDR, vitamin D binding protein and CYP24 as well as mutations in APC distal to codon 850 were strongly associated with Gardners syndrome in a cohort of 457 FAP patients, This work suggests that alterations in the vitamin D/VDR axis are important in Gardner's syndrome, as well as in the etiology of anal cancer.
Tubulin-α1A/1B C-terminal tail (CTT) has seven glutamic acid residues among the last 11 amino acids of its sequence that are potential sites for glutamylation. Cleavage of C-terminal tyrosine ...resulting in the detyrosinated form of tubulin-α1A/1B is another major modification. These modifications among others bring about highly heterogeneous tubulin samples in brain cells and microtubules, play a major role in directing intracellular trafficking, microtubule dynamics, and mitotic events, and can vary depending on the cell and disease state, such as cancer and neurodegenerative disorders. Identified previously using primary mass spectrometry (MS) ions and partial Edman sequencing, tubulin-α1A/1B glutamylation was found exclusively on the E445 residue. We here describe the analysis of tubulin-α1A/1B glutamylation and detyrosination after 2-DE separation, trypsin and proteinase K in-gel digestion, and nanoUPLC-ESI-QqTOF-MS/MS of mouse brain and bovine microtubules. Tyrosinated, detyrosinated, and Δ2-tubulin-α1A/1B CTTs were identified on the basis of a comparison of fragmentation patterns and retention times between endogenous and synthetic peptides. Stringent acceptance criteria were adapted for the identification of novel glutamylation sites. In addition to the previously identified site at E445, glutamylation on mouse and bovine tubulin-α1A/1B CTTs was identified on E441 and E443 with MASCOT Expect values below 0.01. O-Methylation of glutamates was also observed.
The Wnt/β-catenin/Tcf and IκB/NF-κB cascades are independent pathways involved in cell cycle control, cellular differentiation, and inflammation. Constitutive Wnt/β-catenin signaling occurs in ...certain cancers from mutation of components of the pathway and from activating growth factor receptors, including RON and MET. The resulting accumulation of cytoplasmic and nuclear β-catenin interacts with the Tcf/LEF transcription factors to induce target genes. The IκB kinase complex (IKK) that phosphorylates IκB contains IKKα, IKKβ, and IKKγ. Here we show that the cyclin D1 gene functions as a point of convergence between the Wnt/β-catenin and IκB pathways in mitogenic signaling. Mitogenic induction of G
1
-S phase progression and cyclin D1 expression was PI3K dependent, and cyclin D1
−/−
cells showed reduced PI3K-dependent S-phase entry. PI3K-dependent induction of cyclin D1 was blocked by inhibitors of PI3K/Akt/IκB/IKKα or β-catenin signaling. A single Tcf site in the cyclin D1 promoter was required for induction by PI3K or IKKα. In IKKα
−/−
cells, mitogen-induced DNA synthesis, and expression of Tcf-responsive genes was reduced. Reintroduction of IKKα restored normal mitogen induction of cyclin D1 through a Tcf site. In IKKα
−/−
cells, β-catenin phosphorylation was decreased and purified IKKα was sufficient for phosphorylation of β-catenin through its N-terminus in vitro. Because IKKα but not IKKβ induced cyclin D1 expression through Tcf activity, these studies indicate that the relative levels of IKKα and IKKβ may alter their substrate and signaling specificities to regulate mitogen-induced DNA synthesis through distinct mechanisms.
Inhibition of vascular endothelial growth factor receptor-2 (VEGFR2) kinase blocks angiogenesis, the process of generating new capillary blood vessels that are important in tumor growth. To identify ...small molecule inhibitors of the VEGFR2 kinase, we undertook a computer assisted fragment-based screening that used 3-D structural models of the VEGFR2 kinase, and hinge region as a fragment anchor point. Seven novel non-cytotoxic compounds were identified which limited the induction of capillary networks by human umbilical vein endothelial cells in the low micromolar range.
Breast cancer is a progressive and potentially fatal disease that affects women of all ages. Like all progressive diseases, early and reliable diagnosis is the key for successful treatment and ...annihilation. Biomarkers serve as indicators of pathological, physiological, or pharmacological processes. Her2/neu, CA15.3, estrogen receptor (ER), progesterone receptor (PR), and cytokeratins are biomarkers that have been approved by the Food and Drug Administration for disease diagnosis, prognosis, and therapy selection. The structural and functional complexity of protein biomarkers and the heterogeneity of the breast cancer pathology present challenges to the scientific community. Here we review estrogen receptor-related putative breast cancer biomarkers, including those of putative breast cancer stem cells, a minor population of estrogen receptor negative tumor cells that retain the stem cell property of self-renewal. We also review a few promising cytoskeleton targets for ER alpha negative breast cancer.
Structure-based drug repositioning in addition to random chemical screening is now a viable route to rapid drug development. Proteochemometric computational methods coupled with kinase assays showed ...that mebendazole (MBZ) binds and inhibits kinases important in cancer, especially both BRAFWT and BRAFV600E. We find that MBZ synergizes with the MEK inhibitor trametinib to inhibit growth of BRAFWT-NRASQ61K melanoma cells in culture and in xenografts, and markedly decreased MEK and ERK phosphorylation. Reverse Phase Protein Array (RPPA) and immunoblot analyses show that both trametinib and MBZ inhibit the MAPK pathway, and cluster analysis revealed a protein cluster showing strong MBZ+trametinib - inhibited phosphorylation of MEK and ERK within 10 minutes, and its direct and indirect downstream targets related to stress response and translation, including ElK1 and RSKs within 30 minutes. Downstream ERK targets for cell cycle, including cMYC, were down-regulated, consistent with S- phase suppression by MBZ+trametinib, while apoptosis markers, including cleaved caspase-3, cleaved PARP and a sub-G1 population, were all increased with time. These data suggest that MBZ, a well-tolerated off-patent approved drug, should be considered as a therapeutic option in combination with trametinib, for patients with NRASQ61mut or other non-V600E BRAF mutant melanomas.
Retinoic Acid Receptor Responder (RARRES1) initially identified as a novel retinoic acid receptor regulated gene in the skin is a putative tumor suppressor of unknown function. RARRES1 was knocked ...down in immortalized human prostatic epithelial cell line PWR-1E cells and differential protein expression was identified using differential in-gel electrophoresis (DIGE) followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry and western Blot analysis excluding highly abundant proteins routinely identified in almost all proteomics projects. Knock-down of RARRES1: 1- down-regulates PP2A, an enzyme involved in the negative regulation of the growth hormone-stimulated signal transduction pathways; 2- down-regulates Valosin-containing protein causing impaired autophagy; 3- up-regulates the tumor suppressor disks large 2; 4- up-regulates Ankrd26 that belongs to the POTE family of genes that are highly expressed in cancer patients with poor outcome; and 5- down-regulates EB1, a protein that is involved in spindle dynamics and chromosome alignment during mitosis.
Metabolomics is the comprehensive assessment of endogenous metabolites of a biological system. "Oncometabolomics" is a rapidly emerging field with potential for developing specific biomarkers for ...early detection, diagnosis, and disease prognosis. Given the power of this technology, the availability of standardized sample preparation methods for immortalized human cancer cell lines is critical toward augmenting research in this direction. Using MCF-7 cells as a model system, we describe an approach for intracellular metabolite extraction from cell cultures for reproducible and comprehensive metabolite extraction. The samples, when injected onto a reverse-phase 50 × 2.1 mm Acquity 1.7-μm C18 column, using an ultra performance liquid chromatography system (UPLC) coupled with electrospray ionization-quadrupole-time-of-flight-mass spectrometry (ESI-Q-TOF-MS) in positive and negative modes, yielded a data matrix with a total of 2600 features. This method, when compared with a water-extraction procedure described earlier, was found to yield significantly higher coverage and detection of molecular features. Finally, we successfully tested the performance of this method for an array of human cancer cell lines used widely in the cancer research field.
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