Side-population (SP) cells within cancers and cell lines are rare cell populations known to enrich cancer stem-like cells. In this study, we characterized SP cells from the human breast cancer cell line MCF7 as a model for cancer stem-like cells. Compared with non-SP cells, MCF7 SP cells had higher colony-formation ability in vitro and greater tumorigenicity in vivo , suggesting that MCF7 SP cells enrich cancer stem-like cells. cDNA microarray analysis of the SP cells indicated higher expression of ATP-binding cassette transporters and genes involved in quiescence, which were confirmed by quantitative RT-PCR and flow cytometry cell cycle analysis. To identify signal pathways important for cancer stem-like cells, we analyzed cDNA microarray data and identified nine pathways that were altered in the SP cells. To analyze the protein signaling networks, we used reverse-phase signaling pathway protein microarray technology and identified three signaling proteins that are significantly different between MCF7 SP and non-SP cells. Notably, signaling of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR), signal transduction and activator of transcription (STAT3), and phosphatase and tensin homolog (PTEN) was confirmed to be critical for MCF7 SP cell survival and proliferation by pathway specific inhibitors, selected gene knockdown, and in vivo tumorigenicity assay. The STAT3 pathway was found to be positively regulated by mTOR signaling, whereas PTEN served as a negative regulator of both STAT3 and mTOR signaling. This study suggests the existence of prosurvival signaling pathways critical for cancer stem-like cell maintenance, which could be selectively targeted for inhibiting cancer stem-like cells for improved treatment.

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BackgroundWhile it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment.Methodology and Principal FindingsWe examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2–4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patient's breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patient's original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from the organ culture, and abrogated xenograft tumor formation.ConclusionsCytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.

Phosphoprotein driven cellular signaling events represent most of the new molecular targets for cancer treatment. Application of reverse-phase protein microarray technology for the study of ongoing signaling activity within breast tumor specimens holds great potential for elucidating and profiling signaling activity in real-time for patient-tailored therapy. Analysis of laser capture microdissection primary human breast tumors and metastatic lesions reveals pathway specific profiles and a new way to classify cancer based on functional signaling portraits. Moreover, the data demonstrate the requirement of laser capture microdissection for analysis and reveal the metastasis-specific changes that occur within a new microenvironment. Analysis of biopsy material from clinical trials for targeted therapeutics demonstrates the feasibility and utility of comprehensive signal pathway activation profiling for molecular analysis.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC-MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate, and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC-MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately two-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC-MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers.

Purpose Targeting of the HER2 protein in human breast cancer represents a major advance in oncology, but relies on measurements of total HER2 protein and not HER2 signaling network activation. We utilized reverse phase protein microarrays (RPMAs) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity. Experimental Design Three independent study sets, comprising a total of 415 individual patient samples from flash frozen core biopsy samples and FFPE surgical and core samples, were analyzed via RPMA. The phosphorylation and total levels of the HER receptor family proteins and downstream signaling molecules were measured in laser capture microdissected (LCM) enriched tumor epithelium from 127 frozen pre-treatment core biopsy samples and whole tissue lysates from 288 FFPE samples and these results were compared to FISH and IHC. Results RPMA measurements of total HER2 were highly concordant (> 90% all sets) with FISH and/or IHC data, as was phosphorylation of HER2 in the FISH/IHC+ population. Phosphorylation analysis of HER family signaling identified HER2 activation in some FISH/IHC- tumors and, identical to that seen with FISH/IHC+ tumors, the HER2 activation was concordant with EGFR and HER3 phosphorylation and downstream signaling endpoint activation. Conclusions Molecular profiling of HER2 signaling of a large cohort of human breast cancer specimens using a quantitative and sensitive functional pathway activation mapping technique reveals IHC-/FISH-/pHER2+ tumors with HER2 pathway activation independent of total HER2 levels and functional signaling through HER3 and EGFR.

Reverse phase protein microarray analysis was used to identify cell signaling derangements in squamous cell carcinoma (SCC) compared with actinic keratosis (AK) and upper inner arm (UIA). We analyzed two independent tissue sets with isolation and enrichment of epithelial cells by laser capture microdissection. Set 1 served as a pilot and a means to identify protein pathway activation alterations that could be further validated in a second independent set. Set 1 was comprised of 4 AK, 13 SCC, and 20 UIA. Set 2 included 15 AK, 9 SCCs, and 20 UIAs. Activation of 51 signaling proteins, known to be involved in tumorigenesis, were assessed for set 1 and showed that the MEK-ERK [mitogen-activated protein (MAP)/extracellular signalregulated (ERK; MEK)] pathway was activated in SCC compared with AK and UIA, and that epidermal growth factor receptor (EGFR) and mTOR pathways were aberrantly activated in SCC. Unsupervised twoway hierarchical clustering revealed that AK and UIA shared a common signaling network activation architecture while SCC was dramatically different. Statistical analysis found that prosurvival signaling through phosphorylation of ASK and 4EBP1 as well as increased Bax and Bak expression was higher in AK compared with UIA. We expanded pathway network activation mapping in set 2 to 101 key signaling proteins, which corroborated activation of MEK-ERK, EGFR, and mTOR pathways through discovery of a number of upstream and downstream signaling molecules within these pathways to conclude that SCC is indeed a pathway activation-driven disease. Pathway activation mapping of SCC compared with AK revealed several interconnected networks that could be targeted with drug therapy for potential chemoprevention and therapeutic applications. Cancer Prev Res; 5(3); 403-13. Ó2012 AACR.

Accumulating evidence supports a role of the PI3K-AKT pathway in the regulation of cell motility, invasion and metastasis. AKT activation is known to promote metastasis, however under certain circumstances, it also shows an inhibitory activity on metastatic processes, and the cause of such conflicting results is largely unclear. Here we found that AKT1 is an important regulator of metastasis and down-regulation of its activity is associated with increased metastatic potential of A549 cells. Inhibition of AKT1 enhanced migration and invasion in KRAS- or EGFR-mutant non-small cell lung cancer (NSCLC) cells. The allosteric AKT inhibitor MK-2206 promoted metastasis of KRAS-mutated A549 cells in vivo. We next identified that the phosphorylation of Myristoylated alanine-rich C-kinase substrate (MARCKS) and LAMC2 protein level were increased with AKT1 inhibition, and MARCKS or LAMC2 knockdown abrogated migration and invasion induced by AKT1 inhibition. This study unravels an anti-metastatic role of AKT1 in the NSCLC cells with KRAS or EGFR mutations, and establishes an AKT1-MARCKS-LAMC2 feedback loop in this regulation.