Circulating tumour cells (CTCs) can provide valuable prognostic information in a number of epithelial cancers. However, their detection is hampered due to their molecular heterogeneity, which can be induced by the epithelial-mesenchymal transition (EMT) process. Therefore, current knowledge about CTCs from clinical samples is often limited due to an inability to isolate wide spectrum of CTCs phenotypes. In the current work, we aimed at isolation and molecular characterization of CTCs with different EMT status in order to establish their clinical significance in early breast cancer patients. We have obtained CTCs-enriched blood fraction from 83 breast cancer patients in which we have tested the expression of epithelial, mesenchymal and general breast cancer CTCs markers (MGB1/HER2/CK19/CDH1/CDH2/VIM/PLS3), cancer stem cell markers (CD44, NANOG, ALDH1, OCT-4, CD133) and cluster formation gene (plakoglobin). We have shown that in the CTCs-positive patients, epithelial, epithelial-mesenchymal and mesenchymal CTCs markers were detected at a similar rate (in 28%, 24% and 24%, respectively). Mesenchymal CTCs were characterized by the most aggressive phenotype (significantly higher expression of CXCR4, uPAR, CD44, NANOG, p < 0.05 for all), presence of lymph node metastases (p = 0.043), larger tumour size (p = 0.023) and 7.33 higher risk of death in the multivariate analysis (95% CI 1.06–50.41, p = 0.04). Epithelial-mesenchymal subtype, believed to correspond to highly plastic and aggressive state, did not show significant impact on survival. Gene expression profile of samples with epithelial-mesenchymal CTCs group resembled pure epithelial or pure mesenchymal phenotypes, possibly underlining degree of EMT activation in particular patient’s sample. Molecular profiling of CTCs EMT phenotype provides more detailed and clinically informative results, proving the role of EMT in malignant cancer progression in early breast cancer.

Intratumoral heterogeneity of breast cancer remains a major challenge in successful treatment. Failure of cancer therapies can also be accredited to inability to systemically eradicate cancer stem cells (CSCs). Recent evidence points to the role of epithelial-mesenchymal transition (EMT) in expanding the pool of tumor cells with CSCs features. Thus, we assessed expression level as well as heterogeneity of CSCs markers in primary tumors (PT), lymph node metastasis (LNM), and circulating tumor cells (CTCs)-enriched blood fractions in order to correlate them with signs of EMT activation as well as clinicopathological data of breast cancer patients. Level of CSCs markers (ALDH1, CD44, CD133, OCT-4, NANOG) and EMT markers was quantified in PT (N=107), LNM (N=56), and CTCs-enriched blood fractions (N=85). Heterogeneity of CSCs markers expression within each PT and LNM was assessed by calculating Gini Index. Percentage of ALDH1-positive cells was elevated in PT in comparison to LNM (P = .005). However, heterogeneity of the four CSCs markers: ALDH1 (P = .019), CD133 (P = .009), OCT-4 (P = .027), and CD44 (P < .001) was decreased in LNM. Samples classified as mesenchymal (post-EMT) showed elevated expression of CSCs markers (OCT-4 and CD44 in PT; OCT-4 in LNM; ALDH1, OCT-4, NANOG, CD44 in CTCs). Patients with mesenchymal-like CTCs had worse prognosis than patients with epithelial-like or no CTCs (P = .0025). CSCs markers are enriched in PT, LNM, and CTCs with mesenchymal features, but their heterogeneity is decreased in metastatic lymph nodes. Mesenchymal CTCs phenotype correlates with poor prognosis of the patients.

The amplification of estrogen receptor alpha (ERα) encoded by the ESR1 gene has been described as having a prognostic role in breast cancer patients. However, increased dosage of the ESR1 gene (tested by real-time PCR) is also observed in ER-negative breast cancers, which might suggest the expression of alternative isoforms of ERα (other than classical ERα of 66 kDa). In the current work, we have investigated the ESR1 gene dosage in 402 primary breast cancer patients as well as the expression of ERα isoforms—ERα66 and ERα36—on mRNA and protein levels. The obtained results were correlated with clinicopathological data of the patients. Results showed that increased ESR1 gene dosage is not related to ESR1 gene amplification measured by fluorescent in situ hybridization (FISH), but it correlates with the decreased expression of ERα66 isoform (p = 0.01). Interestingly, the short ER isoform ERα36 was expressed in samples with increased ESR1 gene dosage, suggesting that genomic aberration might influence the expression of that particular isoform. Similarly to ESR1 increased gene dosage, high ERα36 expression was linked with the decreased disease-free survival of the patients (p = 0.05), which was independent of the status of the classical ERα66 level in breast tumors.

Neurofibromatosis Type 2 (NF2)-related schwannomatosis is a genetic disorder that causes development of multiple types of nervous system tumors. The primary and diagnostic tumor type is bilateral vestibular schwannoma. There is no cure or drug therapy for NF2. Recommended treatments include surgical resection and radiation, both of which can leave patients with severe neurological deficits or increase the risk of future malignant tumors. Results of our previous pilot high-throughput drug screen identified phosphoinositide 3-kinase (PI3K) inhibitors as strong candidates based on loss of viability of mouse merlin-deficient Schwann cells (MD-SCs). Here we used novel human schwannoma model cells to conduct combination drug screens. We identified a class I PI3K inhibitor, pictilisib and p21 activated kinase (PAK) inhibitor, PF-3758309 as the top combination due to high synergy in cell viability assays. Both single and combination therapies significantly reduced growth of mouse MD-SCs in an orthotopic allograft mouse model. The inhibitor combination promoted cell cycle arrest and apoptosis in mouse merlin-deficient Schwann (MD-SCs) cells and cell cycle arrest in human MD-SCs. This study identifies the PI3K and PAK pathways as potential targets for combination drug treatment of NF2-related schwannomatosis.

Background: Cancer-associated fibroblasts (CAFs) are the most abundant cell type in the tumor microenvironment (TME). Estrogen receptor alpha 36 (ERα36), the alternatively spliced variant of ERα, is described as an unfavorable factor when expressed in cancer cells. ERα can be expressed also in CAFs; however, the role of ERα36 in CAFs is unknown. Methods: Four CAF cultures were isolated from chemotherapy-naïve BC patients and characterized for ERα36 expression and the NanoString gene expression panel using isolated RNA. Conditioned media from CAF cultures were used to assess the influence of CAFs on triple-negative breast cancer (TNBC) cells using a matrigel 3D culture assay. Results: We found that ERα36^high CAFs significantly induced the branching of TNBC cells in vitro (p < 0.001). They also produced a set of pro-tumorigenic cytokines compared to ERα36^low CAFs, among which hepatocyte growth factor (HGF) was the main inducer of TNBC cell invasive phenotype in vitro (p < 0.001). Tumor stroma rich in ERα36^high CAFs was correlated with high Ki67 expression (p = 0.041) and tumor-associated macrophages markers (CD68 and CD163, p = 0.041 for both). HGF was found to be an unfavorable prognostic factor in TCGA database analysis (p = 0.03 for DFS and p = 0.04 for OS). Conclusions: Breast cancer-associated fibroblasts represent distinct subtypes based on ERα36 expression. We propose that ERα36^high CAFs could account for an unfavorable prognosis for TNBC patients.

Isothiocyanates’ precursors (ITCs), including benzyl isothiocyanate (BITC), are considered as cancer chemopreventive agents. ITC derivatives were tested in clinical trials (NCT00005883, NCT01265953, NCT01790204) and preclinical studies aimed to inhibit tumor growth and modulation of their microenvironment. Although efficacy of ITCs was demonstrated with several leukemic cell lines, the final steps of BITC-induced apoptosis were not completely elucidated in the literature. Therefore, we focused on morphological and biochemical events occurring upon treatment of U937 leukemia cells with BITC. Micromolar concentrations of BITC induced cytotoxicity in U937 cells, with major features resembling the hallmarks of apoptosis: phosphatidylserine exposure, low mitochondrial membrane potential, and presence of PARP cleavage by caspases. Disassembly to apoptotic bodies, a final step of classic apoptosis, was not observed. While tracing the signalling pathways, our results showed increased levels of BAG-1 and PUMA proteins, but in contrast to other models of ITCs-induced apoptosis, downregulation of Mcl-1 protein was not noticed. Additionally, BITC–induced dying U937 cells released lower levels of chemoattractants, such as IL-8 and MCP-1, when compared to cells undergoing classical apoptosis. This may disrupt clearance of cell debris by macrophages in vivo (efferocytosis), and in turn affect the inflammatory response. In summary, BITC inhibits tumor growth which makes it a good candidate for supporting cancer treatment. However, atypical apoptosis of leukemic U937 cells induced with BITC may affect the ability of phagocytes to effectively scavenge cellular debris, which poses a question of BITC effectiveness as a chemopreventive agent for leukemias.