Overcoming retinoic acid resistance in HER2-enriched breast cancers: role of MYC.

HER2-enriched (HER2+) breast cancers express high levels of the growth-promoting HER2 protein. Although these cancers are treated with the HER2-targeted drug, trastuzumab, resistance to treatment is common. Retinoic acid (RA) is an anti-cancer agent that has been successfully used for the treatment of leukemia and holds promise for the treatment of solid cancers, including breast cancer. The HER2 gene is frequently co-amplified with RARA, a key determinant of RA sensitivity in breast cancers. It seems surprising, therefore, that HER2+ breast cancers are refractory to RA treatment. Here, we show that MYC mediates RA resistance by suppressing the expression of cellular retinoic acid binding protein 2 (CRABP2), resulting in RARα inactivation. CRABP2 is an intracellular RA transporter that delivers RA to the nuclear receptor RARα for its activation. Our results indicate that response to RA is enhanced by MYC depletion in HER2+ breast cancer cells and that RA treatment enhances trastuzumab responsiveness. Our findings support the use of RA and trastuzumab for the treatment of subsets of patients with breast cancers that are HER2-RARα co-amplified and have low levels of MYC.

Stationary-to-migratory transition in glioblastoma stem-like cells driven by a fatty acid-binding protein 7-RXRα neurogenic pathway.

BACKGROUND: Glioblastoma (GBM) stem-like cells (GSCs) are crucial drivers of treatment resistance and tumor recurrence. While the concept of "migrating" cancer stem cells was proposed a decade ago, the roles and underlying mechanisms of the heterogeneous populations of GSCs remain poorly defined. METHODS: Cell migration using GBM cell lines and patient-derived GSCs was examined using Transwell inserts and the scratch assay. Single-cell RNA sequencing data analysis were used to map GSC drivers to specific GBM cell populations. Xenografted mice were used to model the role of brain-type fatty acid-binding protein 7 (FABP7) in GBM infiltration and expansion. The mechanism by which FABP7 and its fatty acid ligands promote GSC migration was examined by gel shift and luciferase gene reporter assays. RESULTS: A subpopulation of FABP7-expressing migratory GSCs was identified, with FABP7 upregulating SOX2, a key modulator for GBM stemness and plasticity, and ZEB1, a prominent factor in GBM epithelial-mesenchymal transition and invasiveness. Our data indicate that GSC migration is driven by nuclear FABP7 through activation of RXRα, a nuclear receptor activated by polyunsaturated fatty acids (PUFAs). CONCLUSION: Infiltrative progression in GBM is driven by migratory GSCs through activation of a PUFA-FABP7-RXRα neurogenic pathway.

FABP7 Facilitates Uptake of Docosahexaenoic Acid in Glioblastoma Neural Stem-like Cells.

Glioblastoma (GBM) is an aggressive tumor with a dismal prognosis. Neural stem-like cells contribute to GBM's poor prognosis by driving drug resistance and maintaining cellular heterogeneity. GBM neural stem-like cells express high levels of brain fatty acid-binding protein (FABP7), which binds to polyunsaturated fatty acids (PUFAs) ω-6 arachidonic acid (AA) and ω-3 docosahexaenoic acid (DHA). Similar to brain, GBM tissue is enriched in AA and DHA. However, DHA levels are considerably lower in GBM tissue compared to adult brain. Therefore, it is possible that increasing DHA content in GBM, particularly in neural stem-like cells, might have therapeutic value. Here, we examine the fatty acid composition of patient-derived GBM neural stem-like cells grown as neurosphere cultures. We also investigate the effect of AA and DHA treatment on the fatty acid profiles of GBM neural stem-like cells with or without FABP7 knockdown. We show that DHA treatment increases DHA levels and the DHA:AA ratio in GBM neural stem-like cells, with FABP7 facilitating the DHA uptake. We also found that an increased uptake of DHA inhibits the migration of GBM neural stem-like cells. Our results suggest that increasing DHA content in the GBM microenvironment may reduce the migration/infiltration of FABP7-expressing neural stem-like cancer cells.

Trastuzumab Mechanism of Action; 20 Years of Research to Unravel a Dilemma.

Trastuzumab as a first HER2-targeted therapy for the treatment of HER2-positive breast cancer patients was introduced in 1998. Although trastuzumab has opened a new avenue to treat patients with HER2-positive breast cancer and other types of cancer, some patients are not responsive or become resistant to this treatment. So far, several mechanisms have been suggested for the mode of action of trastuzumab; however, the findings regarding these mechanisms are controversial. In this review, we aimed to provide a detailed insight into the various mechanisms of action of trastuzumab.

Super resolution microscopy reveals DHA-dependent alterations in glioblastoma membrane remodelling and cell migration.

Brain fatty acid binding protein (FABP7; B-FABP) promotes glioblastoma (GBM) cell migration and is associated with tumor infiltration, properties associated with a poor prognosis in GBM patients. FABP7-expressing neural stem-like cells are known to drive tumor migration/infiltration and resistance to treatment. We have previously shown that FABP7's effects on cell migration can be reversed when GBM cells are cultured in medium supplemented with the omega-3 fatty acid, docosahexaenoic acid (DHA). Here, we use super-resolution imaging on patient-derived GBM stem-like cells to examine the importance of FABP7 and its fatty acid ligands in mitigating GBM cell migration. As FABPs are involved in fatty acid transport from membrane to cytosol, we focus on the effect of FABP7 and its ligand DHA on GBM membrane remodeling, as well as FABP7 nanoscale domain formation on GBM membrane. Using quantitative plasma membrane lipid order imaging, we show that FABP7 expression in GBM cells correlates with increased membrane lipid order, with DHA dramatically decreasing lipid order. Using super-resolution stimulated emission depletion (STED) microscopy, we observe non-uniform distribution of FABP7 on the surface of GBM cells, with FABP7 forming punctate nanoscale domains of ∼100 nm in diameter. These nanodomains are particularly enriched at the migrating front of GBM cells. Interestingly, FABP7 nanodomains are disrupted when GBM cells are cultured in DHA-supplemented medium. We demonstrate a tight link between cell migration, a higher membrane lipid order and increased FABP7 nanoscale domains. We propose that DHA-mediated disruption of membrane lipid order and FABP7 nanodomains forms the basis of FABP7/DHA-mediated inhibition of cell migration in GBM.

The FABP12/PPARγ pathway promotes metastatic transformation by inducing epithelial-to-mesenchymal transition and lipid-derived energy production in prostate cancer cells.

Early stage localized prostate cancer (PCa) has an excellent prognosis; however, patient survival drops dramatically when PCa metastasizes. The molecular mechanisms underlying PCa metastasis are complex and remain unclear. Here, we examine the role of a new member of the fatty acid-binding protein (FABP) family, FABP12, in PCa progression. FABP12 is preferentially amplified and/or overexpressed in metastatic compared to primary tumors from both PCa patients and xenograft animal models. We show that FABP12 concurrently triggers metastatic phenotypes (induced epithelial-to-mesenchymal transition (EMT) leading to increased cell motility and invasion) and lipid bioenergetics (increased fatty acid uptake and accumulation, increased ATP production from fatty acid ß-oxidation) in PCa cells, supporting increased reliance on fatty acids for energy production. Mechanistically, we show that FABP12 is a driver of PPARγ activation which, in turn, regulates FABP12's role in lipid metabolism and PCa progression. Our results point to a novel role for a FABP-PPAR pathway in promoting PCa metastasis through induction of EMT and lipid bioenergetics.

Upregulation of Myt1 Promotes Acquired Resistance of Cancer Cells to Wee1 Inhibition.

Adavosertib (also known as AZD1775 or MK1775) is a small-molecule inhibitor of the protein kinase Wee1, with single-agent activity in multiple solid tumors, including sarcoma, glioblastoma, and head and neck cancer. Adavosertib also shows promising results in combination with genotoxic agents such as ionizing radiation or chemotherapy. Previous studies have investigated molecular mechanisms of primary resistance to Wee1 inhibition. Here, we investigated mechanisms of acquired resistance to Wee1 inhibition, focusing on the role of the Wee1-related kinase Myt1. Myt1 and Wee1 kinases were both capable of phosphorylating and inhibiting Cdk1/cyclin B, the key enzymatic complex required for mitosis, demonstrating their functional redundancy. Ectopic activation of Cdk1 induced aberrant mitosis and cell death by mitotic catastrophe. Cancer cells with intrinsic adavosertib resistance had higher levels of Myt1 compared with sensitive cells. Furthermore, cancer cells that acquired resistance following short-term adavosertib treatment had higher levels of Myt1 compared with mock-treated cells. Downregulating Myt1 enhanced ectopic Cdk1 activity and restored sensitivity to adavosertib. These data demonstrate that upregulating Myt1 is a mechanism by which cancer cells acquire resistance to adavosertib. SIGNIFICANCE: Myt1 is a candidate predictive biomarker of acquired resistance to the Wee1 kinase inhibitor adavosertib.

NFIB promotes cell survival by directly suppressing p21 transcription in TP53-mutated triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited treatment options and poor prognosis. There is an urgent need to identify and understand the key factors and signalling pathways driving TNBC tumour progression, relapse, and treatment resistance. In this study, we report that gene copy numbers and expression levels of nuclear factor IB (NFIB), a recently identified oncogene in small cell lung cancer, are preferentially increased in TNBC compared to other breast cancer subtypes. Furthermore, increased levels of NFIB are significantly associated with high tumour grade, poor prognosis, and reduced chemotherapy response. Concurrent TP53 mutations and NFIB overexpression (z-scores > 0) were observed in 77.9% of TNBCs, in contrast to 28.5% in non-TNBCs. Depletion of NFIB in TP53-mutated TNBC cell lines promotes cell death, cell cycle arrest, and enhances sensitivity to docetaxel, a first-line chemotherapeutic drug in breast cancer treatment. Importantly, these alterations in growth properties were accompanied by induction of CDKN1A, the gene encoding p21, a downstream effector of p53. We show that NFIB directly interacts with the CDKN1A promoter in TNBC cells. Furthermore, knockdown of combined p21 and NFIB reverses the docetaxel-induced cell growth inhibition observed upon NFIB knockdown, indicating that NFIB's effect on chemotherapeutic drug response is mediated through p21. Our results indicate that NFIB is an important TNBC factor that drives tumour cell growth and drug resistance, leading to poor clinical outcomes. Thus, targeting NFIB in TP53-mutated TNBC may reverse oncogenic properties associated with mutant p53 by restoring p21 activity. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Prolonged mitotic arrest induced by Wee1 inhibition sensitizes breast cancer cells to paclitaxel.

Wee1 kinase is a crucial negative regulator of Cdk1/cyclin B1 activity and is required for normal entry into and exit from mitosis. Wee1 activity can be chemically inhibited by the small molecule MK-1775, which is currently being tested in phase I/II clinical trials in combination with other anti-cancer drugs. MK-1775 promotes cancer cells to bypass the cell-cycle checkpoints and prematurely enter mitosis. In our study, we show premature mitotic cells that arise from MK-1775 treatment exhibited centromere fragmentation, a morphological feature of mitotic catastrophe that is characterized by centromeres and kinetochore proteins that co-cluster away from the condensed chromosomes. In addition to stimulating early mitotic entry, MK-1775 treatment also delayed mitotic exit. Specifically, cells treated with MK-1775 following release from G1/S or prometaphase arrested in mitosis. MK-1775 induced arrest occurred at metaphase and thus, cells required 12 times longer to transition into anaphase compared to controls. Consistent with an arrest in mitosis, MK-1775 treated prometaphase cells maintained high cyclin B1 and low phospho-tyrosine 15 Cdk1. Importantly, MK-1775 induced mitotic arrest resulted in cell death regardless the of cell-cycle phase prior to treatment suggesting that Wee1 inhibitors are also anti-mitotic agents. We found that paclitaxel enhances MK-1775 mediated cell killing. HeLa and different breast cancer cell lines (T-47D, MCF7, MDA-MB-468 and MDA-MB-231) treated with different concentrations of MK-1775 and low dose paclitaxel exhibited reduced cell survival compared to mono-treatments. Our data highlight a new potential strategy for enhancing MK-1775 mediated cell killing in breast cancer cells.