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Dysregulation of the mitogen-activated protein kinase interacting kinases 1/2 (MNK1/2)-eukaryotic initiation factor 4E (eIF4E) signaling axis promotes breast cancer progression. MNK1 is known to influence cancer stem cells (CSCs); self-renewing populations that support metastasis, recurrence, and chemotherapeutic resistance, making them a clinically relevant target. The precise function of MNK1 in regulating CSCs, however, remains unexplored. Here, we generated MNK1 knockout cancer cell lines, resulting in diminished CSC properties in vitro and slowed tumor growth in vivo. Using a multiomics approach, we functionally demonstrated that loss of MNK1 restricts tumor cell metabolic adaptation by reducing glycolysis and increasing dependence on oxidative phosphorylation. Furthermore, MNK1-null breast and pancreatic tumor cells demonstrated suppressed metastasis to the liver, but not the lung. Analysis of The Cancer Genome Atlas (TCGA) data from breast cancer patients validated the positive correlation between MNK1 and glycolytic enzyme protein expression. This study defines metabolic perturbations as a previously unknown consequence of targeting MNK1/2, which may be therapeutically exploited. [ABSTRACT FROM AUTHOR]

This study explored the application of deep learning in second harmonic generation (SHG) microscopy, a rapidly growing area. This study focuses on the impact of glycerol concentration on image noise in SHG microscopy and compares two image restoration techniques: Noise‐to‐Void 2D (N2V 2D, no reference image restoration) and content‐aware image restoration (CARE 2D, full reference image restoration). We demonstrated that N2V 2D effectively restored the images affected by high glycerol concentrations. To reduce sample exposure and damage, this study further addresses low‐power SHG imaging by reducing the laser power by 70% using deep learning techniques. CARE 2D excels in preserving detailed structures, whereas N2V 2D maintains natural muscle structure. This study highlights the strengths and limitations of these models in specific SHG microscopy applications, offering valuable insights and potential advancements in the field. [ABSTRACT FROM AUTHOR]

Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a 'green' xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory. [ABSTRACT FROM AUTHOR]

4'-phosphopantetheinyl (4'PP) groups are essential co-factors added to target proteins by p hospho p antetheinyl transferase (PPTase) enzymes. Although mitochondrial 4'PP-modified proteins have been described for decades, a mitochondrially-localized PPTase has never been found in mammals. We discovered that the cytoplasmic PPTase a mino a dipate s emialdehyde d ehydrogenase p hospho p antetheinyl t ransferase (AASDHPPT) is required for mitochondrial respiration and oxidative metabolism. Loss of AASDHPPT results in failed 4'-PP modification of the mitochondrial acyl carrier protein and blunted activity of the mitochondrial fatty acid synthesis (mtFAS) pathway. We found that in addition to its cytoplasmic localization, AASDHPPT localizes to the mitochondrial matrix via an N-terminal mitochondrial targeting sequence contained within the first 13 amino acids of the protein. Our data show that this novel mitochondrial localization of AASDHPPT is required to support mtFAS activity and oxidative function. We further identify two variants of uncertain significance in AASDHPPT that are likely pathogenic in humans due to loss of mtFAS activity.

Tumors, their microenvironment, and the mechanisms by which collagen morphology changes throughout cancer progression have recently been a topic of interest. Second harmonic generation (SHG) and polarization second harmonic (P-SHG) microscopy are label-free, hallmark methods that can highlight this alteration in the extracellular matrix (ECM). This article uses automated sample scanning SHG and P-SHG microscopy to investigate ECM deposition associated with tumors residing in the mammary gland. We show two different analysis approaches using the acquired images to distinguish collagen fibrillar orientation changes in the ECM. Lastly, we apply a supervised deep-learning model to classify naïve and tumor-bearing mammary gland SHG images. We benchmark the trained model using transfer learning with the well-known MobileNetV2 architecture. By fine-tuning the different parameters of these models, we show a trained deep-learning model that suits such a small dataset with 73% accuracy., Competing Interests: The authors declare no competing interests., (© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

The extracellular matrix (ECM) is a molecular scaffold mainly comprising fibrous proteins, glycoproteins, proteoglycans, and polysaccharides. Aside from acting as a structural support, the ECM's composition dictates cell-matrix interactions at the biochemical and biophysical level. In the context of cancer, the ECM is a critical component of the tumor microenvironment (TME) and dysregulation of its deposition and remodelling has been shown to promote tumor onset, progression, and metastasis. Here, we describe a robust protocol for the isolation and subsequent proteomic analysis of the ECM of murine mammary glands, for downstream assays studying the role of the ECM in breast cancer. The protocol yields sufficient protein amounts to enable not only the global quantitation of protein expression but furthermore the enrichment and quantitative analysis of post-translationally modified peptides to study aberrant signalling., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The extracellular matrix (ECM) plays critical roles in breast cancer development. Whether ECM composition is regulated by the phosphorylation of eIF4E on serine 209, an event required for tumorigenesis, has not been explored. Herein, we used proteomics and mouse modeling to investigate the impact of mutating serine 209 to alanine on eIF4E (i.e., S209A) on mammary gland (MG) ECM. The proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD028953. We discovered that S209A knock-in mice, expressing a non-phosphorylatable form of eIF4E, have less collagen-I deposition in native and tumor-bearing MGs, leading to altered tumor cell invasion. Additionally, phospho-eIF4E deficiency impacts collagen topology; fibers at the tumor-stroma boundary in phospho-eIF4E-deficient mice run parallel to the tumor edge but radiate outwards in wild-type mice. Finally, a phospho-eIF4E-deficient tumor microenvironment resists anti-PD-1 therapy-induced collagen deposition, correlating with an increased anti-tumor response to immunotherapy. Clinically, we showed that collagen-I and phospho-eIF4E are positively correlated in human breast cancer samples, and that stromal phospho-eIF4E expression is influenced by tumor proximity. Together, our work defines the importance of phosphorylation of eIF4E on S209 as a regulator of MG collagen architecture in the tumor microenvironment, thereby positioning phospho-eIF4E as a therapeutic target to augment response to therapy., Competing Interests: Declaration of Competing Interest CHB is the CSO of MRM Proteomics, Inc. RPZ is the CEO of MRM Proteomics, Inc. All other authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Diffuse large B-cell lymphoma (DLBCL) accounts for 40% of non-Hodgkin lymphoma, and 30% to 40% of patients will succumb to relapsed/refractory disease (rrDLBCL). Patients with rrDLBCL generally have low long-term survival rates due to a lack of efficient salvage therapies. Small-molecule inhibitors targeting the histone methyltransferase EZH2 represent an emerging group of novel therapeutics that show promising clinical efficacy in patients with rrDLBCL. The mechanisms that control acquired resistance to this class of targeted therapies, however, remain poorly understood. Here, we develop a model of resistance to the EZH2 inhibitor (EZH2i) GSK343 and use RNA-seq data and in vitro investigation to show that GCB (germinal center B-cell)-DLBCL cell lines with acquired drug resistance differentiate toward an ABC (activated B-cell)-DLBCL phenotype. We further observe that the development of resistance to GSK343 is sufficient to induce cross-resistance to other EZH2i. Notably, we identify the immune receptor SLAMF7 as upregulated in EZH2i-resistant cells, using chromatin immunoprecipitation profiling to uncover the changes in chromatin landscape remodeling that permit this altered gene expression. Collectively, our data reveal a previously unreported response to the development of EZH2i resistance in DLBCL, while providing strong rationale for pursuing investigation of dual-targeting of EZH2 and SLAMF7 in rrDLBCL., (©2022 American Association for Cancer Research.)

Breast cancer diagnosed within 10 years following childbirth is defined as postpartum breast cancer (PPBC) and is highly metastatic. Interactions between immune cells and other stromal cells within the involuting mammary gland are fundamental in facilitating an aggressive tumor phenotype. The MNK1/2-eIF4E axis promotes translation of prometastatic mRNAs in tumor cells, but its role in modulating the function of nontumor cells in the PPBC microenvironment has not been explored. Here, we used a combination of in vivo PPBC models and in vitro assays to study the effects of inactivation of the MNK1/2-eIF4E axis on the protumor function of select cells of the tumor microenvironment. PPBC mice deficient for phospho-eIF4E (eIF4E S209A ) were protected against lung metastasis and exhibited differences in the tumor and lung immune microenvironment compared with wild-type mice. Moreover, the expression of fibroblast-derived IL33, an alarmin known to induce invasion, was repressed upon MNK1/2-eIF4E axis inhibition. Imaging mass cytometry on PPBC and non-PPBC patient samples indicated that human PPBC contains phospho-eIF4E high-expressing tumor cells and CD8 + T cells displaying markers of an activated dysfunctional phenotype. Finally, inhibition of MNK1/2 combined with anti-PD-1 therapy blocked lung metastasis of PPBC. These findings implicate the involvement of the MNK1/2-eIF4E axis during PPBC metastasis and suggest a promising immunomodulatory route to enhance the efficacy of immunotherapy by blocking phospho-eIF4E. SIGNIFICANCE: This study investigates the MNK1/2-eIF4E signaling axis in tumor and stromal cells in metastatic breast cancer and reveals that MNK1/2 inhibition suppresses metastasis and sensitizes tumors to anti-PD-1 immunotherapy., (©2021 American Association for Cancer Research.)

The mechanisms by which breast cancers progress from relatively indolent ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) are not well understood. However, this process is critical to the acquisition of metastatic potential. MAPK-interacting serine/threonine-protein kinase 1 (MNK1) signaling can promote cell invasion. NODAL, a morphogen essential for embryogenic patterning, is often reexpressed in breast cancer. Here we describe a MNK1/NODAL signaling axis that promotes DCIS progression to IDC. We generated MNK1 knockout (KO) or constitutively active MNK1 (caMNK1)-expressing human MCF-10A-derived DCIS cell lines, which were orthotopically injected into the mammary glands of mice. Loss of MNK1 repressed NODAL expression, inhibited DCIS to IDC conversion, and decreased tumor relapse and metastasis. Conversely, caMNK1 induced NODAL expression and promoted IDC. The MNK1/NODAL axis promoted cancer stem cell properties and invasion in vitro . The MNK1/2 inhibitor SEL201 blocked DCIS progression to invasive disease in vivo . In clinical samples, IDC and DCIS with microinvasion expressed higher levels of phospho-MNK1 and NODAL versus low-grade (invasion-free) DCIS. Cumulatively, our data support further development of MNK1 inhibitors as therapeutics for preventing invasive disease. SIGNIFICANCE: These findings provide new mechanistic insight into progression of ductal carcinoma and support clinical application of MNK1 inhibitors to delay progression of indolent ductal carcinoma in situ to invasive ductal carcinoma., (©2019 American Association for Cancer Research.)