Introduction: Acquired tamoxifen resistance involves complex signaling events that are not yet fully understood. Successful therapeutic intervention to delay the onset of hormone resistance depends critically on mechanistic elucidation of viable molecular targets associated with hormone resistance. This study was undertaken to investigate the global proteomic alterations in a tamoxifen resistant MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam). Methods: We cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line. A gel-free, quantitative proteomic method was used to identify and quantify the proteome of the resistant cell line. Nano-flow high-performance liquid chromatography coupled to high resolution Fourier transform mass spectrometry was used to analyze fractionated peptide mixtures that were isobarically labeled from the resistant and control cell lysates. Real time quantitative PCR and Western blots were used to verify selected proteomic changes. Lentiviral vector transduction was used to generate MCF-7 cells stably expressing S100P. Online pathway analysis was performed to assess proteomic signatures in tamoxifen resistance. Survival analysis was done to evaluate clinical relevance of altered proteomic expressions. Results: Quantitative proteomic analysis revealed a wide breadth of signaling events during transition to acquired tamoxifen resistance. A total of 629 proteins were found significantly changed with 364 up-regulated and 265 down-regulated. Collectively, these changes demonstrated the suppressed state of estrogen receptor (ER) and ER-regulated genes, activated survival signaling and increased migratory capacity of the resistant cell line. The protein S100P was found to play a critical role in conferring tamoxifen resistance and enhanced cell motility. Conclusions: Our data demonstrate that the adaptive changes in the proteome of tamoxifen resistant breast cancer cells are characterized by down-regulated ER signaling, activation of alternative survival pathways, and enhanced cell motility through regulation of the actin cytoskeleton dynamics. Evidence also emerged that S100P mediates acquired tamoxifen resistance and migration capacity.

The AKT/mammalian target of rapamycin (mTOR) signaling pathway is regulated by 17α-estradiol (E2) signaling and mediates E2-induced proliferation and progesterone receptor (PgR) expression in breast cancer. Here we use deep sequencing analysis of previously published data from The Cancer Genome Atlas to demonstrate that expression of a key component of mTOR signaling, rapamycin-insensitive companion of mTOR (Rictor), positively correlated with an estrogen receptor-α positive (ERα+) breast tumor signature. Through increased microRNA-155 (miR-155) expression in the ERα+ breast cancer cells we demonstrate repression of Rictor enhanced activation of mTOR complex 1 (mTORC1) signaling with both qPCR and western blot. miR-155-mediated mTOR signaling resulted in deregulated ERα signaling both in cultured cells in vitro and in xenografts in vivo in addition to repressed PgR expression and activity. Furthermore we observed that miR-155 enhanced mTORC1 signaling (observed through western blot for increased phosphorylation on mTOR S2448) and induced inhibition of mTORC2 signaling (evident through repressed Rictor and tuberous sclerosis 1 (TSC1) gene expression). mTORC1 induced deregulation of E2 signaling was confirmed using qPCR and the mTORC1-specific inhibitor RAD001. Co-treatment of MCF7 breast cancer cells stably overexpressing miR-155 with RAD001 and E2 restored E2-induced PgR gene expression. RAD001 treatment of SCID/CB17 mice inhibited E2-induced tumorigenesis of the MCF7 miR-155 overexpressing cell line. Finally we demonstrated a strong positive correlation between Rictor and PgR expression and a negative correlation with Raptor expression in Luminal B breast cancer samples, a breast cancer histological subtype known for having an altered ERα-signaling pathway. miRNA mediated alterations in mTOR and ERα signaling establishes a new mechanism for altered estrogen responses independent of growth factor stimulation.

Extracellular matrix (ECM) components are key regulators in breast cancer progression, as ECM remodeling is essential for breast cancer cells to invade into surrounding tissue. This process is characterized by the alignment of fibrillar collagens, breakdown of basement membrane components, and increased interstitial collagen stiffness. In patients with obesity, pre-existing ECM changes, including excessive collagen deposition and heightened matrix stiffness, mimic alterations detected in breast cancer. Given that obesity is a predictor of poor prognosis and resistance to treatment in breast cancer, it is crucial to understand how ECM conditioned by obesity affects disease outcomes. In this review, we highlight known ECM changes that occur with breast cancer and obesity and describe how these changes impact cancer cell metastasis, disease progression, and the breast cancer tumor microenvironment. We examine how obesity driven ECM remodeling affects treatment response and resistance. Further, we discuss how the compounding factor of age contributes to remodeling and current preclinical models of ECM in breast cancer.

Metastatic breast cancer leads to poor prognoses and worse outcomes in patients due to its invasive behavior and poor response to therapy. It is still unclear what biophysical and biochemical factors drive this more aggressive phenotype in metastatic cancer; however recent studies have suggested that exposure to fluid shear stress in the vasculature could cause this. In this study a modular microfluidic platform capable of mimicking the magnitude of fluid shear stress (FSS) found in human vasculature was designed and fabricated. This device provides a platform to evaluate the effects of FSS on MCF-7 cell line, an estrogen receptor positive (ER⁺) breast cancer cell line, during circulation in the vessels. Elucidation of the effects of FSS on MCF-7 cells was carried out utilizing two approaches: single cell analysis and bulk analysis. For single cell analysis, cells were trapped in a microarray after exiting the serpentine channel and followed by immunostaining on the device (on-chip). Bulk analysis was performed after cells were collected in a microtube at the outlet of the microfluidic serpentine channel for western blotting (off-chip). It was found that cells exposed to an FSS magnitude of 10 dyn/cm² with a residence time of 60 s enhanced expression of the proliferation marker Ki67 in the MCF-7 cell line at a single cell level. To understand possible mechanisms for enhanced Ki67 expression, on-chip and off-chip analyses were performed for pro-growth and survival pathways ERK, AKT, and JAK/STAT. Results demonstrated that after shearing the cells phosphorylation of p-AKT, p-mTOR, and p-STAT3 were observed. However, there was no change in p-ERK1/2. AKT is a mediator of ER rapid signaling, analysis of phosphorylated ERα was carried out and no significant differences between sheared and non-sheared populations were observed. Taken together these results demonstrate that FSS can increase phosphorylation of proteins associated with a more aggressive phenotype in circulating cancer cells. These findings provide additional information that may help inform why cancer cells located at metastatic sites are usually more aggressive than primary breast cancer cells.

Lipedema is a connective tissue disorder characterized by increased dilated blood vessels (angiogenesis), inflammation, and fibrosis of the subcutaneous adipose tissue. This project aims to gain insights into the angiogenic processes in lipedema using human umbilical vein endothelial cells (HUVECs) as an in vitro model. HUVECs were cultured in conditioned media (CM) collected from healthy (non-lipedema, AQH) and lipedema adipocytes (AQL). The impacts on the expression levels of multiple endothelial and angiogenic markers [CD31, von Willebrand Factor (vWF), angiopoietin 2 (ANG2), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMPs), NOTCH and its ligands] in HUVECs were investigated. The data demonstrate an increased expression of CD31 and ANG2 at both the gene and protein levels in HUVECs treated with AQL CM in 2D monolayer and 3D cultures compared to untreated cells. Furthermore, the expression of the vWF, NOTCH 4, and DELTA-4 genes decreased. In contrast, increased VEGF, MMP9, and HGF gene expression was detected in HUVECs treated with AQL CM cultured in a 2D monolayer. In addition, the results of a tube formation assay indicate that the number of formed tubes increased in lipedema-treated HUVECs cultured in a 2D monolayer. Together, the data indicate that lipedema adipocyte-CM promotes angiogenesis through paracrine-driven mechanisms.

The Never-in-Mitosis A-Related Kinase (NEK) family is an important, yet largely understudied, family of protein kinases involved in the regulation of a variety of critical cellular processes. Consequently, dysregulation of NEK function has been linked to the etiology and progression of several disorders, including cancer, ciliopathies, neurodegenerative disorders, inflammatory disorders, and other pervasive diseases. In this review, we have summarized recent findings to provide an overview of the NEK family and their diverse functions within various cellular contexts. In parallel, we have highlighted the emerging roles of NEK family members in human health, identifying potential therapeutic targets within the NEK family and exploring their potential for future clinical applications. Finally, we have addressed ongoing challenges and emerging research directions in this rapidly evolving field, aiming to pave the way for future discoveries and innovations.