MicroRNA Regulation of Breast Cancer Stemness.

Recent advances in our understanding of breast cancer have demonstrated that cancer stem-like cells (CSCs, also known as tumor-initiating cell (TICs)) are central for progression and recurrence. CSCs are a small subpopulation of cells present in breast tumors that contribute to growth, metastasis, therapy resistance, and recurrence, leading to poor clinical outcome. Data have shown that cancer cells can gain characteristics of CSCs, or stemness, through alterations in key signaling pathways. The dysregulation of miRNA expression and signaling have been well-documented in cancer, and recent studies have shown that miRNAs are associated with breast cancer initiation, progression, and recurrence through regulating CSC characteristics. More specifically, miRNAs directly target central signaling nodes within pathways that can drive the formation, maintenance, and even inhibition of the CSC population. This review aims to summarize these research findings specifically in the context of breast cancer. This review also discusses miRNAs as biomarkers and promising clinical therapeutics, and presents a comprehensive summary of currently validated targets involved in CSC-specific signaling pathways in breast cancer.

Enhanced mitochondrial fission suppresses signaling and metastasis in triple-negative breast cancer.

BACKGROUND: Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC). METHODS: We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer. RESULTS: Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer. CONCLUSIONS: In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.

Integrin α9 depletion promotes ß-catenin degradation to suppress triple-negative breast cancer tumor growth and metastasis.

Although integrin α9 (ITGA9) is known to be involved in cell adhesion and motility, its expression in cancer and its role in tumor growth and metastasis remain largely unknown. Our study was designed to investigate the role of ITGA9 in triple-negative breast cancer (TNBC). ITGA9 expression in TNBC cells was knocked out (KO) using CRISPR/Cas9 technology. Four orthotopic mouse mammary xenograft tumor models coupled with cell culture studies were performed to determine the effect of ITGA9 depletion on TNBC tumor growth and metastasis and the underlying mechanism. Bioinformatics analysis showed that ITGA9 level is significantly higher in TNBC than other breast cancer subtypes, and higher ITGA9 level is associated with significantly worse distant metastasis-free survival and recurrence-free survival in TNBC patients. Experimentally, ITGA9 KO significantly reduced TNBC cell cancer stem cell (CSC)-like property, tumor angiogenesis, tumor growth and metastasis by promoting ß-catenin degradation. Further mechanistic studies revealed that ITGA9 KO causes integrin-linked kinase (ILK) relocation from the membrane region to the cytoplasm, where it interacts with protein kinase A (PKA) and inhibits PKA activity leading to increased activity of glycogen synthase kinase 3 (GSK3) and subsequent ß-catenin degradation. Overexpressing ß-catenin in ITGA9 KO cells reversed the inhibitory effect of ITGA9 KO on tumor growth and metastasis. Furthermore, ITGA9 downregulation in TNBC tumors by nanoparticle-mediated delivery of ITGA9 siRNA drastically decreased tumor angiogenesis, tumor growth and metastasis. These findings indicate that ITGA9 depletion suppresses TNBC tumor growth and metastasis by promoting ß-catenin degradation through the ILK/PKA/GSK3 pathway.

Single-cell RNA-sequencing of migratory breast cancer cells: discovering genes associated with cancer metastasis.

Considerable evidence suggests breast cancer metastasis arises from cells undergoing epithelial-to-mesenchymal-transition (EMT) and cancer stem-like cells (CSCs). Using a microfluidic device that enriches migratory breast cancer cells with enhanced capacity for tumor formation and metastasis, we identified genes differentially expressed in migratory cells by high-throughput single-cell RNA-sequencing. Migratory cells exhibited overall signatures of EMT and CSCs with variable expression of marker genes, and they retained expression profiles of EMT over time. With single-cell resolution, we discovered intermediate EMT states and distinct epithelial and mesenchymal sub-populations of migratory cells, indicating breast cancer cells can migrate rapidly while retaining an epithelial state. Migratory cells showed differential profiles for regulators of oxidative stress, mitochondrial morphology, and the proteasome, revealing potential vulnerabilities and unexpected consequences of drugs. We also identified novel genes correlated with cell migration and outcomes in breast cancer as potential prognostic biomarkers and therapeutic targets to block migratory cells in metastasis.

Upregulation of histone-lysine methyltransferases plays a causal role in hexavalent chromium-induced cancer stem cell-like property and cell transformation.

While hexavalent chromium [Cr(VI)] is generally considered as a genotoxic environmental carcinogen, studies showed that Cr(VI) exposure also causes epigenetic changes. However, whether Cr(VI)-caused epigenetic dysregulations plays an important role in Cr(VI) carcinogenicity remain largely unknown. The aim of this study was to determine if chronic low dose Cr(VI) exposure causes epigenetic changes, the underlying mechanism and whether chronic low dose Cr(VI) exposure-caused epigenetic dysregulation contributes causally to Cr(VI)-induced cancer stem cell (CSC)-like property and cell transformation. Two immortalized human bronchial epithelial cell lines (BEAS-2B and 16HBE) were exposed to 0.25 µM of K2Cr2O7 for 20 and 40 weeks to induce cell transformation, respectively. Cr(VI)-induced epigenetic changes were examined in Cr(VI)-transformed cells and Cr(VI) exposure-caused human lung cancer tissues. Pharmacological inhibitors and gene knockdown experiments were used to determine the role of epigenetic dysregulation in Cr(VI) carcinogenicity. We found that chronic Cr(VI) exposure causes epigenetic dysregulation as evidenced by the increased levels of histone H3 repressive methylation marks (H3K9me2 and H3K27me3) and the related histone-lysing methyltransferases (HMTases). Pharmacological inhibition or knockdown of HMTases reduces H3 repressive methylation marks and malignant phenotypes of Cr(VI)-transformed cells. Moreover, knockdown of HMTases in parental cells significantly reduces chronic Cr(VI) exposure-induced CSC-like property and cell transformation. Further mechanistic study revealed that knockdown of HMTases decreases Cr(VI) exposure-caused DNA damage. Our findings indicate that chronic Cr(VI) exposure increases H3 repressive methylation marks by increasing the related HMTases expression; and that increased expression of HMTases plays a causal role in Cr(VI)-induced CSC-like property and cell transformation.

MicroRNA-200b suppresses arsenic-transformed cell migration by targeting protein kinase Cα and Wnt5b-protein kinase Cα positive feedback loop and inhibiting Rac1 activation.

MicroRNA-200b (miR-200b) is a member of miR-200 family that has been found to inhibit cell migration and cancer metastasis; however, the underlying mechanism is not well understood. We previously reported that miR-200 expression is depleted in arsenic-transformed human bronchial epithelial cells with highly migratory and invasive characteristics, whereas stably re-expressing miR-200b strongly suppresses arsenic-transformed cell migration. This study was performed to investigate how miR-200b inhibits arsenic-transformed cell migration. We found that protein kinase Cα (PKCα) is significantly up-regulated in arsenic-transformed cells. Combining bioinformatics analysis with PKCα 3'-untranslated region vector luciferase reporter assays, we showed that PKCα is a direct target of miR-200b. Inhibiting PKCα activity or knocking down PKCα expression drastically reduced cell migration, phenocoping the inhibitory effect of overexpressing miR-200b. In contrast, forced expression of PKCα in miR-200b overexpressing cells impaired the inhibitory effect of miR-200b on cell migration. In addition, we also found a positive feedback loop between Wnt5b and PKCα in arsenic-transformed cells. Knocking down Wnt5b expression reduced phospho-PKC levels and cell migration; and knocking down PKCα expression decreased Wnt5b level and cell migration. Moreover, forced expression of PKCα increased Wnt5b and phospho-PKC levels and cell migration. Further mechanistic studies revealed that Rac1 is highly activated in arsenic-transformed cells and stably expressing miR-200b abolishes Rac1 activation changing actin cytoskeleton organization. Manipulating PKCα or Wnt5b expression levels significantly altered the level of active Rac1. Together, these findings indicate that miR-200b suppresses arsenic-transformed cell migration by targeting PKCα and Wnt5b-PKCα positive feedback loop and subsequently inhibiting Rac1 activation.

MicroRNA-200b targets protein kinase Cα and suppresses triple-negative breast cancer metastasis.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis and lacks effective targeted therapies. The microRNA-200 (miR-200) family is found to inhibit or promote breast cancer metastasis; however, the underlying mechanism is not well understood. This study was performed to investigate the effect and mechanism of miR-200b on TNBC metastasis and identify targets for developing more efficient treatment for TNBC. We found that miR-200 family expression levels are significantly lower in highly migratory TNBC cells and metastatic TNBC tumors than other types of breast cancer cells and tumors. Ectopically expressing a single member (miR-200b) of the miR-200 family drastically reduces TNBC cell migration and inhibits tumor metastasis in an orthotopic mouse mammary xenograft tumor model. We identified protein kinase Cα (PKCα) as a new direct target of miR-200b and found that PKCα protein levels are inversely correlated with miR-200b levels in 12 kinds of breast cancer cells. Inhibiting PKCα activity or knocking down PKCα levels significantly reduces TNBC cell migration. In contrast, forced expression of PKCα impairs the inhibitory effect of miR-200b on cell migration and tumor metastasis. Further mechanistic studies revealed that PKCα downregulation by miR-200b results in a significant decrease of Rac1 activation in TNBC cells. These results show that loss of miR-200b expression plays a crucial role in TNBC aggressiveness and that miR-200b suppresses TNBC cell migration and tumor metastasis by targeting PKCα. Our findings suggest that miR-200b and PKCα may serve as promising therapeutic targets for metastatic TNBC.

Spermatozoon ultrastructure of hangingflies, Bittacus strigosus and Bittacus stigmaterus.

In the present study, spermatozoon ultrastructure was documented in two species of hangingflies, Bittacus strigosus Hagen (Mecoptera: Bittacidae) and B. stigmaterus Say. Structures considered important to phylogenetic assessment that were observed in B. strigosus and B. stigmaterus included a short bilayered acrosome, elongated nucleus, tube-like glycocalyx, centriole adjunct material, accessory bodies, two mitochondrial derivatives, extra axonemal rods, globular units, and 9+2 arrangement of microtubules in the axoneme. Comparisons were made to Bittacus planus Cheng, which was previously examined by electron microscopy (Xie and Hua 2010). Similarities among the ultrastructural characteristics of the three Bittacus species support the monophyly of this genus. Displacement of a mitochondrial derivative by an accessory body was documented for the first time. This paper includes clarifications on differences between accessory bodies and extra axonemal rods, which are issues important to phylogenetic placement.

Characterizing heterogeneous single-cell dose responses computationally and experimentally using threshold inhibition surfaces and dose-titration assays.

Single cancer cells within a tumor exhibit variable levels of resistance to drugs, ultimately leading to treatment failures. While tumor heterogeneity is recognized as a major obstacle to cancer therapy, standard dose-response measurements for the potency of targeted kinase inhibitors aggregate populations of cells, obscuring intercellular variations in responses. In this work, we develop an analytical and experimental framework to quantify and model dose responses of individual cancer cells to drugs. We first explore the connection between population and single-cell dose responses using a computational model, revealing that multiple heterogeneous populations can yield nearly identical population dose responses. We demonstrate that a single-cell analysis method, which we term a threshold inhibition surface, can differentiate among these populations. To demonstrate the applicability of this method, we develop a dose-titration assay to measure dose responses in single cells. We apply this assay to breast cancer cells responding to phosphatidylinositol-3-kinase inhibition (PI3Ki), using clinically relevant PI3Kis on breast cancer cell lines expressing fluorescent biosensors for kinase activity. We demonstrate that MCF-7 breast cancer cells exhibit heterogeneous dose responses with some cells requiring over ten-fold higher concentrations than the population average to achieve inhibition. Our work reimagines dose-response relationships for cancer drugs in an emerging paradigm of single-cell tumor heterogeneity.

Epithelial to mesenchymal transition in arsenic-transformed cells promotes angiogenesis through activating ß-catenin-vascular endothelial growth factor pathway.

Arsenic exposure represents a major health concern increasing cancer risks, yet the mechanism of arsenic carcinogenesis has not been elucidated. We and others recently reported that cell malignant transformation by arsenic is accompanied by epithelial to mesenchymal transition (EMT). However, the role of EMT in arsenic carcinogenesis is not well understood. Although previous studies showed that short term exposure of endothelial cells to arsenic stimulated angiogenesis, it remains to be determined whether cells that were malignantly transformed by long term arsenic exposure have a pro-angiogenic effect. The objective of this study was to investigate the effect of arsenic-transformed human bronchial epithelial cells that underwent EMT on angiogenesis and the underlying mechanism. It was found that the conditioned medium from arsenic-transformed cells strongly stimulated tube formation by human umbilical vein endothelial cells (HUVECs). Moreover, enhanced angiogenesis was detected in mouse xenograft tumor tissues resulting from inoculation of arsenic-transformed cells. Mechanistic studies revealed that ß-catenin was activated in arsenic-transformed cells up-regulating its target gene expression including angiogenic-stimulating vascular endothelial growth factor (VEGF). Stably expressing microRNA-200b in arsenic-transformed cells that reversed EMT inhibited ß-catenin activation, decreased VEGF expression and reduced tube formation by HUVECs. SiRNA knockdown ß-catenin decreased VEGF expression. Adding a VEGF neutralizing antibody into the conditioned medium from arsenic-transformed cells impaired tube formation by HUVECs. Reverse transcriptase-PCR analysis revealed that the mRNA levels of canonical Wnt ligands were not increased in arsenic-transformed cells. These findings suggest that EMT in arsenic-transformed cells promotes angiogenesis through activating ß-catenin-VEGF pathway.

Enhanced mitochondrial fission inhibits triple-negative breast cancer cell migration through an ROS-dependent mechanism.

Mitochondria produce reactive oxygen species (ROS), which function in signal transduction. Mitochondrial dynamics, encompassing morphological shifts between fission and fusion, can directly impact ROS levels in cancer cells. In this study, we identified an ROS-dependent mechanism for how enhanced mitochondrial fission inhibits triple negative breast cancer (TNBC) cell migration. We found that enforcing mitochondrial fission in TNBC resulted in an increase in intracellular ROS levels and reduced cell migration and the formation of actin-rich migratory structures. Consistent with mitochondrial fission, increasing ROS levels in cells inhibited cell migration. Conversely, reducing ROS levels with either a global or mitochondrially targeted scavenger overcame the inhibitory effects of mitochondrial fission. Mechanistically, we found that the ROS sensitive SHP-1/2 phosphatases partially regulate inhibitory effects of mitochondrial fission on TNBC migration. Overall, our work reveals the inhibitory effects of ROS in TNBC and supports mitochondrial dynamics as a potential therapeutic target for cancer.

Bone Marrow Mesenchymal Stem Cells Induce Metabolic Plasticity in Estrogen Receptor-Positive Breast Cancer.

Cancer cells reprogram energy metabolism through metabolic plasticity, adapting ATP-generating pathways in response to treatment or microenvironmental changes. Such adaptations enable cancer cells to resist standard therapy. We employed a coculture model of estrogen receptor-positive (ER+) breast cancer and mesenchymal stem cells (MSC) to model interactions of cancer cells with stromal microenvironments. Using single-cell endogenous and engineered biosensors for cellular metabolism, coculture with MSCs increased oxidative phosphorylation, intracellular ATP, and resistance of cancer cells to standard therapies. Cocultured cancer cells had increased MCT4, a lactate transporter, and were sensitive to the MCT1/4 inhibitor syrosingopine. Combining syrosingopine with fulvestrant, a selective estrogen receptor degrading drug, overcame resistance of ER+ breast cancer cells in coculture with MSCs. Treatment with antiestrogenic therapy increased metabolic plasticity and maintained intracellular ATP levels, while MCT1/4 inhibition successfully limited metabolic transitions and decreased ATP levels. Furthermore, MCT1/4 inhibition decreased heterogenous metabolic treatment responses versus antiestrogenic therapy. These data establish MSCs as a mediator of cancer cell metabolic plasticity and suggest metabolic interventions as a promising strategy to treat ER+ breast cancer and overcome resistance to standard clinical therapies. IMPLICATIONS: This study reveals how MSCs reprogram metabolism of ER+ breast cancer cells and point to MCT4 as potential therapeutic target to overcome resistance to antiestrogen drugs.

Fiber density and matrix stiffness modulate distinct cell migration modes in a 3D stroma mimetic composite hydrogel.

The peritumoral stroma is a complex 3D tissue that provides cells with myriad biophysical and biochemical cues. Histologic observations suggest that during metastatic spread of carcinomas, these cues influence transformed epithelial cells, prompting a diversity of migration modes spanning single cell and multicellular phenotypes. Purported consequences of these variations in tumor escape strategies include differential metastatic capability and therapy resistance. Therefore, understanding how cues from the peritumoral stromal microenvironment regulate migration mode has both prognostic and therapeutic value. Here, we utilize a synthetic stromal mimetic in which matrix fiber density and bulk hydrogel mechanics can be orthogonally tuned to investigate the contribution of these two key matrix attributes on MCF10A migration mode phenotypes, epithelial-mesenchymal transition (EMT), and invasive potential. We develop an automated computational image analysis framework to extract migratory phenotypes from fluorescent images and determine 3D migration metrics relevant to metastatic spread. Using this analysis, we find that matrix fiber density and bulk hydrogel mechanics distinctly contribute to a variety of MCF10A migration modes including amoeboid, single mesenchymal, clusters, and strands. We identify combinations of physical and soluble cues that induce a variety of migration modes originating from the same MCF10A spheroid and use these settings to examine a functional consequence of migration mode -resistance to apoptosis. We find that cells migrating as strands are more resistant to staurosporine-induced apoptosis than either disconnected clusters or individual invading cells. Improved models of the peritumoral stromal microenvironment and understanding of the relationships between matrix attributes and cell migration mode can aid ongoing efforts to identify effective cancer therapeutics that address cell plasticity-based therapy resistances. STATEMENT OF SIGNIFICANCE: Stromal extracellular matrix structure dictates both cell homeostasis and activation towards migratory phenotypes. However decoupling the effects of myriad biophysical cues has been difficult to achieve. Here, we encapsulate electrospun fiber segments within an amorphous hydrogel to create a fiber-reinforced hydrogel composite in which fiber density and hydrogel stiffness can be orthogonally tuned. Quantification of 3D cell migration reveal these two parameters uniquely contribute to a diversity of migration phenotypes spanning amoeboid, single mesenchymal, multicellular cluster, and collective strand. By tuning biophysical and biochemical cues to elicit heterogeneous migration phenotypes, we find that collective strands best resist apoptosis. This work establishes a composite approach to modulate fibrous topography and bulk hydrogel mechanics and identified biomaterial parameters to direct distinct 3D cell migration phenotypes.

Root-localized phytochrome chromophore synthesis is required for photoregulation of root elongation and impacts root sensitivity to jasmonic acid in Arabidopsis.

Plants exhibit organ- and tissue-specific light responses. To explore the molecular basis of spatial-specific phytochrome-regulated responses, a transgenic approach for regulating the synthesis and accumulation of the phytochrome chromophore phytochromobilin (PΦB) was employed. In prior experiments, transgenic expression of the BILIVERDIN REDUCTASE (BVR) gene was used to metabolically inactivate biliverdin IXα, a key precursor in the biosynthesis of PΦB, and thereby render cells accumulating BVR phytochrome deficient. Here, we report analyses of transgenic Arabidopsis (Arabidopsis thaliana) lines with distinct patterns of BVR accumulation dependent upon constitutive or tissue-specific, promoter-driven BVR expression that have resulted in insights on a correlation between root-localized BVR accumulation and photoregulation of root elongation. Plants with BVR accumulation in roots and a PΦB-deficient elongated hypocotyl2 (hy2-1) mutant exhibit roots that are longer than those of wild-type plants under white illumination. Additional analyses of a line with root-specific BVR accumulation generated using a GAL4-dependent bipartite enhancer-trap system confirmed that PΦB or phytochromes localized in roots directly impact light-dependent root elongation under white, blue, and red illumination. Additionally, roots of plants with constitutive plastid-localized or root-specific cytosolic BVR accumulation, as well as phytochrome chromophore-deficient hy1-1 and hy2-1 mutants, exhibit reduced sensitivity to the plant hormone jasmonic acid (JA) in JA-dependent root inhibition assays, similar to the response observed for the JA-insensitive mutants jar1 and myc2. Our analyses of lines with root-localized phytochrome deficiency or root-specific phytochrome depletion have provided novel insights into the roles of root-specific PΦB, or phytochromes themselves, in the photoregulation of root development and root sensitivity to JA.

Ultrasound-Induced Mechanical Compaction in Acoustically Responsive Scaffolds Promotes Spatiotemporally Modulated Signaling in Triple Negative Breast Cancer.

Cancer cells continually sense and respond to mechanical cues from the extracellular matrix (ECM). Interaction with the ECM can alter intracellular signaling cascades, leading to changes in processes that promote cancer cell growth, migration, and survival. The present study used a recently developed composite hydrogel composed of a fibrin matrix and phase-shift emulsion, termed an acoustically responsive scaffold (ARS), to investigate effects of local mechanical properties on breast cancer cell signaling. Treatment of ARSs with focused ultrasound drives acoustic droplet vaporization (ADV) in a spatiotemporally controlled manner, inducing local compaction and stiffening of the fibrin matrix adjacent to the matrix-bubble interface. Combining ARSs and live single cell imaging of triple-negative breast cancer cells, it is discovered that both basal and growth-factor stimulated activities of protein kinase B (also known as Akt) and extracellular signal-regulated kinase (ERK), two major kinases driving cancer progression, negatively correlate with increasing distance from the ADV-induced bubble both in vitro and in a mouse model. Together, these data demonstrate that local changes in ECM compaction regulate Akt and ERK signaling in breast cancer and support further applications of the novel ARS technology to analyze spatial and temporal effects of ECM mechanics on cell signaling and cancer biology.

Effects of iron modulation on mesenchymal stem cell-induced drug resistance in estrogen receptor-positive breast cancer.

Patients with estrogen receptor-positive (ER+) breast cancer, the most common subtype, remain at risk for lethal metastatic disease years after diagnosis. Recurrence arises partly because tumor cells in bone marrow become resistant to estrogen-targeted therapy. Here, we utilized a co-culture model of bone marrow mesenchymal stem cells (MSCs) and ER+ breast cancer cells to recapitulate interactions of cancer cells in bone marrow niches. ER+ breast cancer cells in direct contact with MSCs acquire cancer stem-like (CSC) phenotypes with increased resistance to standard antiestrogenic drugs. We confirmed that co-culture with MSCs increased labile iron in breast cancer cells, a phenotype associated with CSCs and disease progression. Clinically approved iron chelators and in-house lysosomal iron-targeting compounds restored sensitivity to antiestrogenic therapy. These findings establish iron modulation as a mechanism to reverse MSC-induced drug resistance and suggest iron modulation in combination with estrogen-targeted therapy as a promising, translatable strategy to treat ER+ breast cancer.

Multiparametric MRI to quantify disease and treatment response in mice with myeloproliferative neoplasms.

Histopathology, the standard method to assess BM in hematologic malignancies such as myeloproliferative neoplasms (MPNs), suffers from notable limitations in both research and clinical settings. BM biopsies in patients fail to detect disease heterogeneity, may yield a nondiagnostic sample, and cannot be repeated frequently in clinical oncology. Endpoint histopathology precludes monitoring disease progression and response to therapy in the same mouse over time, missing likely variations among mice. To overcome these shortcomings, we used MRI to measure changes in cellularity, macromolecular constituents, and fat versus hematopoietic cells in BM using diffusion-weighted imaging (DWI), magnetization transfer, and chemical shift-encoded fat imaging. Combining metrics from these imaging parameters revealed dynamic alterations in BM following myeloablative radiation and transplantation. In a mouse MPLW515L BM transplant model of MPN, MRI detected effects of a JAK2 inhibitor, ruxolitinib, within 5 days of initiating treatment and identified differing kinetics of treatment responses in subregions of the tibia. Histopathology validated the MRI results for BM composition and heterogeneity. Anatomic MRI scans also showed reductions in spleen volume during treatment. These findings establish an innovative, clinically translatable MRI approach to quantify spatial and temporal changes in BM in MPN.

Overstretched and overlooked: solving challenges faced by early-career investigators after the pandemic.

The coronavirus disease 2019 (COVID-19) pandemic has had a detrimental effect on research. However, little has been done to identify and solve the unique challenges faced by early career investigators (ECIs). As a group of American Cancer Society-funded ECIs, we provide recommendations for solving these challenges in the aftermath of the pandemic.

Pre-existing Cell States Control Heterogeneity of Both EGFR and CXCR4 Signaling.

INTRODUCTION: CXCR4 and epidermal growth factor receptor (EGFR) represent two major families of receptors, G-protein coupled receptors and receptor tyrosine kinases, with central functions in cancer. While utilizing different upstream signaling molecules, both CXCR4 and EGFR activate kinases ERK and Akt, although single-cell activation of these kinases is markedly heterogeneous. One hypothesis regarding the origin of signaling heterogeneity proposes that intercellular variations arise from differences in pre-existing intracellular states set by extrinsic noise. While pre-existing cell states vary among cells, each pre-existing state defines deterministic signaling outputs to downstream effectors. Understanding causes of signaling heterogeneity will inform treatment of cancers with drugs targeting drivers of oncogenic signaling. METHODS: We built a single-cell computational model to predict Akt and ERK responses to CXCR4- and EGFR-mediated stimulation. We investigated signaling heterogeneity through these receptors and tested model predictions using quantitative, live-cell time-lapse imaging. RESULTS: We show that the pre-existing cell state predicts single-cell signaling through both CXCR4 and EGFR. Computational modeling reveals that the same set of pre-existing cell states explains signaling heterogeneity through both EGFR and CXCR4 at multiple doses of ligands and in two different breast cancer cell lines. The model also predicts how phosphatidylinositol-3-kinase (PI3K) targeted therapies potentiate ERK signaling in certain breast cancer cells and that low level, combined inhibition of MEK and PI3K ablates potentiated ERK signaling. CONCLUSIONS: Our data demonstrate that a conserved motif exists for EGFR and CXCR4 signaling and suggest potential clinical utility of the computational model to optimize therapy.

MicroRNA Regulation of the Small Rho GTPase Regulators-Complexities and Opportunities in Targeting Cancer Metastasis.

The small Rho GTPases regulate important cellular processes that affect cancer metastasis, such as cell survival and proliferation, actin dynamics, adhesion, migration, invasion and transcriptional activation. The Rho GTPases function as molecular switches cycling between an active GTP-bound and inactive guanosine diphosphate (GDP)-bound conformation. It is known that Rho GTPase activities are mainly regulated by guanine nucleotide exchange factors (RhoGEFs), GTPase-activating proteins (RhoGAPs), GDP dissociation inhibitors (RhoGDIs) and guanine nucleotide exchange modifiers (GEMs). These Rho GTPase regulators are often dysregulated in cancer; however, the underlying mechanisms are not well understood. MicroRNAs (miRNAs), a large family of small non-coding RNAs that negatively regulate protein-coding gene expression, have been shown to play important roles in cancer metastasis. Recent studies showed that miRNAs are capable of directly targeting RhoGAPs, RhoGEFs, and RhoGDIs, and regulate the activities of Rho GTPases. This not only provides new evidence for the critical role of miRNA dysregulation in cancer metastasis, it also reveals novel mechanisms for Rho GTPase regulation. This review summarizes recent exciting findings showing that miRNAs play important roles in regulating Rho GTPase regulators (RhoGEFs, RhoGAPs, RhoGDIs), thus affecting Rho GTPase activities and cancer metastasis. The potential opportunities and challenges for targeting miRNAs and Rho GTPase regulators in treating cancer metastasis are also discussed. A comprehensive list of the currently validated miRNA-targeting of small Rho GTPase regulators is presented as a reference resource.