Faculty Bibliography

BACKGROUND:Patients with breast cancer exhibit muscle weakness, which is associated with increased mortality risk and reduced quality of life. Muscle weakness is experienced even in the absence of loss of muscle mass in breast cancer patients, indicating intrinsic muscle dysfunction. Physical activity is correlated with reduced cancer mortality and disease recurrence. However, the molecular processes underlying breast cancer-induced muscle weakness and the beneficial effect of exercise are largely unknown. METHODS:Eight-week-old breast cancer (MMTV-PyMT, PyMT) and control (WT) mice had access to active or inactive in-cage voluntary running wheels for 4 weeks. Mice were also subjected to a treadmill test. Muscle force was measured ex vivo. Tumour markers were determined with immunohistochemistry. Mitochondrial biogenesis and function were assessed with transcriptional analyses of PGC-1α, the electron transport chain (ETC) and antioxidants superoxide dismutase (Sod) and catalase (Cat), combined with activity measurements of SOD, citrate synthase (CS) and β-hydroxyacyl-CoA-dehydrogenase (βHAD). Serum and intramuscular stress levels were evaluated by enzymatic assays, immunoblotting, and transcriptional analyses of, for example, tumour necrosis factor-α (TNF-α) and p38 mitogen-activated protein kinase (MAPK) signalling. RESULTS:PyMT mice endured shorter time and distance during the treadmill test (~30%, P < 0.05) and ex vivo force measurements revealed ~25% weaker slow-twitch soleus muscle (P < 0.001). This was independent of cancer-induced alteration of muscle size or fibre type. Inflammatory stressors in serum and muscle, including TNF-α and p38 MAPK, were higher in PyMT than in WT mice (P < 0.05). Cancer-induced decreases in ETC (P < 0.05, P < 0.01) and antioxidant gene expression were observed (P < 0.05). The exercise intervention counteracted the cancer-induced muscle weakness and was accompanied by a less aggressive, differentiated tumour phenotype, determined by increased CK8 and reduced CK14 expression (P < 0.05). In PyMT mice, the exercise intervention led to higher CS activity (P = 0.23), enhanced β-HAD and SOD activities (P < 0.05), and reduced levels of intramuscular stressors together with a normalization of the expression signature of TNFα-targets and ETC genes (P < 0.05, P < 0.01). At the same time, the exercise-induced PGC-1α expression, and CS and β-HAD activity was blunted in muscle from the PyMT mice as compared with WT mice, indicative that breast cancer interfere with transcriptional programming of mitochondria and that the molecular adaptation to exercise differs between healthy mice and those afflicted by disease. CONCLUSIONS:Four-week voluntary wheel running counteracted muscle weakness in PyMT mice which was accompanied by reduced intrinsic stress and improved mitochondrial and antioxidant profiles and activities that aligned with muscles of healthy mice.

Despite the significant advancements made in targeted anti-cancer therapy, drug resistance constitutes a multifaceted phenomenon leading to therapy failure and ultimately mortality. Emerging experimental evidence highlight a role of cholesterol metabolism in facilitating drug resistance in cancer. This review aims to describe the role of cholesterol in facilitating multi-drug resistance in cancer. We focus on specific signaling pathways that contribute to drug resistance and the link between these pathways and cholesterol. Additionally, we briefly discuss the molecular mechanisms related to the epithelial-mesenchymal transition (EMT), and the documented link between EMT, metastasis and drug resistance. We illustrate this by specifically focusing on hypoxia and the role it plays in influencing cellular cholesterol content following EMT induction. Finally, we provide a proposed model delineating the crucial role of cholesterol in EMT and discuss whether targeting cholesterol could serve as a novel means of combatting drug resistance in cancer progression and metastasis.

Children suffering from neurologic cancers undergoing chemotherapy and radiotherapy are at high risk of reduced neurocognitive abilities likely via damage to proliferating neural stem cells (NSC). Therefore, strategies to protect NSCs are needed. We argue that induced cell-cycle arrest/quiescence in NSCs during cancer treatment can represent such a strategy. Here, we show that hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are dynamically expressed over the cell cycle in NSCs, depolarize the membrane potential, underlie spontaneous calcium oscillations and are required to maintain NSCs in the actively proliferating pool. Hyperpolarizing pharmacologic inhibition of HCN channels during exposure to ionizing radiation protects NSCs cells in neurogenic brain regions of young mice. In contrast, brain tumor-initiating cells, which also express HCN channels, remain proliferative during HCN inhibition. IMPLICATIONS: Our finding that NSCs can be selectively rescued while cancer cells remain sensitive to the treatment, provide a foundation for reduction of cognitive impairment in children with neurologic cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

SIGNIFICANCE:Machine learning is increasingly being applied to the classification of microscopic data. In order to detect some complex and dynamic cellular processes, time-resolved live-cell imaging might be necessary. Incorporating the temporal information into the classification process may allow for a better and more specific classification. AIM:We propose a methodology for cell classification based on the time-lapse quantitative phase images (QPIs) gained by digital holographic microscopy (DHM) with the goal of increasing performance of classification of dynamic cellular processes. APPROACH:The methodology was demonstrated by studying epithelial-mesenchymal transition (EMT) which entails major and distinct time-dependent morphological changes. The time-lapse QPIs of EMT were obtained over a 48-h period and specific novel features representing the dynamic cell behavior were extracted. The two distinct end-state phenotypes were classified by several supervised machine learning algorithms and the results were compared with the classification performed on single-time-point images. RESULTS:In comparison to the single-time-point approach, our data suggest the incorporation of temporal information into the classification of cell phenotypes during EMT improves performance by nearly 9% in terms of accuracy, and further indicate the potential of DHM to monitor cellular morphological changes. CONCLUSIONS:Proposed approach based on the time-lapse images gained by DHM could improve the monitoring of live cell behavior in an automated fashion and could be further developed into a tool for high-throughput automated analysis of unique cell behavior.

Non-communicable diseases (NCDs) are medical conditions that, by definition, are non-infectious and non-transmissible among people. Much of current NCDs are generally due to genetic, behavioral, and metabolic risk factors that often include excessive alcohol consumption, smoking, obesity, and untreated elevated blood pressure, and share many common signal transduction pathways. Alterations in cell and physiological signaling and transcriptional control pathways have been well studied in several human NCDs, but these same pathways also regulate expression and function of the protein synthetic machinery and mRNA translation which have been less well investigated. Alterations in expression of specific translation factors, and disruption of canonical mRNA translational regulation, both contribute to the pathology of many NCDs. The two most common pathological alterations that contribute to NCDs discussed in this review will be the regulation of eukaryotic initiation factor 2 (eIF2) by the integrated stress response (ISR) and the mammalian target of rapamycin complex 1 (mTORC1) pathways. Both pathways integrally connect mRNA translation activity to external and internal physiological stimuli. Here, we review the role of ISR control of eIF2 activity and mTORC1 control of cap-mediated mRNA translation in some common NCDs, including Alzheimer's disease, Parkinson's disease, stroke, diabetes mellitus, liver cirrhosis, chronic obstructive pulmonary disease (COPD), and cardiac diseases. Our goal is to provide insights that further the understanding as to the important role of translational regulation in the pathogenesis of these diseases.

The kinase mTOR complex 1 (mTORC1) promotes cellular growth and is frequently dysregulated in cancers. In response to nutrients, mTORC1 is activated on lysosomes by Rag and Rheb guanosine triphosphatases (GTPases) and drives biosynthetic processes. How limitations in nutrients suppress mTORC1 activity remains poorly understood. We find that when amino acids are limited, the Rap1-GTPases confine lysosomes to the perinuclear region and reduce lysosome abundance, which suppresses mTORC1 signaling. Rap1 activation, which is independent of known amino acid signaling factors, limits the lysosomal surface available for mTORC1 activation. Conversely, Rap1 depletion expands the lysosome population, which markedly increases association between mTORC1 and its lysosome-borne activators, leading to mTORC1 hyperactivity. Taken together, we establish Rap1 as a critical coordinator of the lysosomal system, and propose that aberrant changes in lysosomal surface availability can impact mTORC1 signaling output.

Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ERα) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.

Tight junctions (TJ) act as hubs for intracellular signaling pathways controlling epithelial cell fate and function. Deregulation of TJ is a hallmark of epithelial-mesenchymal transition (EMT), which contributes to carcinoma progression and metastasis. However, the signaling mechanisms linking TJ to the induction of EMT are not understood. Here, we identify a TJ-based signalosome, which controls AKT signaling and EMT in breast cancer. The coxsackie and adenovirus receptor (CXADR), a TJ protein with an essential yet uncharacterized role in organogenesis and tissue homeostasis, was identified as a key component of the signalosome. CXADR regulated the stability and function of the phosphatases and AKT inhibitors PTEN and PHLPP2. Loss of CXADR led to hyperactivation of AKT and sensitized cells to TGFβ1-induced EMT. Conversely, restoration of CXADR stabilized PHLPP2 and PTEN, inhibited AKT, and promoted epithelial differentiation. Loss of CXADR in luminal A breast cancer correlated with loss of PHLPP2 and PTEN and poor prognosis. These results show that CXADR promotes the formation of an AKT-inhibitory signalosome at TJ and regulates epithelial-mesenchymal plasticity in breast cancer cells. Moreover, loss of CXADR might be used as a prognostic marker in luminal breast cancer. SIGNIFICANCE: The tight junction protein CXADR controls epithelial-mesenchymal plasticity in breast cancer by stabilizing the AKT regulators PTEN and PHLPP2.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/47/F1.large.jpg.