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Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by reduced expression of the survival motor neuron (SMN) protein. In addition to motor neuron survival, SMN deficiency affects the integrity and function of afferent synapses that provide glutamatergic excitatory drive essential for motor neuron firing and muscle contraction. However, it is unknown whether deficits in the metabolism of excitatory amino acids and their precursors contribute to neuronal dysfunction in SMA. To address this issue, we measured the levels of the main neuroactive D- and L-amino acids acting on glutamatergic receptors in the central nervous system of SMNΔ7 mice as well as the cerebrospinal fluid (CSF) of SMA patients of varying severity before and after treatment with the SMN-inducing drug Nusinersen. Our findings reveal that SMN deficiency disrupts glutamate and serine metabolism in the CSF of severe SMA patients, including decreased concentration of L-glutamate, which is partially corrected by Nusinersen therapy. Moreover, we identify dysregulated L-glutamine to L-glutamate conversion as a shared neurochemical signature of altered glutamatergic synapse metabolism that implicates astrocyte dysfunction in both severe SMA patients and mouse models. Lastly, consistent with a correlation of higher CSF levels of D-serine with better motor function in severe SMA patients, we show that daily supplementation with the NMDA receptor co-agonist D-serine improves neurological deficits in SMNΔ7 mice. Altogether, these findings provide direct evidence for dysregulation of D- and L-amino acid metabolism linked to glutamatergic neurotransmission in severe SMA and have potential implications for treating this neurological disorder., Competing Interests: Declaration of Competing Interest C.B. received advisory board honoraria from Avexis, Biogen, Novartis and Roche. The other authors declare no competing interests.

Stasimon/Tmem41b is a transmembrane protein with phospholipid scrambling activity that resides in the endoplasmic reticulum and has been implicated in autophagy, lipid metabolism, and viral replication. Stasimon/Tmem41b has also been linked to the function of sensory-motor circuits and the pathogenesis of spinal muscular atrophy. However, the early embryonic lethality of constitutive knockout in mice has hindered the analysis of spatial and temporal requirements of Stasimon/Tmem41b in vivo. To address this, we developed a novel mouse line harboring a conditional knockout allele of the Stasimon/Tmem41b gene in which exon 4 has been flanked by loxP sites (Stas/Tmem41b CKO ). Cre-mediated recombination of Stas/Tmem41b CKO generates a functionally null allele (Stas/Tmem41b Δ4 ) resulting in loss of protein expression and embryonic lethality in the homozygous mouse mutant. Here, using a ubiquitously expressed, tamoxifen inducible Cre recombinase in the homozygous Stas/Tmem41b CKO mice, we demonstrate that postnatal depletion of Stasimon/Tmem41b rapidly arrests weight gain in adult mice and causes motor dysfunction and death approximately three weeks after tamoxifen treatment. Moreover, we show that depletion of Stasimon/Tmem41b severely affects cell proliferation in mouse embryonic fibroblasts. This study provides new insights into the essential requirement of Stasimon/Tmem41b for cellular and organismal fitness and expands the experimental toolkit to investigate its functions in the mammalian system., Competing Interests: Declaration of competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of motor neurons and skeletal muscle atrophy which is caused by ubiquitous deficiency in the survival motor neuron (SMN) protein. Several cellular defects contribute to sensory-motor circuit pathology in SMA mice, but the underlying mechanisms have often been studied in one mouse model without validation in other available models. Here, we used Smn2B/- mice to investigate specific behavioral, morphological, and functional aspects of SMA pathology that we previously characterized in the SMNΔ7 model. Smn2B/- SMA mice on a pure FVB/N background display deficits in body weight gain and muscle strength with onset in the second postnatal week and median survival of 19 days. Morphological analysis revealed severe loss of proprioceptive synapses on the soma of motor neurons and prominent denervation of neuromuscular junctions (NMJs) in axial but not distal muscles. In contrast, no evidence of cell death emerged from analysis of several distinct pools of lumbar motor neurons known to be lost in the disease. Moreover, SMA motor neurons from Smn2B/- mice showed robust nuclear accumulation of p53 but lack of phosphorylation of serine 18 at its amino-terminal, which selectively marks degenerating motor neurons in the SMNΔ7 mouse model. These results indicate that NMJ denervation and deafferentation, but not motor neuron death, are conserved features of SMA pathology in Smn2B/- mice., Competing Interests: The authors have declared that no competing interests exist.

Cancer cells can disseminate during very early and sometimes asymptomatic stages of tumor progression. Though biological barriers to tumorigenesis have been identified and characterized, the mechanisms that limit early dissemination remain largely unknown. We report here that the orphan nuclear receptor nuclear receptor subfamily 2, group F, member 1 (NR2F1)/COUP-TF1 serves as a barrier to early dissemination. NR2F1 expression was decreased in patient ductal carcinoma in situ (DCIS) samples. High-resolution intravital imaging of HER2+ early-stage cancer cells revealed that loss of function of NR2F1 increased in vivo dissemination and was accompanied by decreased E-cadherin expression, activation of wingless-type MMTV integration site family, member 1 (WNT)-dependent β-catenin signaling, disorganized laminin 5 deposition, and increased expression of epithelial-mesenchymal transition (EMT) genes such as twist basic helix-loop-helix transcription factor 1 (TWIST1), zinc finger E-box binding homeobox 1 (ZEB1), and paired related homeobox 1 (PRRX1). Furthermore, downregulation of NR2F1 promoted a hybrid luminal/basal phenotype. NR2F1 expression was positively regulated by p38α signaling and repressed by HER2 and WNT4 pathways. Finally, early cancer cells with NR2F1LOW/PRRX1HIGH staining were observed in DCIS samples. Together, these findings reveal the existence of an inhibitory mechanism of dissemination regulated by NR2F1 in early-stage breast cancer cells., Significance: During early stages of breast cancer progression, HER2-mediated suppression of NR2F1 promotes dissemination by inducing EMT and a hybrid luminal/basal-like program., (©2022 American Association for Cancer Research.)

Background: Progesterone receptor (PR) is expressed from a single gene as two isoforms, PRA and PRB. In normal breast human tissue, PRA and PRB are expressed in equimolar ratios, but isoform ratio is altered during malignant progression, usually leading to high PRA:PRB ratios. We took advantage of a transgenic mouse model where PRA isoform is predominant (PRA transgenics) and identified the key transcriptional events and associated pathways underlying the preneoplastic phenotype in mammary glands of PRA transgenics as compared with normal wild-type littermates., Methods: The transcriptomic profiles of PRA transgenics and wild-type mammary glands were generated using microarray technology. We identified differentially expressed genes and analyzed clustering, gene ontology (GO), gene set enrichment analysis (GSEA), and pathway profiles. We also performed comparisons with publicly available gene expression data sets of human breast cancer., Results: We identified a large number of differentially expressed genes which were mainly associated with metabolic pathways for the PRA transgenics phenotype while inflammation- related pathways were negatively correlated. Further, we determined a significant overlap of the pathways characterizing PRA transgenics and those in breast cancer subtypes Luminal A and Luminal B and identified novel putative biomarkers, such as PDHB and LAMB3., Conclusion: The transcriptional targets identified in this study should facilitate the formulation or refinement of useful molecular descriptors for diagnosis, prognosis, and therapy of breast cancer.

PHLDA1 (pleckstrin homology-like domain, family A, member 1) is a multifunctional protein that plays distinct roles in several biological processes including cell death and therefore its altered expression has been identified in different types of cancer. Progressively loss of PHLDA1 was found in primary and metastatic melanoma while its overexpression was reported in intestinal and pancreatic tumors. Previous work from our group showed that negative expression of PHLDA1 protein was a strong predictor of poor prognosis for breast cancer disease. However, the function of PHLDA1 in mammary epithelial cells and the tumorigenic process of the breast is unclear. To dissect PHLDA1 role in human breast epithelial cells, we generated a clone of MCF10A cells with stable knockdown of PHLDA1 and performed functional studies. To achieve reduced PHLDA1 expression we used shRNA plasmid transfection and then changes in cell morphology and biological behavior were assessed. We found that PHLDA1 downregulation induced marked morphological alterations in MCF10A cells, such as changes in cell-to-cell adhesion pattern and cytoskeleton reorganization. Regarding cell behavior, MCF10A cells with reduced expression of PHLDA1 showed higher proliferative rate and migration ability in comparison with control cells. We also found that MCF10A cells with PHLDA1 knockdown acquired invasive properties, as evaluated by transwell Matrigel invasion assay and showed enhanced colony-forming ability and irregular growth in low attachment condition. Altogether, our results indicate that PHLDA1 downregulation in MCF10A cells leads to morphological changes and a more aggressive behavior.

Our understanding of the minimal residual disease (MRD) in solid cancers indicates that it can persist in the system for years or even decades. We now know that the persistence of MRD might depend on the dormancy of the disseminated cancer cells (DCCs). Once DCCs exit dormancy, they become metastatic and the survival rates of the patients inevitably decrease. Thus, innovative treatments are required to extend the asymptomatic phase of MRD after the initial therapeutic intervention. With the latest advances in cancer research, there is a greater need to explore and understand the biology, timing of dissemination, and origin of DCCs during tumor progression. These important aspects of DCCs impact the selection, design, administration, and timing of effective therapies. Herein, we summarize the current understanding of MRD biology in solid tumors, with a focus on epigenetics and pluripotency, presenting an overall view of the direction the field is taking to reach the goal of reducing cancer-related mortalities that result from metastasis.

Background: Identification of biomarkers in lung cancer, a leading cause of cancer-related mortality, has a meaningful clinical relevance in the quest of novel prognostic factors and therapeutic targets. The glycan-binding protein galectin-1 (Gal-1) modulates tumor progression by mediating cell-cell and cell-extracellular matrix interactions, as well as angiogenesis and tumor immune-escape. Previous works reported the expression of Gal-1 in lung cancer, although its clinical significance remains uncertain., Objective: To assess the clinicopathologic relevance and prognostic value of Gal-1 expression in a cohort of 103 Stage I-III non-small cell lung cancer (NSCLC) patients., Methods: Gal-1 expression was determined by immunohistochemistry in tumor tissue samples. The percentage of immunoreactive tumor cells and stroma, as well as the presence of blood vessels with positively stained endothelium in the tumor and surrounding normal tissue, were recorded. Results were correlated with the clinicopathologic factors of the patients (Spearman's rank correlation coefficient, chi-square test) and overall survival by univariate (Kaplan Meier) and multivariate analyses (Cox regression hazard model)., Results: We did not observe significant associations between Gal-1 expression and relevant clinicopathologic features at diagnosis of NSCLC. However, Kaplan Meier analysis revealed a significant association between Gal-1 expression and overall survival, when Gal-1 expression was analyzed on tumor cells alone ("tumor cell percentage") or when an integrated score accounting for tumor cell as well as stromal expression of Gal-1 ("total score") was assessed. Patients showing high Gal-1 expression evidenced a poorer clinical outcome. Furthermore, "total score" remained significantly associated with survival by multivariate Cox regression analysis in the whole cohort of patients, even when controlling for the classical predictors and prognostic factors of NSCLC., Conclusion: We conclude that Gal-1 expression may be a useful biomarker for better prediction of the clinical outcome and management of NSCLC patients., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Disruption of adenylyl cyclase type 5 (AC5) knockout (KO) is a novel model for longevity. Because malignancy is a major cause of death and reduced lifespan in mice, the goal of this investigation was to examine the role of AC5KO in protecting against cancer. There have been numerous discoveries in genetically engineered mice over the past several decades, but few have been translated to the bedside. One major reason is that it is difficult to alter a gene in patients, but rather a pharmacological approach is more appropriate. The current investigation employs a parallel construction to examine the extent to which inhibiting AC5, either in a genetic knockout (KO) or by a specific pharmacological inhibitor protects against cancer. This study is unique, not only because a combined genetic and pharmacological approach is rare, but also there are no prior studies on the extent to which AC5 affects cancer. We found that AC5KO delayed age-related tumor incidence significantly, as well as protecting against mammary tumor development in AC5KO × MMTV-HER-2 neu mice, and B16F10 melanoma tumor growth, which can explain why AC5KO is a model of longevity. In addition, a Food and Drug Administration approved antiviral agent, adenine 9-β-D-arabinofuranoside (Vidarabine or AraAde), which specifically inhibits AC5, reduces LP07 lung and B16F10 melanoma tumor growth in syngeneic mice. Thus, inhibition of AC5 is a previously unreported mechanism for prevention of cancers associated with aging and that can be targeted by an available pharmacologic inhibitor, with potential consequent extension of lifespan., (© 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Lung cancer is the most frequent and one of the most deadly cancer types and is classified into small cell lung cancer and non-small cell lung cancer (NSCLC). Transforming growth factor beta (TGFβ) regulates a wide array of cell functions and plays a major role in lung diseases, including NSCLC. TGFβ signals through the complex of TGFβ type I and type II receptors, triggering Smad and non-Smad signaling pathways such as PI3K/Akt and MEK1/ERK. We investigated the role of TGFβ1 on the progression of the murine lung adenocarcinoma cell line LP07. Furthermore, we undertook a retrospective study with tissue samples from stage I and II NSCLC patients to assess the clinical pathologic role and prognostic significance of TβRI expression. We demonstrated that although lung cancer cell monolayers responded to TGFβ1 anti-mitogenic effects and TGFβ1 pulse (24 h treatment) delayed tumor growth at primary site; a switch towards malignant progression upon TGFβ1 treatment was observed at the metastatic site. In our model, TGFβ1 modulated in vitro clonogenicity, protected against stress-induced apoptosis and increased adhesion, spreading, lung retention and metastatic outgrowth. PI3K and MEK1 signaling pathways were involved in TGFβ1-mediated metastasis stimulation. Several of these TGFβ responses were also observed in human NSCLC cell lines. In addition, we found that a higher expression of TβRI in human lung tumors is associated with poor patient's overall survival by univariate analysis, while multivariate analysis did not reach statistical significance. Although additional detailed analysis of the endogenous signaling in vivo and in vitro is needed, these studies may provide novel molecular targets for the treatment of lung cancer.

Colorectal cancer (CRC) is the third most common malignant neoplasm worldwide. The objective of this study was to examine whether carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., would inhibit the cell viability of three CRC cell lines: Caco-2, HT29 and LoVo in a dose-dependent manner, with IC₅₀ values in the range of 24-96 µM. CA induced cell death by apoptosis in Caco-2 line after 24 h of treatment and inhibited cell adhesion and migration, possibly by reducing the activity of secreted proteases such as urokinase plasminogen activator (uPA) and metalloproteinases (MMPs). These effects may be associated through a mechanism involving the inhibition of the COX-2 pathway, because we have determined that CA downregulates the expression of COX-2 in Caco-2 cells at both the mRNA and protein levels. Therefore, CA modulates different targets involved in the development of CRC. These findings indicate that carnosic acid may have anticancer activity and may be useful as a novel chemotherapeutic agent.

This review presents recent information about the cross-talk between the tumor cells and the microenvironment in the target organ of metastasis at the premetastatic and metastatic stage. The development of metastatic foci is driven not only by the tumor cells intrinsic properties, but also by the interplay with resident and foreign cells located at particular niches in the target organ. The primary tumor modulates the metastatic target through the production of soluble factors that mobilize cells from distant organs like the bone marrow, which in turn localize in the metastatic niche. There is also strong evidence indicating that some primary tumors induce a fertile ground for the tumor cell at the target organ even before the arrival of the disseminated tumor cell (premetastatic niche). The relationship between the players of the metastatic setting is dynamic and shows a high degree of plasticity. Tumor cells change through the acquisition of genetic and/or epigenetic alterations that provide adaptive advantages and the metastatic niche is remodeled by incoming cell types or newly secreted soluble mediators, as a result a reciprocal dialogue is established that invokes new levels of molecular and cellular complexity. Unraveling the mechanisms that sustain the metastatic niche will allow a better understanding of the biology of the disseminated tumor cell, the design of new therapeutic approaches and, hopefully, the improvement of cancer patients' survival.

As 20% of stage I NSCLC patients develop recurrent and often incurable cancer, the identification of prognostic markers has a meaningful clinical application. The biological significance of steroid hormone and EGF receptors, able to regulate key physiological functions, remains elusive in NSCLC. Our aim was to investigate the prognostic input of estrogen receptors (ERα, ERβ), progesterone receptors (PR) and EGFR in tumors from 58 stage I NSCLC patients. Antigen expression was analyzed by immunohistochemistry. Prognostic evaluation was performed with the multivariate Cox model. We found that about 70 and 40% of samples expressed ERα or ERβ at cytoplasmic or nuclear level, respectively. Besides, only 12.1% of samples weakly expressed nuclear PR and 62.7% showed membrane EGFR staining. Correlation studies indicated an inverse association between EGFR expression and smoking status (p<0.01). Multivariate studies showed that the lack of nuclear ERβ or the loss of EGFR expression were independent prognosis markers associated with shorter overall survival. We also found that patients whose tumors were negative for these two biomarkers presented the worst outcome. In conclusion, our findings could be useful for selecting stage I NSCLC patients with poor prognosis to apply an earlier treatment that impacts on survival.

The impact of interstitial inflammatory cells, such as mast cells, and angiogenesis on the prognosis of cancer patients has been reported in many solid tumors, although there is disagreement about their role. We undertook a retrospective study with tissue samples from 65 patients with stage I and II non-small cell lung cancer to assess the clinical pathologic role and prognostic significance of mast cells. Mast cell phenotypes were identified by immunohistochemistry for tryptase and chymase. In addition, we identified microvessels using the endothelial marker CD34. Mast cell and microvessel density was quantified by assessing immunopositive cells in the intratumoral and peritumoral zones of tumor specimens. Both mast cell and microvessel density was higher in the peritumoral zone than the intratumoral zone (P