Elimination of unfit cells maintains tissue health and prolongs lifespan.

Viable yet damaged cells can accumulate during development and aging. Although eliminating those cells may benefit organ function, identification of this less fit cell population remains challenging. Previously, we identified a molecular mechanism, based on "fitness fingerprints" displayed on cell membranes, which allows direct fitness comparison among cells in Drosophila. Here, we study the physiological consequences of efficient cell selection for the whole organism. We find that fitness-based cell culling is naturally used to maintain tissue health, delay aging, and extend lifespan in Drosophila. We identify a gene, azot, which ensures the elimination of less fit cells. Lack of azot increases morphological malformations and susceptibility to random mutations and accelerates tissue degeneration. On the contrary, improving the efficiency of cell selection is beneficial for tissue health and extends lifespan.

Cell mixing induced by myc is required for competitive tissue invasion and destruction.

Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. Here we use the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Here we show, using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. We further show that elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. Our results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues.

Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production.

Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: (i) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of Gαs and BepA in Escherichia coli; (ii) in vitro AC assays with membrane-anchored full-length human AC and recombinant BepA and Gαs; (iii) surface plasmon resonance experiments; and (iv) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the Gαs-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein α-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released Gαs. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium.

CBA (4-chloro-2-(2-chlorophenoxy)acetamido) benzoic acid) inhibits TMEM206 mediated currents and TMEM206 does not contribute to acid-induced cell death in colorectal cancer cells.

Introduction: Upon activation at low pH, TMEM206 conducts Cl- ions across plasma and vesicular membranes. In a (patho)physiological context, TMEM206 was reported to contribute to acid-induced cell death in neurons, kidney and cervical epithelial cells. We investigated the role of TMEM206 in acid-induced cell death in colorectal cancer cells. In addition, we studied CBA as a new small molecule inhibitor for TMEM206. Methods: The role of TMEM206 in acid-induced cell death was studied with CRISPR/Cas9-mediated knockout and FACS analysis. The pharmacology of TMEM206 was determined with the patch clamp technique. Results: In colorectal cancer cells, TMEM206 is not a critical mediator of acid-induced cell death. CBA is a small molecule inhibitor of TMEM206 (IC50 = 9.55 µM) at low pH, at pH 6.0 inhibition is limited. Conclusion: CBA demonstrates effective and specific inhibition of TMEM206; however, its inhibitory efficacy is limited at pH 6.0. Despite this limitation, CBA is a potent inhibitor for functional studies at pH 4.5 and may be a promising scaffold for the development of future TMEM206 inhibitors.

p53 alters intracellular Ca2+ signaling through regulation of TRPM4.

Altered expression of transient receptor potential channel melastatin 4 (TRPM4) contributes to several diseases, including cardiac conduction disorders, immune diseases, and cancer. Yet the underlying mechanisms of TRPM4 expression changes remain elusive. In this study, we report that loss of tumor suppressor protein p53 or p63γ function or mutation of a putative p53 response element in the TRPM4 promoter region increase TRPM4 promoter activity in the colorectal cancer cell line HCT 116. In cells that lack p53 expression, we observed increased TRPM4 mRNA and protein levels and TRPM4-mediated Na+ currents. This phenotype can be reversed by transient overexpression of p53. In the prostate cancer cell line LNCaP, which expresses p53 endogenously, p53 overexpression decreases TRPM4-mediated currents. As in other cancer cells, CRISPR-Cas9 mediated knockout of TRPM4 in p53 deficient HCT 116 cells results in increased store-operated Ca2+entry. The effect of the TRPM4 knockout is mimicked by p53 mediated suppression of TRPM4 in the parental cell line expressing TRPM4. In addition, a TRPM4 knockout-mediated shift in cell cycle is abolished upon loss of p53. Taken together, these findings indicate that p53 represses TRPM4 expression, thereby altering cellular Ca2+ signaling and that TRPM4 adds to cell cycle shift dependent on p53 signaling. One sentence summary: TRPM4 is repressed in the p53 pathway leading to reduced currents and increased calcium signaling.

Investigation of Novel Small Molecular TRPM4 Inhibitors in Colorectal Cancer Cells.

(1) Background: Transient receptor potential melastatin (TRPM4) ion channel aberrant expression or malfunction contributes to different types of cancer, including colorectal cancer (CRC). However, TRPM4 still needs to be validated as a potential target in anti-cancer therapy. Currently, the lack of potent and selective TRPM4 inhibitors limits further studies on TRPM4 in cancer disease models. In this study, we validated novel TRPM4 inhibitors, CBA, NBA, and LBA, in CRC cells. (2) Methods: The potency to inhibit TRPM4 conductivity in CRC cells was assessed with the whole-cell patch clamp technique. Furthermore, the impact of TRPM4 inhibitors on cellular functions, such as viability, proliferation, and cell cycle, were assessed in cellular assays. (3) Results: We show that in CRC cells, novel TRPM4 inhibitors irreversibly block TRPM4 currents in a low micromolar range. NBA decreases proliferation and alters the cell cycle in HCT116 cells. Furthermore, NBA reduces the viability of the Colo205 cell line, which highly expresses TRPM4. (4) Conclusions: NBA is a promising new TRPM4 inhibitor candidate, which could be used to study the role of TRPM4 in cancer disease models and other diseases.

Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells, with Limited Impact on Cancer Hallmark Functions.

Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca2+ activated monovalent cation channel that contributes to the pathophysiology of several diseases. For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells. Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays. To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na+. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype. In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.

TRPM4 is highly expressed in human colorectal tumor buds and contributes to proliferation, cell cycle, and invasion of colorectal cancer cells.

Transient receptor potential melastatin-4 channel (TRPM4) dysregulation contributes to heart conditions, immune diseases, and cervical and prostate cancer. Up to now, the involvement of TRPM4 in colorectal cancer (CRC) pathophysiology remains unknown. Here, we investigated tumor tissue microarrays from 379 CRC patients and analyzed TRPM4 protein expression, tumor characteristics, and clinical outcome. High TRPM4 protein expression was associated with unfavorable tumor features characteristic for epithelial-mesenchymal transition and infiltrative growth patterns, that is, a high number of tumor buds and a low percentage in tumor border configuration. Compared to CRC cells representing early cancer stages, TRPM4 protein expression was the highest in cells representing late-stage metastatic cancer. Investigation of CRC cell line HCT116 and five CRISPR/cas9 TRPM4 knockout clones demonstrated that TRPM4 exhibited large Na+ current densities (~ 60 pA/pF). In addition, CRISPR/cas9 TRPM4 knockout clones showed a tendency toward decreased migration and invasion, cell viability, and proliferation and exhibited a shift in cell cycle when compared to HCT116. Stable overexpression of TRPM4 (TRPM4 wild-type) in two CRISPR/cas9 TRPM4 knockout clones rescued the decrease in cell viability and cell cycle shift. Stable overexpression of a nonconducting, dominant-negative TRPM4 mutant (TRPM4 D894A) did not rescue the decrease in viability or cell cycle shift. Taken together, these findings pointed to TRPM4 ion channel conductivity as the underlying mechanism for decreased viability and cell cycle shift in the TRPM4 knockout clones. Together with previous findings, our present data suggest that TRPM4 plays a versatile role in cancer cell proliferation, cell cycle, and invasion.

Culling Less Fit Neurons Protects against Amyloid-ß-Induced Brain Damage and Cognitive and Motor Decline.

Alzheimer's disease (AD) is the most common form of dementia, impairing cognitive and motor functions. One of the pathological hallmarks of AD is neuronal loss, which is not reflected in mouse models of AD. Therefore, the role of neuronal death is still uncertain. Here, we used a Drosophila AD model expressing a secreted form of human amyloid-ß42 peptide and showed that it recapitulates key aspects of AD pathology, including neuronal death and impaired long-term memory. We found that neuronal apoptosis is mediated by cell fitness-driven neuronal culling, which selectively eliminates impaired neurons from brain circuits. We demonstrated that removal of less fit neurons delays ß-amyloid-induced brain damage and protects against cognitive and motor decline, suggesting that contrary to common knowledge, neuronal death may have a beneficial effect in AD.