Tctex-1 controls ciliary resorption by regulating branched actin polymerization and endocytosis.

The primary cilium is a plasma membrane-protruding sensory organelle that undergoes regulated assembly and resorption. While the assembly process has been studied extensively, the cellular machinery that governs ciliary resorption is less well understood. Previous studies showed that the ciliary pocket membrane is an actin-rich, endocytosis-active periciliary subdomain. Furthermore, Tctex-1, originally identified as a cytoplasmic dynein light chain, has a dynein-independent role in ciliary resorption upon phosphorylation at Thr94. Here, we show that the remodeling and endocytosis of the ciliary pocket membrane are accelerated during ciliary resorption. This process depends on phospho(T94)Tctex-1, actin, and dynamin. Mechanistically, Tctex-1 physically and functionally interacts with the actin dynamics regulators annexin A2, Arp2/3 complex, and Cdc42. Phospho(T94)Tctex-1 is required for Cdc42 activation before the onset of ciliary resorption. Moreover, inhibiting clathrin-dependent endocytosis or suppressing Rab5GTPase on early endosomes effectively abrogates ciliary resorption. Taken together with the epistasis functional assays, our results support a model in which phospho(T94)Tctex-1-regulated actin polymerization and periciliary endocytosis play an active role in orchestrating the initial phase of ciliary resorption.

Disruption of the crypt niche promotes outgrowth of mutated colorectal tumor stem cells.

Recent data establish a logarithmic expansion of leucine rich repeat containing G protein coupled receptor 5-positive (Lgr5+) colonic epithelial stem cells (CESCs) in human colorectal cancer (CRC). Complementary studies using the murine 2-stage azoxymethane-dextran sulfate sodium (AOM-DSS) colitis-associated tumor model indicate early acquisition of Wnt pathway mutations drives CESC expansion during adenoma progression. Here, subdivision of the AOM-DSS model into in vivo and in vitro stages revealed DSS induced physical separation of CESCs from stem cell niche cells and basal lamina, a source of Wnt signals, within hours, disabling the stem cell program. While AOM delivery in vivo under non-adenoma-forming conditions yielded phenotypically normal mucosa and organoids derived thereof, niche injury ex vivo by progressive DSS dose escalation facilitated outgrowth of Wnt-independent dysplastic organoids. These organoids contained 10-fold increased Lgr5+ CESCs with gain-of-function Wnt mutations orthologous to human CRC driver mutations. We posit CRC originates by niche injury-induced outgrowth of normally suppressed mutated stem cells, consistent with models of adaptive oncogenesis.

Retinal pigment epithelium-specific CLIC4 mutant is a mouse model of dry age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Dry AMD has unclear etiology and no treatment. Lipid-rich drusen are the hallmark of dry AMD. An AMD mouse model and insights into drusenogenesis are keys to better understanding of this disease. Chloride intracellular channel 4 (CLIC4) is a pleomorphic protein regulating diverse biological functions. Here we show that retinal pigment epithelium (RPE)-specific Clic4 knockout mice exhibit a full spectrum of functional and pathological hallmarks of dry AMD. Multidisciplinary longitudinal studies of disease progression in these mice support a mechanistic model that links RPE cell-autonomous aberrant lipid metabolism and transport to drusen formation.

Colorectal Cancer Develops Inherent Radiosensitivity That Can Be Predicted Using Patient-Derived Organoids.

Identifying colorectal cancer patient populations responsive to chemotherapy or chemoradiation therapy before surgery remains a challenge. Recently validated mouse protocols for organoid irradiation employ the single hit multi-target (SHMT) algorithm, which yields a single value, the D0, as a measure of inherent tissue radiosensitivity. Here, we translate these protocols to human tissue to evaluate radioresponsiveness of patient-derived organoids (PDO) generated from normal human intestines and rectal tumors of patients undergoing neoadjuvant therapy. While PDOs from adenomas with a logarithmically expanded Lgr5+ intestinal stem cell population retain the radioresistant phenotype of normal colorectal PDOs, malignant transformation yields PDOs from a large patient subpopulation displaying marked radiosensitivity due to reduced homologous recombination-mediated DNA repair. A proof-of-principle pilot clinical trial demonstrated that rectal cancer patient responses to neoadjuvant chemoradiation, including complete response, correlate closely with their PDO D0 values. Overall, upon transformation to colorectal adenocarcinoma, broad radiation sensitivity occurs in a large subset of patients that can be identified using SHMT analysis of PDO radiation responses. SIGNIFICANCE: Analysis of inherent tissue radiosensitivity of patient-derived organoids may provide a readout predictive of neoadjuvant therapy response to radiation in rectal cancer, potentially allowing pretreatment stratification of patients likely to benefit from this approach.

Disabling the Fanconi Anemia Pathway in Stem Cells Leads to Radioresistance and Genomic Instability.

Fanconi anemia is an inherited genome instability syndrome characterized by interstrand cross-link hypersensitivity, congenital defects, bone marrow failure, and cancer predisposition. Although DNA repair mediated by Fanconi anemia genes has been extensively studied, how inactivation of these genes leads to specific cellular phenotypic consequences associated with Fanconi anemia is not well understood. Here we report that Fanconi anemia stem cells in the C. elegans germline and in murine embryos display marked nonhomologous end joining (NHEJ)-dependent radiation resistance, leading to survival of progeny cells carrying genetic lesions. In contrast, DNA cross-linking does not induce generational genomic instability in Fanconi anemia stem cells, as widely accepted, but rather drives NHEJ-dependent apoptosis in both species. These findings suggest that Fanconi anemia is a stem cell disease reflecting inappropriate NHEJ, which is mutagenic and carcinogenic as a result of DNA misrepair, while marrow failure represents hematopoietic stem cell apoptosis. SIGNIFICANCE: This study finds that Fanconi anemia stem cells preferentially activate error-prone NHEJ-dependent DNA repair to survive irradiation, thereby conferring generational genomic instability that is instrumental in carcinogenesis.

Sex Differences in Colon Cancer Metabolism Reveal A Novel Subphenotype.

Women have a lower incidence of colorectal cancer (CRC) than men, however, they have a higher incidence of right-sided colon cancer (RCC). This is of concern as patients with RCC have the poorest clinical outcomes among all CRC patients. Aberrant metabolism is a known hallmark and therapeutic target for cancer. We propose that metabolic subphenotypes exist between CRCs due to intertumoral molecular and genomic variation, and differences in environmental milieu of the colon which vary between the sexes. Metabolomics analysis of patient colon tumors (n = 197) and normal tissues (n = 39) revealed sex-specific metabolic subphenotypes dependent on anatomic location. Tumors from women with RCC were nutrient-deplete, showing enhanced energy production to fuel asparagine synthesis and amino acid uptake. The clinical importance of our findings were further investigated in an independent data set from The Cancer Genomic Atlas, and demonstrated that high asparagine synthetase (ASNS) expression correlated with poorer survival for women. This is the first study to show a unique, nutrient-deplete metabolic subphenotype in women with RCC, with implications for tumor progression and outcomes in CRC patients.

Tumor Tissue-Specific Biomarkers of Colorectal Cancer by Anatomic Location and Stage.

The progress in the discovery and validation of metabolite biomarkers for the detection of colorectal cancer (CRC) has been hampered by the lack of reproducibility between study cohorts. The majority of discovery-phase biomarker studies have used patient blood samples to identify disease-related metabolites, but this pre-validation phase is confounded by non-specific disease influences on the metabolome. We therefore propose that metabolite biomarker discovery would have greater success and higher reproducibility for CRC if the discovery phase was conducted in tumor tissues, to find metabolites that have higher specificity to the metabolic consequences of the disease, that are then validated in blood samples. This would thereby eliminate any non-tumor and/or body response effects to the disease. In this study, we performed comprehensive untargeted metabolomics analyses on normal (adjacent) colon and tumor tissues from CRC patients, revealing tumor tissue-specific biomarkers (n = 39/group). We identified 28 highly discriminatory tumor tissue metabolite biomarkers of CRC by orthogonal partial least-squares discriminant analysis (OPLS-DA) and univariate analyses (VIP > 1.5, p < 0.05). A stepwise selection procedure was used to identify nine metabolites that were the most predictive of CRC with areas under the curve (AUCs) of >0.96, using various models. We further identified five biomarkers that were specific to the anatomic location of tumors in the colon (n = 236). The combination of these five metabolites (S-adenosyl-L-homocysteine, formylmethionine, fucose 1-phosphate, lactate, and phenylalanine) demonstrated high differentiative capability for left- and right-sided colon cancers at stage I by internal cross-validation (AUC = 0.804, 95% confidence interval, CI 0.670-0.940). This study thus revealed nine discriminatory biomarkers of CRC that are now poised for external validation in a future independent cohort of samples. We also discovered a discrete metabolic signature to determine the anatomic location of the tumor at the earliest stage, thus potentially providing clinicians a means to identify individuals that could be triaged for additional screening regimens.

Organoids Reveal That Inherent Radiosensitivity of Small and Large Intestinal Stem Cells Determines Organ Sensitivity.

Tissue survival responses to ionizing radiation are nonlinear with dose, rather yielding tissue-specific descending curves that impede straightforward analysis of biologic effects. Apoptotic cell death often occurs at low doses, while at clinically relevant intermediate doses, double-strand break misrepair yields mitotic death that determines outcome. As researchers frequently use a single low dose for experimentation, such strategies may inaccurately depict inherent tissue responses. Cutting edge radiobiology has adopted full dose survival profiling and devised mathematical algorithms to fit curves to observed data to generate highly reproducible numerical data that accurately define clinically relevant inherent radiosensitivities. Here, we established a protocol for irradiating organoids that delivers radiation profiles simulating the organ of origin. This technique yielded highly similar dose-survival curves of small and large intestinal crypts in vivo and their cognate organoids analyzed by the single-hit multi-target (SHMT) algorithm, outcomes reflecting the inherent radiation profile of their respective Lgr5+ stem cell populations. As this technological advance is quantitative, it will be useful for accurate evaluation of intestinal (patho)physiology and drug screening. SIGNIFICANCE: These findings establish standards for irradiating organoids that deliver radiation profiles that phenocopy the organ of origin.See related commentary by Muschel et al., p. 927.

CLIC4 regulates late endosomal trafficking and matrix degradation activity of MMP14 at focal adhesions in RPE cells.

Dysregulation in the extracellular matrix (ECM) microenvironment surrounding the retinal pigment epithelium (RPE) has been implicated in the etiology of proliferative vitreoretinopathy and age-related macular degeneration. The regulation of ECM remodeling by RPE cells is not well understood. We show that membrane-type matrix metalloproteinase 14 (MMP14) is central to ECM degradation at the focal adhesions in human ARPE19 cells. The matrix degradative activity, but not the assembly, of the focal adhesion is regulated by chloride intracellular channel 4 (CLIC4). CLIC4 is co-localized with MMP14 in the late endosome. CLIC4 regulates the proper sorting of MMP14 into the lumen of the late endosome and its proteolytic activation in lipid rafts. CLIC4 has the newly-identified "late domain" motif that binds to MMP14 and to Tsg101, a component of the endosomal sorting complex required for transport (ESCRT) complex. Unlike the late domain mutant CLIC4, wild-type CLIC4 can rescue the late endosomal sorting defect of MMP14. Finally, CLIC4 knockdown inhibits the apical secretion of MMP2 in polarized human RPE monolayers. These results, taken together, demonstrate that CLIC4 is a novel matrix microenvironment modulator and a novel regulator for late endosomal cargo sorting. Moreover, the late endosomal sorting of MMP14 actively regulates its surface activation in RPE cells.

D-501036, a novel selenophene-based triheterocycle derivative, exhibits potent in vitro and in vivo antitumoral activity which involves DNA damage and ataxia telangiectasia-mutated nuclear protein kinase activation.

D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-N-methylpyrrole] is herein identified as a novel antineoplastic agent with a broad spectrum of antitumoral activity against several human cancer cells and an IC(50) value in the nanomolar range. The IC(50) values for D-501036 in the renal proximal tubule, normal bronchial epithelial, and fibroblast cells were >10 mumol/L. D-501036 exhibited no cross-resistance with vincristine- and paclitaxel-resistant cell lines, whereas a low level of resistance toward the etoposide-resistant KB variant was observed. Cell cycle analysis established that D-501036 treatment resulted in a dose-dependent accumulation in S phase with concomitant loss of both the G(0)-G(1) and G(2)-M phase in both Hep 3B and A-498 cells. Pulsed-field gel electrophoresis showed D-501036-induced, concentration-dependent DNA breaks in both Hep 3B and A-498 cells. These breaks did not involve interference with either topoisomerase-I and topoisomerase-II function or DNA binding. Rapid reactive oxygen species production and formation of Se-DNA adducts were evident following exposure of cells to D-501036, indicating that D-501036-mediated DNA breaks were attributable to the induction of reactive oxygen species and DNA adduct formation. Moreover, D-501036-induced DNA damage activated ataxia telangiectasia-mutated nuclear protein kinase, leading to hyperphosphorylation of Chk1, Chk2, and p53, decreased expression of CDC25A, and up-regulation of p21(WAF1) in both p53-proficient and p53-deficient cells. Collectively, the results indicate that D-501036-induced cell death was associated with DNA damage-mediated induction of ataxia telangiectasia-mutated activation, and p53-dependent and -independent apoptosis pathways. Notably, D-501036 shows potent activity against the growth of xenograft tumors of human renal carcinoma A-498 cells. Thus, D-501036 is a promising anticancer compound that has strong potential for the management of human cancers.

The Biology of Ciliary Dynamics.

The cilium is an evolutionally conserved apical membrane protrusion that senses and transduces diverse signals to regulate a wide range of cellular activities. The cilium is dynamic in length, structure, and protein composition. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases. The cilium undergoes cell-cycle-dependent assembly and disassembly, with ciliary resorption linked with G1-S transition and cell-fate choice. In the resting cell, the cilium remains sensitive to environmental cues for remodeling during tissue homeostasis and repair. Recent findings further reveal an interplay between the cilium and extracellular vesicles and identify bioactive cilium-derived vesicles, posing a previously unrecognized role of cilia for sending signals. The photoreceptor outer segment is a notable dynamic cilium. A recently discovered protein transport mechanism in photoreceptors maintains light-regulated homeostasis of ciliary length.

7-Aroyl-aminoindoline-1-sulfonamides as a novel class of potent antitubulin agents.

A novel series of 7-aroyl-aminoindoline-1-benzenesulfonamides showed excellent activity as inhibitors of tubulin polymerization through binding with the colchicine binding site of microtubules. Compound 15 and 16 display IC50 values of 1.1 and 1.2 microM, respectively. Compound 15 inhibited the human cancer cell growth of KB, MKN45, H460, HT29, and TSGH, as well as one human-resistant cancer line of KB-vin 10, with an IC50 of 9.6, 8.8, 9.4, 8.6, 10.8, and 8.9 nM, respectively.

CLIC4 regulates apical exocytosis and renal tube luminogenesis through retromer- and actin-mediated endocytic trafficking.

Chloride intracellular channel 4 (CLIC4) is a mammalian homologue of EXC-4 whose mutation is associated with cystic excretory canals in nematodes. Here we show that CLIC4-null mouse embryos exhibit impaired renal tubulogenesis. In both developing and developed kidneys, CLIC4 is specifically enriched in the proximal tubule epithelial cells, in which CLIC4 is important for luminal delivery, microvillus morphogenesis, and endolysosomal biogenesis. Adult CLIC4-null proximal tubules display aberrant dilation. In MDCK 3D cultures, CLIC4 is expressed on early endosome, recycling endosome and apical transport carriers before reaching its steady-state apical membrane localization in mature lumen. CLIC4 suppression causes impaired apical vesicle coalescence and central lumen formation, a phenotype that can be rescued by Rab8 and Cdc42. Furthermore, we show that retromer- and branched actin-mediated trafficking on early endosome regulates apical delivery during early luminogenesis. CLIC4 selectively modulates retromer-mediated apical transport by negatively regulating the formation of branched actin on early endosomes.

The internal loop of fission yeast Ndc80 binds Alp7/TACC-Alp14/TOG and ensures proper chromosome attachment.

The Ndc80 outer kinetochore complex plays a critical role in kinetochore-microtubule attachment, yet our understanding of the mechanism by which this complex interacts with spindle microtubules for timely and accurate chromosome segregation remains limited. Here we address this issue using an ndc80 mutant (ndc80-NH12) from fission yeast that contains a point mutation within a ubiquitous internal loop. This mutant is normal for assembly of the Ndc80 complex and bipolar spindle formation yet defective in proper end-on attachment to the spindle microtubule, with chromosome alignment defects and missegregation happening later during mitosis. We find that ndc80-NH12 exhibits impaired localization of the microtubule-associated protein complex Alp7/transforming acidic coiled coil (TACC)-Alp14/tumor-overexpressed gene (TOG) to the mitotic kinetochore. Consistently, wild-type Ndc80 binds these two proteins, whereas the Ndc80-NH12 mutant protein displays a substantial reduction of interaction. Crucially, forced targeting of Alp7-Alp14 to the outer kinetochore rescues ndc80-NH12-mutant phenotypes. The loop was previously shown to bind Dis1/TOG, by which it ensures initial chromosome capture during early mitosis. Strikingly, ndc80-NH12 is normal in Dis1 localization. Genetic results indicate that the loop recruits Dis1/TOG and Alp7/TACC-Alp14/TOG independently. Our work therefore establishes that the Ndc80 loop plays sequential roles in spindle-kinetochore attachment by connecting the Ndc80 complex to Dis1/TOG and Alp7/TACC-Alp14/TOG.

Ndc80 internal loop interacts with Dis1/TOG to ensure proper kinetochore-spindle attachment in fission yeast.

The Ndc80 complex, a conserved outer kinetochore complex, comprising four components (Ndc80/Hec1, Nuf2, Spc24, and Spc25), constitutes one of the core microtubule-binding sites within the kinetochore. Despite this knowledge, molecular mechanisms by which this complex contributes to establishment of correct bipolar attachment of the kinetochore to the spindle microtubule remain largely elusive. Here we show that the conserved internal loop of fission yeast Ndc80 directly binds the Dis1/TOG microtubule-associated protein, thereby coupling spindle microtubule dynamics with kinetochore capture. Ndc80 loop mutant proteins fail to recruit Dis1 to kinetochores, imposing unstable attachment and frequent spindle collapse. In these mutants, mitotic progression is halted attributable to spindle assembly checkpoint activation, and chromosomes remain in the vicinity of the spindle poles without congression. dis1 deletion precisely phenocopies the loop mutants. Intriguingly, forced targeting of Dis1 to the Ndc80 complex rescues loop mutant's defects. We propose that Ndc80 comprises two microtubule-interacting interfaces: the N-terminal region directly binds the microtubule lattice, while the internal loop interacts with the plus end of microtubules via Dis1/TOG. Therefore, our results provide a crucial insight into how the Ndc80 complex establishes stable bipolar attachment to the spindle microtubule.

A novel oral indoline-sulfonamide agent, N-[1-(4-methoxybenzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-isonicotinamide (J30), exhibits potent activity against human cancer cells in vitro and in vivo through the disruption of microtubule.

We have previously synthesized a series of 7-aroylaminoindoline-1-sulfonamides as a novel class of antitubulin agents. Here we show that one of these new compounds, N-[1-(4-methoxybenzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-isonicotinamide (J30), is potently effective against various resistant and nonresistant cancer cell lines despite the status of multidrug resistance, multidrug-resistance associated protein, or other resistance factors in vitro. J30 inhibits assembly of purified tubulin by strongly binding to the colchicine-binding site. Western blot and immunofluorescence experiments demonstrate that J30 depolymerizes microtubules in the KB cell line, resulting in an accumulation of G2/M phase cells. Further studies indicate that J30 causes cell cycle arrest, as assessed by flow analyses and the appearance of MPM-2 (a specific mitotic marker), and is associated with up-regulation of cyclin B1, phosphorylation of Cdc25C, and dephosphorylation of Cdc2. J30 also causes Bcl-2 phosphorylation, cytochrome c translocation, and activation of the caspase-9 and caspase-3 cascades. These findings suggest that the J30-mediated apoptotic signaling pathway depends on caspases and mitochondria. Finally, we show that oral administration of J30 significantly inhibits tumor growth in NOD/scid mice bearing human oral, gastric, and drug-resistant xenografts. Together, our results suggest that J30 has potential as a chemotherapeutic agent for treatment of various malignancies.