Two distinct epithelial-to-mesenchymal transition programs control invasion and inflammation in segregated tumor cell populations.

Epithelial-to-mesenchymal transition (EMT) triggers cell plasticity in embryonic development, adult injured tissues and cancer. Combining the analysis of EMT in cell lines, embryonic neural crest and mouse models of renal fibrosis and breast cancer, we find that there is not a cancer-specific EMT program. Instead, cancer cells dedifferentiate and bifurcate into two distinct and segregated cellular trajectories after activating either embryonic-like or adult-like EMTs to drive dissemination or inflammation, respectively. We show that SNAIL1 acts as a pioneer factor in both EMT trajectories, and PRRX1 drives the progression of the embryonic-like invasive trajectory. We also find that the two trajectories are plastic and interdependent, as the abrogation of the EMT invasive trajectory by deleting Prrx1 not only prevents metastasis but also enhances inflammation, increasing the recruitment of antitumor macrophages. Our data unveil an additional role for EMT in orchestrating intratumor heterogeneity, driving the distribution of functions associated with either inflammation or metastatic dissemination.

Protection of animals against devastating RNA viruses using CRISPR-Cas13s.

The intrinsic nature of CRISPR-Cas in conferring immunity to bacteria and archaea has been repurposed to combat pathogenic agents in mammalian and plant cells. In this regard, CRISPR-Cas13 systems have proved their remarkable potential for single-strand RNA viruses targeting. Here, different types of Cas13 orthologs were applied to knockdown foot-and-mouth disease virus (FMDV), a highly contagious disease of a wide variety of species with genetically diverse strains and is widely geographically distributed. Using programmable CRISPR RNAs capable of targeting conserved regions of the viral genome, all Cas13s from CRISPR system type VI (subtype A/B/D) could comprehensively target and repress different serotypes of FMDV virus. This approach has the potential to destroy all strains of a virus as targets the ultra-conserved regions of genome. We experimentally compared the silencing efficiency of CRISPR and RNAi by designing the most effective short hairpin RNAs according to our developed scoring system and observed comparable results. This study showed successful usage of various Cas13 enzymes for suppression of FMDV, which provides a flexible strategy to battle with other animal infectious RNA viruses, an underdeveloped field in the biotechnology scope.

In vivo interaction screening reveals liver-derived constraints to metastasis.

It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.

Ciliated, Mitochondria-Rich Postmitotic Cells are Immune-privileged, and Mimic Immunosuppressive Microenvironment of Tumor-Initiating Stem Cells: From Molecular Anatomy to Molecular Pathway.

Cancer whose major problems are metastasis, treatment resistance, and recurrence is the leading cause of death worldwide. Tumor-initiating stem cells (TiSCs) are a subset of the tumor population responsible for tumor resistance and relapse. Understanding the characteristics and shared features between tumor-initiating stem cells (TiSCs) and long-lived postmitotic cells may hold a key to better understanding the biology of cancer. Postmitotic cells have exited the cell cycle and are transitioned into a non-dividing and terminally differentiated state with a specialized function within a tissue. Conversely, a cancer cell with TiSC feature can divide and produce a variety of progenies, and is responsible for disease progression, tumor resistance to therapy and immune system and disease relapse. Surprisingly, our comprehensive evaluation of TiSCs suggests common features with long-lived post-mitotic cells. They are similar in structure (primary cilia, high mitochondrial content, and being protected by a barrier), metabolism (autophagy and senescence), and function (immunoescape and/or immune-privileged by a blood barrier). In-depth exploration showed how mitochondrial metabolism contributes to these shared features, including high energy demands arising from ciliary and microtubular functionality, increased metabolic activity, and movement. These findings can assist in decoding the remaining properties which offer insights into the biology of TiSCs, with potential implications for enhancing cancer treatment strategies and patient prognosis.

Reactivation of embryonic genetic programs in tissue regeneration and disease.

Embryonic genetic programs are reactivated in response to various types of tissue damage, providing cell plasticity for tissue regeneration or disease progression. In acute conditions, these programs remedy the damage and then halt to allow a return to homeostasis. In chronic situations, including inflammatory diseases, fibrosis and cancer, prolonged activation of embryonic programs leads to disease progression and tissue deterioration. Induction of progenitor identity and cell plasticity, for example, epithelial-mesenchymal plasticity, are critical outcomes of reactivated embryonic programs. In this Review, we describe molecular players governing reactivated embryonic genetic programs, their role during disease progression, their similarities and differences and lineage reversion in pathology and discuss associated therapeutics and drug-resistance mechanisms across many organs. We also discuss the diversity of reactivated programs in different disease contexts. A comprehensive overview of commonalities between development and disease will provide better understanding of the biology and therapeutic strategies.

Terminal differentiation of villus tip enterocytes is governed by distinct Tgfß superfamily members.

The protective and absorptive functions of the intestinal epithelium rely on differentiated enterocytes in the villi. The differentiation of enterocytes is orchestrated by sub-epithelial mesenchymal cells producing distinct ligands along the villus axis, in particular Bmps and Tgfß. Here, we show that individual Bmp ligands and Tgfß drive distinct enterocytic programs specific to villus zonation. Bmp4 is expressed from the centre to the upper part of the villus and activates preferentially genes connected to lipid uptake and metabolism. In contrast, Bmp2 is produced by villus tip mesenchymal cells and it influences the adhesive properties of villus tip epithelial cells and the expression of immunomodulators. Additionally, Tgfß induces epithelial gene expression programs similar to those triggered by Bmp2. Bmp2-driven villus tip program is activated by a canonical Bmp receptor type I/Smad-dependent mechanism. Finally, we establish an organoid cultivation system that enriches villus tip enterocytes and thereby better mimics the cellular composition of the intestinal epithelium. Our data suggest that not only a Bmp gradient but also the activity of individual Bmp drives specific enterocytic programs.

Tracing colonic embryonic transcriptional profiles and their reactivation upon intestinal damage.

We lack a holistic understanding of the genetic programs orchestrating embryonic colon morphogenesis and governing damage response in the adult. A window into these programs is the transcriptomes of the epithelial and mesenchymal cell populations in the colon. Performing unbiased single-cell transcriptomic analyses of the developing mouse colon at different embryonic stages (embryonic day 14.5 [E14.5], E15.5, and E18.5), we capture cellular and molecular profiles of the stages before, during, and after the appearance of crypt structures, as well as in a model of adult colitis. The data suggest most adult lineages are established by E18.5. We find embryonic-specific gene expression profiles and cell populations that reappear in response to tissue damage. Comparison of the datasets from mice and human colitis suggests the processes are conserved. In this study, we provide a comprehensive single-cell atlas of the developing mouse colon and evidence for the reactivation of embryonic genes in disease.

Distinct populations of crypt-associated fibroblasts act as signaling hubs to control colon homeostasis.

Despite recent progress in recognizing the importance of mesenchymal cells for the homeostasis of the intestinal system, the current picture of how these cells communicate with the associated epithelial layer remains unclear. To describe the relevant cell populations in an unbiased manner, we carried out a single-cell transcriptome analysis of the adult murine colon, producing a high-quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt-associated colonic fibroblast populations that are demarcated by different strengths of platelet-derived growth factor receptor A (Pdgfra) expression. Crypt-bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt-top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Our findings enhance our understanding of the crosstalk between various colonic epithelial cells and their associated mesenchymal signaling hubs along the crypt axis-placing differential Pdgfra expression levels in the spotlight of intestinal fibroblast identity.

A gene regulatory network to control EMT programs in development and disease.

The Epithelial to Mesenchymal Transition (EMT) regulates cell plasticity during embryonic development and in disease. It is dynamically orchestrated by transcription factors (EMT-TFs), including Snail, Zeb, Twist and Prrx, all activated by TGF-ß among other signals. Here we find that Snail1 and Prrx1, which respectively associate with gain or loss of stem-like properties and with bad or good prognosis in cancer patients, are expressed in complementary patterns during vertebrate development and in cancer. We show that this complementarity is established through a feedback loop in which Snail1 directly represses Prrx1, and Prrx1, through direct activation of the miR-15 family, attenuates the expression of Snail1. We also describe how this gene regulatory network can establish a hierarchical temporal expression of Snail1 and Prrx1 during EMT and validate its existence in vitro and in vivo, providing a mechanism to switch and select different EMT programs with important implications in development and disease.

MicroRNAs Establish the Right-Handed Dominance of the Heart Laterality Pathway in Vertebrates.

Despite their external bilateral symmetry, vertebrates have internal left/right (L/R) asymmetries required for optimal organ function. BMP-induced epithelial to mesenchymal transition (EMT) in the lateral plate mesoderm (LPM) triggers L/R asymmetric cell movements toward the midline, higher from the right, which are crucial for heart laterality in vertebrates. However, how the L/R asymmetric levels of EMT factors are achieved is not known. Here, we show that the posterior-to-anterior Nodal wave upregulates several microRNAs (miRNAs) to transiently attenuate the levels of EMT factors (Prrx1a and Snail1) on the left LPM in a Pitx2-independent manner in the fish and mouse. These data clarify the role of Nodal in heart laterality and explain how Nodal and BMP exert their respective dominance on the left and right sides through the mutual inhibition of their respective targets, ensuring the proper balance of L/R information required for heart laterality and morphogenesis.

Variants in GNPTAB, GNPTG and NAGPA genes are associated with stutterers.

Non-syndromic stuttering is a neurodevelopmental disorder characterized by disruptions in normal flow of speech in the form of repetition, prolongation and involuntary halts. Previously, mutations with more severe effects on GNPTAB and GNPTG have been reported to cause Mucolipidosisll (ML-ll) and Mucolipidosislll (ML-lll), two lysosomal storage disorders with multiple pathologies. We used homozygosity mapping and Sanger sequencing to investigate variants of the three genes in 25 Iranian families with at least two first degree related non-syndromic stutterers. Bioinformatic evaluation and Segregation analysis of the found variants helped us define probable consequences. We also compared our findings with those related to Mucolipidosis. 14 variations were found in the three genes 3 of which, including a novel variant within intronic region of GNPTG and a heterozygous 2-bp deletion in coding region of GNPTAB, co-segregated with stuttering in the families they were found. Bioinformatics analysis predicted all three variants causing deleterious effects on gene functioning. Our findings support the role of these three variants in non-syndromic stuttering. This finding may challenge the current belief that variations causing stuttering are at different sites and have less severe consequences than genetic changes that cause ML-ll and ML-lll.

Clinical significance of NDRG3 in patients with breast cancer.

AIM: The expression level of NDRG3 gene is investigated among breast cancer (BC) patients. METHODS: Real-time quantitative PCR was performed. RESULTS:  NDRG3 was downregulated in BC patients particularly in advanced stage of the disease. HER2 status was significantly correlated with the expression of NDRG3. Also, triple-negative BC patients showed low levels of NDRG3 expression in comparison to other subtypes. Lastly, the expression of NDRG3 had significant impact on survival, with NDRG3 downregulated patients having the worst event-free survival rate among others. CONCLUSION: We have presented that NDRG3 might be a tumor suppressor candidate. NDRG3 downregulation might be involved in the tumorigenesis and development of invasive BC in an advanced phase of the disease.

High expression of CEACAM19, a new member of carcinoembryonic antigen gene family, in patients with breast cancer.

Carcinoembryonic antigen (CEA) family members play important roles in malignancies and are introduced as biomarkers in different types of cancers. Among them CEACAM19 (CEAL1) gene, a new member of the CEA family, remains to be fully elucidated. The aim of this study was investigating the mRNA expression level of CEACAM19 in tumor samples of breast cancer patients compared to breast tissue of normal individuals. We evaluated the expression level of this gene in 75 breast tumors by using real-time quantitative PCR. Also, we studied the correlation between CEACAM19 expression and clinicopathological features and hormone receptors status, including estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 of patients. Out of the enrolled patients, six of them (7.9%) showed low expression, ten (13.2%) showed normal expression and 59 (77.6%) showed high expression of CEACAM19. There was a significant correlation between high expression of CEACAM19 gene in tumor samples compared to normal tissues (P = 0.039). No significant correlation was seen between clinicopathological factors and disease-free survival with mRNA levels of CEACAM19 in tumor samples, while the difference between the expression of CEACAM19 in ER/PR-positive and ER/PR-negative breast cancer patients was statistically significant (P = 0.046). In conclusion, CEACAM19 showed high expression in tumor samples compared to normal mammary tissue. In addition, CEACAM19 may represent as a novel therapeutic target in certain subgroups of breast cancer patients such as ER/PR-negative. Critical roles of CEA proteins in tumor progression may nominate them as robust potential targets for therapeutic intervention in near future.

Adenosine kinase deficiency with neurodevelopemental delay and recurrent hepatic dysfunction: A case report.

Hypermethioninemia may be benign, present as a nonspecific sign of nongenetic conditions such as liver failure and prematurity, or a severe, progressive inborn error of metabolism. Genetic causes of hypermethioninemia include mitochondrial depletion syndromes caused by mutations in the MPV17 and DGUOK genes and deficiencies of cystathionine ß-synthase, methionine adenosyltransferase types I and III, glycine N-methyltransferase, S-adenosylhomocysteine hydrolase, citrin, fumarylacetoacetate hydrolase, and adenosine kinase. Here we present a 3-year old girl with a history of poor feeding, irritability, respiratory infections, cholestasis, congenital heart disease, neurodevelopmental delay, hypotonia, sparse hair, facial dysmorphisms, liver dysfunction, severe hypermethioninemia and mild homocystinemia. Genetic analysis of the adenosine kinase (ADK) gene revealed a previously unreported variant (c.479-480 GA>TG) resulting in a stop codon (p.E160X) in ADK. A methionine-restricted diet normalized the liver function test results and improved her hypotonia.

First report of inherited thyroxine-binding globulin deficiency in Iran caused by a known de novo mutation in SERPINA7.

BACKGROUND: Thyroxine-binding globulin (TBG) is the main transporter of thyroid hormones in human serum, encoded by the gene TBG (SERPINA7), located in long arm of X-chromosome (Xq21-q22). Deficiency of SERPINA7 (serum protease inhibitor, clade A [alpha-1 antiproteinase, antitrypsin], member 7) leads to inherited TBG deficiency. Several mutations have been reported in the coding and noncoding regions of SERPINA7 in association with TGB deficiency. METHODS: Automated chemiluminescence immunoassays were used to determine TSH, free and total T4 and T3 (fT4, TT4, TT3) and TBG. Direct DNA sequencing identified the mutation in SERPINA7. RESULTS: We present a 3 and 4/12 year old boy, born premature, who was mismanaged as hypothyroidism before referral to our center, and was diagnosed with TBG deficiency at our center with a hemizygous substitution in exon 1, position c.347T > A, leading to replacement of isoleucine for arginine in position 96 (considering the first 20 amino acid signal peptide). CONCLUSION: This known mutation, reported as the first SERPINA7 mutation in Iran, emphasizes the point that endocrinologists should pay more attention to inherited TBG to prevent unnecessary treatment.

SLUG and SOX9 Cooperatively Regulate Tumor Initiating Niche Factors in Breast Cancer.

Presence of tumor initiating cells and a proper niche is essential for metastatic colonization. SLUG and SOX9 transcription factors play essential roles in induction and maintenance of tumor initiating capacity in breast cancer cells. On the other hand, Tenascin-C and Periostin are crucial factors in metastatic niche that support tumor initiating capability in breast cancer. In this study, regulatory effect of SLUG and SOX9 transcription factors on the expression of Tenascin-C and Periostin was examined. SLUG and SOX9 were overexpressed and knocked-down in MCF7 and MDA-MB-231 cells, respectively. The cells as little and highly invasive breast cancer-derived cells were infected by inducing and shRNA lentivirus constructs. Then, Tenascin-C and Periostin as well as SLUG and SOX9 expression levels were measured in the cells via Real-Time PCR. Simultaneous overexpression of SLUG and SOX9 significantly induced Tenascin-C and Periostin expression. SLUG and SOX9 knock-down also significantly reduced the expression of Tenascin-C and Periostin. In this analysis Periostin showed the most deviation in both up- and down-regulation levels. This regulatory effect might shed light to a crosstalk between factors involved in the tumor initiating capacity and metastatic niche of the breast cancer.

A novel mutation pattern of kidney anion exchanger 1 gene in patients with distal renal tubular acidosis in Iran.

INTRODUCTION: Mutations of the anion exchanger 1 (AE1) gene encoding the kidney anion exchanger 1 can result in autosomal dominant or autosomal recessive form of distal renal tubular acidosis (DRTA). This study aimed to report deletion mutations of the AE1 and its impact on Iranian children with DRTA. MATERIALS AND METHODS: Twelve children with DRTA referred to Ali Asghar Children Hospital were investigated for all AE1 gene exons through polymerase chain reaction amplification, DNA sequencing, and bioinformatics analysis. RESULTS: Eleven of 12 patients (91.7%) showed an alteration in AE1 gene with a real hot spot in its exons 11 or 15. Homozygote and heterozygote deletions were confirmed in exon 15 in 5 (41.7%) and 3 (25.0%), respectively. Two patients (16.7%) showed homozygote deletions in exon 11 of AE1 gene, and 1 patient (8.3%) showed point mutation in exon 11. The 3-dimensional structures of the native and these mutant kidney AE1 proteins were determined by the multitemplate method using the Phyre and Hidden Markov Model algorithms. CONCLUSIONS: Parents' consanguinity of these patients reveals that cousins are at a high risk for DRTA. This study is considered as a pilot study showing the importance of AE1 mutations in Iranian children with DRTA and further studies is recommended in this geographic region of the world. These models suggest that alteration in the structures leads to alteration in function and change in the current role of AE1.

The perivascular niche governs an autoregulatory network to support breast cancer metastasis.

Unravelling the autoregulatory network that induces and maintains cancer stem cell state may provide novel effective therapies against breast cancer metastasis. The perivascular niche develops elements that initiate the autoregulatory machine to induce and maintain cancer stem cells, but not EMT, among newly arrived tumour cells. Inhibition of one or more primary key elements that trigger this circuit may result in the prevention or cure of breast cancer metastasis.