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• Research Paper Volume 13, Issue 8 pp 12207-12223

  Anti-oncogenic effects of SOX2 silencing on hepatocellular carcinoma achieved by upregulating miR-222-5p-dependent CYLD via the long noncoding RNA CCAT1

  Relevance score: 7.6818542

  Jian Pu, Xianjian Wu, Yi Wu, Zesheng Shao, Chunying Luo, Qianli Tang, Jianchu Wang, Huamei Wei, Yuan Lu

  Keywords: SOX2, CCAT1, EGFR, miR-222-5p, CYLD

  Published in Aging on March 22, 2021

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  In this study, we determined the involvement of SOX2 and its downstream signaling molecules in hepatocellular carcinoma (HCC) progression. We carried out lentiviral transfection in HepG2 cells to determine the roles of SOX2, CCAT1, EGFR, miR-222-5p, and CYLD in HepG2 cells. We first determined the interaction between SOX2 and CCAT1 and that between miR-222-5p and CYLD and their effect on tumor growth in vivo was analyzed in HCC-xenograft bearing nude mice xenografts. SOX2 and CCAT1 were highly expressed in HCC tissues and HepG2 cells. SOX2 bound to the regulatory site of CCAT1. Silencing of SOX2 or CCAT1 inhibited HepG2 cell proliferation, migration, and invasion as well as decreased the expression of CCAT1 and EGFR. CCAT1 silencing reduced EGFR expression, but EGFR expression was increased in HCC tissues and HepG2 cells, which promoted proliferation, migration, and invasion in vitro. EGFR upregulated miR-222-5p, leading to downregulation of CYLD. miR-222-5p inhibition or CYLD overexpression repressed cell functions in HepG2 cells. SOX2 silencing decreased CCAT1, EGFR, and miR-222-5p expression but increased CYLD expression. Loss of SOX2 also reduced the growth rate of tumor xenografts. In summary, SOX2-mediated HCC progression through an axis involving CCAT1, EGFR, and miR-222-5p upregulation and CYLD downregulation.

• Research Paper Volume 11, Issue 1 pp 127-159

  Premature aging and cancer development in transgenic mice lacking functional CYLD

  Relevance score: 6.3702703

  Josefa P. Alameda, Ángel Ramírez, Rosa A. García-Fernández, Manuel Navarro, Angustias Page, José C. Segovia, Rebeca Sanchez, Cristian Suárez-Cabrera, Jesús M. Paramio, Ana Bravo, M. Jesús Fernández-Aceñero, M. Llanos Casanova

  Keywords: CYLD, premature aging, tumor suppressor, NF-κB, keratinocyte differentiation, skin, inflammation

  Published in Aging on January 10, 2019

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  CYLD is a deubiquitinating enzyme known for its role as a tumor suppressor whose mutation leads to skin appendages tumors and other cancers. In this manuscript we report that the tumor suppressor CYLD, similarly to other renowned tumor suppressor genes, protects from premature aging and cancer. We have generated transgenic mice expressing the mutant CYLD^C/S protein, lacking its deubiquitinase function, under the control of the keratin 5 promoter, the K5-CYLD^C/S mice. These mice express the transgene in different organs, including those considered to be more susceptible to aging, such as skin and thymus. Our results show that K5-CYLD^C/S mice exhibit epidermal, hair follicle, and sebaceous gland alterations; and, importantly, they show signs of premature aging from an early age. Typically, 3-month-old K5-CYLD^C/S mice exhibit a phenotype characterized by alopecia and kyphosis, and, the histological examination reveals that transgenic mice show signs of accelerated aging in numerous organs such as skin, thymus, pancreas, liver and lung. Additionally, they spontaneously develop tumors of diverse origin. Over-activation of the NF-κB pathway, along with hyperactivation of Akt, JNK and c-Myc, and chronic inflammation, appear as the mechanisms responsible for the premature aging of the K5-CYLD^C/S mice.

  

  Analysis of the expression of the endogenous and the mutant CYLD protein in the K5-CYLD^C/S mice. (A) Scheme of the construction used to obtain the K5-CYLD^C/S mice. (B) Representative image showing the expression of K5 in the back skin of Control mice. Arrow: sebaceous gland; arrow head: ORS. (C) Analysis by WB of the expression of HA and CYLD in total protein extracts from the back skin of 30 day-old control and transgenic mice. Both lines of K5-CYLD^C/S mice express higher levels of CYLD than Controls. HA was not detected in Control mice. GAPDH was used as a loading control. Immunostaining -with HA (D, F, H) and CYLD (E, G, I) antibodies- of back skin samples from Control (D, E) and transgenic (K5-CYLD^C/S-X and K5-CYLD^C/S-A) mice (F-G, H-I, respectively). HA is not expressed in Control mice (D). In the K5-CYLD^C/S mice both, the expression of HA (F, H) and CYLD (G, I) follows the expression pattern of K5. Scale bars: 250μm (B); 150μm (D-I).

  
  
  

  

  Analysis of the hair regrowth in Control and K5-CYLD^C/S mice. (A) Representative image of the patches of diffuse alopecia (arrows) of the transgenic mice (7-month-old mice are shown). (B, C) Representative images of the skin of 1-month-old mice. (D-L) Representative images of a hair growth experiment. The back of 7-week-old mice was shaved and the skin was collected 16 days after depilation. (D-F) Hair regrowth in Control mice. (G-L) Hair regrowth in the K5-CYLD^C/S mice. (D, G, J) Representative images of freshly depilated mice of the corresponding phenotypes (day 0 of the experiment). (E, F) 16 days after shaving Control HFs were in the anagen-catagen I phase (E) and mice exhibited a homogeneous hair regrowth (F). (H) Histology showing initiation of the anagen phase of the hair cycle 16 days after shaving the back of a transgenic mouse. The macroscopic view of this mouse showed that hair was very short and hardly visible (I). (K) Histology of a section of the back skin of a K5-CYLD^C/S mouse showing a delay in the growth of the new hair, so that 16 days after shaving it still remains in the telogen phase. (L) Macroscopically these areas correspond to those lacking hair in the back skin of the transgenic mouse (white asterisks). Red arrows show differences in the thickness of the skin between Control (E) and transgenic mice (H, K). Scale bars: 280 μm.

  
  
  

  

  Histological alterations in the back skin of 1 and 3-month-old K5-CYLD^C/S mice. (A-E) Histology of the back skin of 1-month-old Control (A, D) and K5-CYLD^C/S mice (B, C, E). (A) Observe small sebaceous glands (white arrows) and HFs in the anagen phase in Control mice. (B, C) Note in transgenic mice the presence of moderately hyperplastic sebaceous glands, epidermal ridges and HFs initiating the anagen phase of the second hair growth cycle. (D, E) Slight thinning of the epidermis of K5-CYLD^C/S mice. (F-I) Histology of the back skin of 3-month-old Control (F) and K5-CYLD^C/S mice (G-I). (F) Note small sebaceous glands and telogenic HFs in Control mice. (G-I) Observe marked epidermal atrophy; abundant epidermal ridges of pyknotic keratinocytes, increased hyperplasia of the sebaceous glands and areas with orphan sebaceous glands lacking hair follicles in transgenic mice. White arrows: sebaceous glands; black arrows: epidermal ridges of pyknotic keratinocytes; double-headed red arrows: areas of epidermal atrophy. Scale bars: 250 μm (C, F-H); 200 μm (A, B); 180 μm (I); 150 μm (D, E).

  
  
  

  

  Histopathological signs of premature aging in the back skin of young (5- and 8-month-old) transgenic mice. Representative histology of the back skin of 5-month-old Control and transgenic mice (A-G). (A) Histology of the back skin of a Control mouse. Observe the presence of small sebaceous glands (white arrow) and 3 layers of keratinocytes in the interfollicular epidermis (higher magnification is showed in the inset). (B-E) The epidermis of the K5-CYLD^C/S mice shows frequent and extensive areas of atrophy (double-headed red arrows in B and C; also compare the inset in B with that of A); as well as papillomatous hyperplasia (red arrow in C) and epidermal ridges (black arrows in E and G). Abundant hyperplastic sebaceous glands -often orphan, were detected (white arrows in C-E). (F-G) Observe the scarce adipose tissue present in the skin of the transgenic mice (compare the length of the double-headed black arrows). Representative histology of the back skin of Control (H) and transgenic mice (I, J) of 8-month-old. Observe in the K5-CYLD^C/S mice the presence of papillomatous hyperplasia (red arrows in I); epidermal ridges (black arrows in J); abundant hyperplastic sebaceous glands (white arrows), some of them orphan (without HF) (I), and patchy epidermal atrophy associated to moderate hyperkeratosis (compare the inset in H with those of I and J). Representative histological images of the tail skin of Control (K) and transgenic mice (L-N). Note in the skin of transgenic mice the presence of hyperplastic sebaceous glands, most of them orphan (white arrows), and epidermal atrophy (compare the insets in K with those of M and N). Images of the histology of both K5-CYLD^C/S-X and K5-CYLD^C/S-A are shown. White arrows, sebaceous glands; black arrows: epidermal ridges; double-headed red arrows: areas of epidermal atrophy. Scale bars: 150 μm (C, E); 180 μm (A, B, D, I, J, L-N); 200 μm (H, K) and (F, G) 350 μm.

  
  
  

  

  Histological and molecular signs of premature aging in the back skin of transgenic mice. (A-H) Representative histological images showing the back skin of 20-month-old Control mice (A) and the severe aging phenotype of the back skin of 20-month-old transgenic mice (B-D). (B-D) Note severe epidermal atrophy (compare insets in A with those of B and C; double-headed red arrow in D); foci of papillomatous hyperplasia (red arrows in C and D); numerous hyperplastic sebaceous glands, most of them orphan and grouped in the dermis (D); reduced number of HFs, and scarce or even lack of adipose tissue (compare A with B-D) in the back skin of the K5-CYLD^C/S mice. (E-H) Tail skin of Control (E) and transgenic (F-H) mice. Note the presence of hyperplastic sebaceous glands and extensive epidermal atrophy (compare inset in E with those in F and G) in the tail of the K5-CYLD^C/S mice. (I, J) WB of total protein extracts from skin of 12-month-old (I) and 6-month-old (J) showing elevated levels of p16, p19 and γH2AX in the K5-CYLD^C/S mice. Tubulin and Actin are used as control loading. (K) WB of total protein extracts from the skin of Control mice from 1 to 33 months of age showing the decreased expression of CYLD as mice age. GAPDH is used as a control loading. White arrows: sebaceous glands; red arrows: papillomatous hyperplasia; double-headed red arrows: areas of epidermal atrophy. Scale bars: 250 μm (A-D); 200 μm (E-H).

  
  
  

  

  Deficient differentiation in the skin of K5-CYLD^C/S mice. Representative immunostainings of the back skin of 20-month-old mice (A-J). Observe the strong expression of the epidermal differentiation proteins Involucrin, Loricrin and Filaggrin in the suprabasal layers of the epidermis of Control mice (A,C,E,G); and the weak and discontinuous expression of these proteins in the epidermis of K5-CYLD^C/S mice (B,D,F,H), specially faint in the areas of epidermal atrophy (red brackets). (I, J) Representative images corresponding to the immunostaining of the back skin of Control (I) and transgenic (J) mice with the K5 specific antibody. (I) Strong K5 staining in basal keratinocytes of Control mice. A faint and patched expression is detected in the epidermis of the transgenic mice, especially in the regions of atrophic epidermis (indicated by red brackets). Scale bars: 180 μm (A-H); 150 μm (I, J).

  
  
  

  

  Overactivation of the NF-κB, and other pro-aging pathways, along with increased IL6 and TNF-α expression in the skin of the K5-CYLD^C/S mice. (A) p65 and IκBα phosphorylation kinetics in the back skin of 3-day-old Control and transgenic mice treated with TNF-α for the indicated times. (B) WB showing over-activation of the classical NF-κB signaling pathway (P-p65 and P-IκBα) in the skin of 20-month-old transgenic mice. (C) Increased expression of the inflammatory cytokines TNF-α and IL6 in the skin of the K5-CYLD^C/S mice. (D-F) WB showing the hyperactivation (phosphorylation) of Akt (D), JNK (E) and c-Myc (F) in the skin of adult K5-CYLD^C/S mice. Graphic representations of the densitometric analysis of western blots corresponding to extracts from 5-7 animals of each genotype are shown. Mann-Whitney U test was used for statistical analysis. (*p<0.05; **p<0.01; ***p<0.001).

  
  
  

  

  Premature thymic involution and over-activation of NF-κB in the thymus of K5-CYLD^C/S mice. (A-C) Analysis of the expression of the transgene by immunostaining with a specific antibody against the HA tag. Expression of HA is detected in the medulla of the thymus of the K5-CYLD^C/S mice (B), following the expression pattern of the K5 (C), while it is not detected in the Controls (A). (D) Analysis by WB of the expression of the transgene in protein extracts from isolated thymic cells of mice of 3.5-month-old. Note the overactivation of NF-κB (increased levels of P-p65) in the K5-CYLD^C/S mice. Mann-Whitney U test was used for statistical analysis. (*p<0.05). (E, F) Histological analysis of the thymus of 2.5-month-old mice. Observe the expansion of the cortical zone and reduction of the medullar region in the thymus of transgenic mice (F). (G, H) H&E staining of 3.5-month-old Control (G) and K5-CYLD^C/S mice (H) thymus. A representative image of the thymic atrophy and infiltration of white adipose tissue in the thymus of transgenic mice (H) is shown. (I) Western blot showing the decreased expression of CYLD with age in the thymus of control mice. M, medulla. C, cortex. a, adipose tissue. Scale bars: 200 μm (A, B); 300 μm (C); 350 μm (E-H).

  
  
  

  

  Alterations found in the pancreas, liver, lung and stomach of the K5-CYLD^C/S mice suggestive of early aging of the K5-CYLD^C/S mice. (A-H) Histopathologic analysis by H&E staining of pancreas from 5-month-old (B, F) and 12-month-old Control and transgenic mice. (A, E, F) Pancreas from Control mice: note the presence of Islets of Langerhans (L) of heterogeneous but moderate size. (B-D; G, H) Histology sections representatives of pancreas from K5-CYLD^C/S mice. Note the hyperplasia of the Islets of Langerhans (B, C, G, H). (D) Extrapancreatic location of the Islets of Langerhans, in the peripancreatic fat, observed in the K5-CYLD^C/S mice. (H) Foci of inflammation (asterisk) in the pancreas of K5-CYLD^C/S mice. (I-N) Histopathological analysis of liver, lung and stomach sections from different organs of 20-month-old K5-CYLD^C/S mice. (I-K) Representative images showing anisokariosis (I), eosinophilic intracytoplasmic inclusions (arrow in J), intranuclear eosinophilic inclusions (arrow in K), and inflammation foci (asterisks in L) in the liver. (M) Example of inflammation foci observed in the lung. (N) Stomach with an inflammation focus. The pancreas of 4 Control and 4 transgenic mice of 5- and 12-month-old were analyzed. Number of animals whose liver, lung and stomach has been analyzed is showed in Table 1. Asterisks: Inflammation. Scale bars: 250 μm (A, B, D-F); 350 μm (C); 500 μm (G); 150 μm (H; L-N); 40 μm (I-K).

  
  
  

  

  K5-CYLD^C/S mice develop spontaneous tumors in many organs. (A-C) Skin tumors. Infiltrating SCC arisen in the back skin of 8-month-old transgenic mouse (A, B). (C) Hair follicle derived tumor (trichofolliculoma) developed in the snout of a K5-CYLD^C/S mouse. (D) Macroscopic appearance of a lung adenocarcinoma. (E) Lung acinar adenocarcinoma. (F) Lung papillary adenocarcinoma. (G, H) Hepatocellular carcinoma (HCC); liver (Lv). (I) Mammary adenoepithelioma (asterisk). (J) Well differentiated gastric adenocarcinoma (asterisk). (K) In situ gastric carcinoma (asterisk). Scale bars: 500 μm (A); 300 μm (C, J, K); 200 μm (E, F); 250 μm (H, J); 100 μm (B).

  
  
  

  

  Analysis of the expression of the transgene in the tumors developed in the K5-CYLD^C/S mice and in their matched non tumoral tissue. Immunohistochemical staining with K5 and HA antibodies. (A, B) Snout sections from Control mice. K5 expression in the basal layer of the epidermis, HF and the immature cells of the sebaceous glands (A); HA is not detected (B). (C) HA expression in the snout of transgenic mice following the K5 expression pattern. HA expression in the tricofolliculoma of the snout (D) and in the SCC of the back skin of K5-CYLD^C/S mice (E). (F-G) K5 expression in the basal layer of the epithelium of bronchia and bronchioles of Control mice (F); no HA staining was observed (G). (H, I) HA in bronchia and bronchioles of transgenic mice (H) and in alveolar cells (I). (J) HA expression in the lung ADC. (K, L) K5 expression in the myoepithelial cells around the mammary secretory acini of Control mice (K); HA is not detected (L). (M) HA in the mammary secretory acini of lactating transgenic mice following the K5 expression patter. (N) HA expression in the mammary adenomyoepithelioma. (O, P) Stomach from a Control mice showing K5 expression in the aglandular epithelia (forestomach, fs), plica (pl), and in scattered glands (g) (O); HA is not expressed (P). (Q) Expression of HA in the stomach of transgenic mice following the K5 expression pattern. (R) Gastric carcinoma in situ expressing HA. (S, T) Neither K5 nor HA are expressed in the liver of Control mice. (U) HA is not detected in hepatocytes of K5-CYLD^C/S mice. (V) HA is not expressed in the hepatocarcinomas (HCC) of transgenic animals. Scale bars: 300 μm (A-C; N, R); 150 μm (D, E, I, J); 70 μm (K-M); 250 μm (G, O, P); 200 μm (F, H, Q, V); 100 μm (S-U). ADC: adenocarcinoma.

  
  
  

  

  The reduction of the NF-κB overactivation in keratinocytes expressing the CYLD^C/S mutant decreases the expression of the biomarker of aging p16 and TNFα. HaCaT (Control and CYLD^C/S) cells were treated with sodium salicylate for 48h when indicated (+). WB shows that HaCaT-CYLD^C/S cells exhibit increased levels of expression of P-p65, p16 and TNFα, but the treatment with sodium salicylate, which reduced P-p65 levels, also decreases p16 and TNF-α levels in these cells. Actin was used as a control loading.