Erratum: The WW domains dictate isoform-specific regulation of YAP1 stability and pancreatic cancer cell malignancy: Erratum.

[This corrects the article DOI: 10.7150/thno.42795.].

The WW domains dictate isoform-specific regulation of YAP1 stability and pancreatic cancer cell malignancy.

YAP1 is a key mediator of the Hippo pathway capable of exerting a profound effect on organ size as well as tumorigenesis. Alternative mRNA splicing of human YAP1 results in at least 8 protein isoforms that differ within the 2nd WW motif and the transcriptional activation domain. Methods: To investigate the isoform-specific differences in their mRNA expression, transcriptional activity and tumor-promoting function, we cloned cDNA encoding all of the eight YAP1 protein isoforms. Then, we examined their mRNA expression, subcellular localization, transcriptional regulation properties, interactions with key regulatory partners, and protein stability in response to changes in cell density, as well as their effects on pancreatic cancer cell malignancy both in vitro and in vivo. Results: Multiple YAP1 mRNA isoforms are expressed in commonly used pancreatic cancer lines as well as human pancreatic cancer PDX lines. Based on the analysis of heterologous reporter and endogenous target genes, all YAP1 isoforms are capable of activating transcription, albeit to a different extent. Importantly, we unveiled a marked discrepancy between the mRNA and protein expression levels of the YAP1-1 and YAP1-2 isoforms. We further discovered that the YAP1-2 isoform, which contains two tandem WW motifs, is less stable at the protein level, particularly at high cell densities. Mechanistically, we found that the presence of the 2nd WW motif in YAP1-2 facilitates the de novo formation of the YAP1-2/AMOT/LATS1 complex and contributes to a stronger binding of YAP1-2 to LATS1 and subsequently increased YAP1-2 ubiquitination and degradation by ß-TRCP. Conclusion: Our data reveals a potent effect of YAP1-1 on pancreatic cancer malignancy in vitro and in vivo and provides novel mechanistic insight into isoform-specific and cell density-dependent regulation of YAP1 stability, as well as its impact on cancer malignancy.

The expression of S100A8/S100A9 is inducible and regulated by the Hippo/YAP pathway in squamous cell carcinomas.

BACKGROUND: S100A8 and S100A9, two heterodimer-forming members of the S100 family, aberrantly express in a variety of cancer types. However, little is known about the mechanism that regulates S100A8/S100A9 co-expression in cancer cells. METHODS: The expression level of S100A8/S100A9 measured in three squamous cell carcinomas (SCC) cell lines and their corresponding xenografts, as well as in 257 SCC tissues. The correlation between S100A8/S100A9, Hippo pathway and F-actin cytoskeleton were evaluated using western blot, qPCR, ChIP and Immunofluorescence staining tests. IncuCyte ZOOM long time live cell image monitoring system, qPCR and Flow Cytometry measured the effects of S100A8/S100A9 and YAP on cell proliferation, cell differentiation and apoptosis. RESULTS: Here, we report that through activation of the Hippo pathway, suspension and dense culture significantly induce S100A8/S100A9 co-expression and co-localization in SCC cells. Furthermore, these expressional characteristics of S100A8/S100A9 also observed in the xenografts derived from the corresponding SCC cells. Importantly, Co-expression of S100A8/S100A9 detected in 257 SCC specimens derived from five types of SCC tissues. Activation of the Hippo pathway by overexpression of Lats1, knockdown of YAP, as well as disruption of F-actin indeed obviously results in S100A8/S100A9 co-expression in attached SCC cells. Conversely, inhibition of the Hippo pathway leads to S100A8/S100A9 co-expression in a manner opposite of cell suspension and dense. In addition, we found that TEAD1 is required for YAP-induced S100A8/S100A9-expressions. The functional studies provide evidence that knockdown of S100A8/S100A9 together significantly inhibit cell proliferation but promote squamous differentiation and apoptosis. CONCLUSIONS: Our findings demonstrate for the first time that the expression of S100A8/S100A9 is inducible by changes of cell shape and density through activation of the Hippo pathway in SCC cells. Induced S100A8/S100A9 promoted cell proliferation, inhibit cell differentiation and apoptosis.

miR-137 inhibits melanoma cell proliferation through downregulation of GLO1.

Late-stage melanoma is refractory to current therapies. MicroRNAs (miRNAs) can modulate many physiological and pathological processes of melanoma. Studies have demonstrated that miR-137 acts as a tumor suppressor by inhibiting the proliferation of melanoma cells through targeting multiple mRNAs. The glyoxalase system member glyoxalase 1 (GLO1) is the principal scavenging enzyme of methylglyoxal (MG), a toxic byproduct of glycolysis. Using 35S in vivo/vitro labelling analysis for dynamic proteomics (SiLAD), we found that miR-137 downregulated the expression of GLO1 in melanoma cells. Bioinformatics analysis predicted that GLO1 is a direct target of miR-137. This was validated by dual luciferase reporter assay. Quantitative RT-PCR (qRT-PCR) and western blot analysis indicated that miR-137 could decrease endogenous GLO1 expression. Furthermore, siRNA targeting of GLO1 mimicked inhibition of melanoma cell proliferation caused by miR-137 overexpression. Re-expression of GLO1 was able to restore miR-137-mediated suppression of melanoma cell proliferation. Therefore, these results suggest that miR-137 inhibits the proliferation of melanoma cells by targeting GLO1.

YAP Expression and Activity Are Suppressed by S100A7 via p65/NFκB-mediated Repression of ΔNp63.

In several squamous cell carcinoma (SCC) cells, it has been previously observed that induction of the S100 calcium-binding protein A7 (S100A7) is repressed by YAP via the Hippo pathway. This report now demonstrates that S100A7 also represses YAP expression and activity by ΔNp63 in cancer cells. Stable overexpression of S100A7 activates the NFκB pathway and inhibits the expression of ΔNp63. Caffeic acid phenethyl ester (CAPE), as a specific inhibitor of NFκB, counteracts the inhibitory effect of S100A7 on the expression of ΔNp63 and its target genes. Depletion of S100A7 significantly promotes ΔNp63 expression. These data indicate that S100A7 acts as a suppressor of ΔNp63. Mechanistic examination finds that ΔNp63 not only directly binds to the region of YAP promoter and induces its expression, but also inhibits the Hippo pathway and enhances YAP activity. Importantly, either the positive correlation between S100A7 and YAP phosphorylation at S127 or the negative correlation between S100A7 and ΔNp63 is also observed in skin SCC tissues. Chemosensitivity analysis reveals that S100A7 enhances cancer cells' resistance by inhibition of YAP expression and activity. These results demonstrate that S100A7 is an upstream modulator of the Hippo pathway and extend our understanding of S100A7 functions in cancer.Implications: S100A7 is a new upstream regulator of the Hippo signaling pathway and reduces chemosensitivity of SCC cells through inhibitions of YAP expression and activity. Mol Cancer Res; 15(12); 1752-63. ©2017 AACR.

S100A7 promotes lung adenocarcinoma to squamous carcinoma transdifferentiation, and its expression is differentially regulated by the Hippo-YAP pathway in lung cancer cells.

Our previous study revealed that S100A7 was selectively expressed in lung squamous cell carcinoma tissues but not in adenocarcinoma. Thus far, the functions of S100A7 in lung cancer have remained largely unknown. Here, we reveal that S100A7 overexpression facilitates the transdifferentiation from adenocarcinoma (ADC) to squamous carcinoma (SCC) in several lung cancer cells, which is confirmed by an increase in DNp63 expression and a decrease in thyroid transcription factor 1 (TTF1) and aspartic proteinase napsin (napsin A) expression. Further study finds that activation of the Hippo pathway induces S100A7 expression and further confirms that nuclear YAP acts as a repressor of S100A7 in H292 cells. Subsequently, we verify that TEAD1 is required for YAP transcriptional repression of S100A7. More importantly, we determine that S100A7 overexpression partially rescues lung ADC to SCC transdifferentiation inhibited by YAP overexpression in all tested cells, suggesting that S100A7 and YAP have the opposite effects on lung ADC to SCC conversion. Taken together, our study demonstrates for the first time that S100A7 not only functions as a facilitator of adenous-squamous carcinoma phenotypic transition in lung cancer cells but also that its expression is differentially regulated by the Hippo-YAP pathway.

The Characteristic of S100A7 Induction by the Hippo-YAP Pathway in Cervical and Glossopharyngeal Squamous Cell Carcinoma.

S100A7 is expressed in many squamous cell carcinomas (SCCs). Our previous study revealed that S100A7 was dramatically induced in several SCC cells and activation of the Hippo pathway significantly promoted S100A7 in epidermoid carcinoma cells. However, whether the Hippo pathway regulates S100A7 expression in SCCs remains largely unknown. Here, we uncover that S100A7 induction by the Hippo-YAP pathway displays different characteristic in cervical and glossopharyngeal SCC. In well differentiated HCC94 cervical cells and FaDu pharyngeal cells, S100A7 is easily induced by both suspension and dense culture, which is accompanied by an increase in YAP phosphorylation and a decrease in nuclear YAP. Strikingly, these correlations of S100A7 and YAP reverse after recovery of cell attachment or relief from dense culture. Further examination finds that S100A7 induction is significantly repressed by nuclear YAP, which is validated by activation or inhibition of the Hippo pathway via loss- and/or gain-of- LATS1 and MST1 function. Subsequently, we prove that TEAD1 is required for YAP transcriptional repression of S100A7. However, S100A7 is hardly induced in poorly differentiated SiHa cervical cells and NCI-H226 pulmonary cells even in suspension or activation of the Hippo pathway. More importantly, cervical and lingual SCC tissues array analyses show that S100A7 expression displays the positive correlation with pYAP-S127 and the negative correlation with nuclear YAP in the majority of well differentiated but not in poorly differentiated tissues. Collectively, our findings demonstrate that the different induction of S100A7 toward activation of the Hippo pathway mainly depends on the degree of cell differentiation in cervical and glossopharyngeal SCC.

S100A7 induction is repressed by YAP via the Hippo pathway in A431 cells.

YAP is an oncogenic transcriptional co-activator and is inhibited by the Hippo pathway. Recent studies have revealed that YAP is also a sensor of cell morphology and cell density and can be phosphorylated by cytoskeleton reorganization. Our previous study demonstrated that S100A7 was upregulated in several squamous cell carcinoma (SCC) specimens and was dramatically induced in SCC cells by suspension and dense culture as well as in xenografts. However, little is known about how S100A7 induction occurs in cancer cells. Here, we identify that S100A7 induction is accompanied by YAP phosphorylation in both suspended and dense A431 cells. This correlation reverses after recovery of cell attachment or relief from dense culture. Further examination finds that S100A7 induction is repressed by nuclear YAP, which is further validated by activation or inhibition of the Hippo pathway via loss- and/or gain-of- LATS1 and MST1 function. Strikingly, disruption of the F-actin promotes S100A7 expression via YAP by activation of the Hippo pathway. Furthermore, we demonstrate that repression of S100A7 by YAP required TEAD1 transcriptional factor. Taken together, our findings demonstrate for the first time that S100A7 is repressed by YAP via the Hippo pathway.

Selective expression of transthyretin in subtypes of lung cancer.

Transthyretin (TTR) is expressed primarily in liver, choroid plexus of brain and pancreatic islet A and B cells. It is also synthesized in some endocrine tumors. In the present study, the protein expression of TTR in lung cancer tissues and cell lines was investigated by western blot. The mRNA expression of TTR in 24 pairs of frozen lung cancer tissues was examined by RT-PCR. The specific expression and cellular distribution of TTR were also evaluated in 104 paraffin-embedded lung cancer samples and 3 normal lung tissues by immunohistochemistry. Similarly, the subcellular localization and expression of TTR were further analyzed in lung cancer cell lines. With the exception of mucinous adenocarcinoma, the expression of TTR protein was observed in all tested subtypes of lung carcinoma. Adenocarcinoma displayed the highest positive expression rate of TTR, accounting for 84.4 %, and the positive expression rate of TTR was up to 85.7 % at stages III and IV. The secretory bubbles with strong TTR staining were observed in luminal cells of lung cancer. Furthermore, the localization of TTR in the cytoplasm of lung cancer cells and the secretion of TTR into extracellular milieu were also confirmed. Taken together, TTR is selectively synthesized in lung cancer cells and can be secreted extracellularly.

Depletion of CABYR-a/b sensitizes lung cancer cells to TRAIL-induced apoptosis through YAP/p73-mediated DR5 upregulation.

Our previous study revealed that knockdown of CABYR-a/b increases the chemosensitivity of lung cancer cells through inactivation of Akt. Here, we demonstrated that depletion of CABYR-a/b significantly increased DR5 expression and sensitized lung cancer cells to TRAIL-induced apoptosis in vitro and/or in vivo. Importantly, treatment with AD5-10, a DR5-specific agonistic monoclonal antibody, was able to mimic TRAIL-induced apoptosis in CABYR-a/b-silenced cells. Strikingly, we identified that depletion of CABYR-a/b not only increased the expressions of p73 and DR5 but also decreased the phosphorylation of YAP S127. Loss- or gain-of-function studies of YAP and p73 revealed that double deletions of YAP and p73 effectively decreased the expression of DR5 and abolished TRAIL-induced apoptosis in CABYR-a/b knockdown cells. Conversely, the co-overexpression of YAP and p73 promoted the expression of DR5 and sensitized cells to TRAIL-induced apoptosis. Taken together, our results demonstrate that depletion of CABYR-a/b sensitizes lung cancer cells to TRAIL-induced apoptosis through YAP/p73-mediated DR5 upregulation.

miR-137 inhibits proliferation of melanoma cells by targeting PAK2.

MicroRNAs (miRNA) are key players in a variety of cancers including malignant melanoma. miR-137 has been reported to be a tumor suppressor in melanoma and several targets have been identified for this miRNA. We previously developed a novel proteomics technology, (35) S in vivo/vitro labelling analysis for dynamic proteomics (SiLAD). Because of its high sensitivity in analysing protein expression rates, SiLAD has the potential to unravel miRNA effects on mRNAs coding for proteins with long half-lives or high abundance. Using SiLAD, we discovered that miR-137 significantly downregulated the expression rate of p21-activated kinase 2 (PAK2) in melanoma cells. Bioinformatics analysis predicted PAK2 as a direct target of miR-137, which was confirmed by luciferase reporter assay and Western blot analysis. We found that overexpression of miR-137 inhibited the proliferation of melanoma cells, which could be phenocopied by knockdown of PAK2 using siRNAs. Furthermore, overexpression of PAK2 restored miR-137-mediated suppression of cell proliferation. These findings indicate that miR-137 could inhibit proliferation through targeting PAK2 in melanoma cells.

The Characteristics and Function of S100A7 Induction in Squamous Cell Carcinoma: Heterogeneity, Promotion of Cell Proliferation and Suppression of Differentiation.

S100A7 is highly expressed in squamous cell carcinomas (SCC) and is related to the terminal differentiation of keratinocytes. However, its characteristic and function in SCC is not very known. In this present study, we used immunohistochemistry to examine the expression of S100A7 in 452 SCC specimens, including the lung, esophagus, oral cavity, skin, cervix, bladder, and three SCC cell lines. We found that S100A7-positive staining showed significant heterogeneity in six types of SCC specimen and three SCC cell lines. Further examination found that S100A7-positive cells and its expression at mRNA and protein levels could be induced in HCC94, FaDu, and A-431 cells both in vitro and in vivo using immunohistochemistry, real-time PCR, and Western blotting. Notably, the upregulation of squamous differentiation markers, including keratin-4, keratin-13, TG-1, and involucrin, also accompanied S100A7 induction, and a similar staining pattern of S100A7 and keratin-13 was found in HCC94 cells both in vitro and in vivo. Further study revealed that the overexpression of S100A7 significantly increased proliferation and inhibited squamous differentiation in A-431 cells both in vitro and in vivo. Conversely, silencing S100A7 inhibited cell growth and survival and increased the expression of keratin-4, keratin-13, TG-1, and involucrin in HCC94 cells. Therefore, these results demonstrate that S100A7 displays heterogeneous and inducible characteristic in SCC and also provide novel evidence that S100A7 acts as a dual regulator in promoting proliferation and suppressing squamous differentiation of SCC.

Chronological protein synthesis in regenerating rat liver.

Liver regeneration has been studied for decades; however, its regulation remains unclear. In this study, we report a dynamic tracing of protein synthesis in rat regenerating liver with a new proteomic technique, (35) S in vivo labeling analysis for dynamic proteomics (SiLAD). Conventional proteomic techniques typically measure protein alteration in accumulated amounts. The SiLAD technique specifically detects protein synthesis velocity instead of accumulated amounts of protein through (35) S pulse labeling of newly synthesized proteins, providing a direct way for analyzing protein synthesis variations. Consequently, protein synthesis within short as 30 min was visualized and protein regulations in the first 8 h of regenerating liver were dynamically traced. Further, the 3.5-5 h post partial hepatectomy (PHx) was shown to be an important regulatory turning point by acute regulation of many proteins in the initiation of liver regeneration.

S100A7 acts as a dual regulator in promoting proliferation and suppressing squamous differentiation through GATA-3/caspase-14 pathway in A431 cells.

S100A7 is expressed in many squamous cell carcinomas (SCCs), such as SCC of the skin, and well-differentiated SCC always displays stronger staining of this protein. A431 cells, an epidermal cancer cell line, were selected as a cell model to investigate the roles and mechanism of S100A7 in SCC of the skin. In this study, we demonstrated that the overexpression of S100A7 in A431 cells significantly promoted cell proliferation in vitro and tumor growth in vivo, whereas it suppressed the expression of GATA-3, caspase-14 and three squamous differentiation markers, keratin-1, TG-1 and involucrin. Conversely, the overexpression of caspase-14 not only significantly decreased cell proliferation and delayed tumor growth but also markedly induced the expression of three squamous differentiation markers, whereas S100A7 and GATA-3 were not influenced. Further evidence showed that silencing GATA-3 greatly inhibited the expression of caspase-14 and three differentiation markers, while the expression of S100A7 was not changed; contrary results were obtained when overexpressing GATA-3. Importantly, restoring the expression of GATA-3 and caspase-14 in A431-S100A7 cells could bypass the ability of S100A7 to increase cell viability and repress squamous differentiation. These data suggested that S100A7 expression in SCC may play an important role in the maintenance of SCC cell dedifferentiation, at least in SCC of the skin.

Absence of the first two zinc fingers in BCL6 causes the loss of inhibitory effects on cell growth.

BCL6ZF is a novel transcript of BCL6, which lacks the first two zinc fingers of BCL6. It has been established that BCL6 acts as a sequence­specific transcriptional repressor, however, the functions of BCL6ZF remain undefined. By generating stably overexpressed BCL6 and BCL6ZF in NCI­H1299 lung cancer cells, it was found that BCL6 suppressed the levels of cell growth associated with impaired G1 phase progression compared with those of the mock control cells. However, the effects of BCL6ZF on cell growth and the cell cycle were negligible. Further study of these results demonstrated that eight genes downstream of BCL6 were markedly downregulated by the overexpression of BCL6, whereas BCL6ZF suppressed only TGFBI, indicating that the loss of the first two zinc fingers caused the loss of the inhibitory effects on cell growth and transcriptional repression. In addition, it was determined that the BCL6ZF protein was not degraded as easily as BCL6 protein by the ubiquitin/proteasome pathway, implying that the loss of the first two zinc fingers changes the three­dimensional structure of BCL6ZF. The results demonstrated that BCL6 and BCL6ZF had different role in H1299 cells both in vitro and in vivo. The loss of its inhibitory effects on cell growth and transcriptional repressions.

Knockdown of CABYR-a/b increases chemosensitivity of human non-small cell lung cancer cells through inactivation of Akt.

UNLABELLED: CABYR is a calcium-binding tyrosine phosphorylation-regulated protein that was identified as a novel cancer testis antigen in lung cancer in our previous study. However, the role of CABYR as a driver of disease progression or as a chemosensitizer is poorly understood. This study sought to investigate the relationship between the expression levels of CABYR-a/b, which are the two predominant isoforms of the five isoform proteins encoded by CABYR, and chemosensitivity in non-small cell lung cancer cells. We found that the short hairpin RNA-mediated knockdown of CABYR-a/b significantly inhibited the proliferation of NCI-H460 and A549 cells and resulted in the attenuation of Akt phosphorylation, which is constitutively active in lung cancer cells. The silencing of CABYR-a/b expression notably impacted the downstream components of the Akt pathways: decreasing the phospho-GSK-3ß (Ser9) levels and increasing the expression of the p53 and p27 proteins. Furthermore, CABYR-a/b knockdown led to a significant increase in chemosensitivity in response to chemotherapeutic drugs and drug-induced apoptosis, both in vitro and in vivo. Conversely, the transient transfection of CABYR-a/b-depleted cells with constitutively active Akt partially restored the resistance to cisplatin and paclitaxel and significantly decreased the activation of GSK-3ß and cleaved PARP. Taken together, our results suggest that the inhibition of CABYR-a/b is a novel method to improve the apoptotic response and chemosensitivity in lung cancer and that this cancer testis antigen is an attractive target for lung cancer drug development. IMPLICATIONS: Suppression of CABYR-a/b expression increases chemosensitivity of lung cancer cells by inhibiting Akt activity.

High-confidence de novo peptide sequencing using positive charge derivatization and tandem MS spectra merging.

De novo peptide sequencing holds great promise in discovering new protein sequences and modifications but has often been hindered by low success rate of mass spectra interpretation, mainly due to the diversity of fragment ion types and insufficient information for each ion series. Here, we describe a novel methodology that combines highly efficient on-tip charge derivatization and tandem MS spectra merging, which greatly boosts the performance of interpretation. TMPP-Ac-OSu (succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide) was used to derivatize peptides at N-termini on tips to reduce mass spectra complexity. Then, a novel approach of spectra merging was adopted to combine the benefits of collision-induced dissociation (CID) and electron transfer dissociation (ETD) fragmentation. We applied this methodology to rat C6 glioma cells and the Cyprinus carpio and searched the resulting peptide sequences against the protein database. Then, we achieved thousands of high-confidence peptide sequences, a level that conventional de novo sequencing methods could not reach. Next, we identified dozens of novel peptide sequences by homology searching of sequences that were fully backbone covered but unmatched during the database search. Furthermore, we randomly chose 34 sequences discovered in rat C6 cells and verified them. Finally, we conclude that this novel methodology that combines on-tip positive charge derivatization and tandem MS spectra merging will greatly facilitate the discovery of novel proteins and the proteome analysis of nonmodel organisms.

Multifunctional core-shell upconversion nanoparticles for targeted tumor cells induced by near-infrared light.

Here we synthesize silica-coated NaYF4:Yb/Er nanocomposites with a photosensitizer hypericin covalently bound to silica shells (UCNPs@SiO2@hypericin) successfully, exhibiting precise size-control, good water dispersity and excellent biocompatibility. Under near-infrared light (NIR) irradiation, UCNPs convert NIR light to strong green light which agrees well with the absorbance peak of the photosensitizer hypericin, and triggers hypericin to generate singlet oxygen effectively. The cell apoptosis studies by flow cytometry, fluorescence microscope imaging with Annexin V-FITC/PI and caspase-3 western blotting demonstrate that UCNPs@SiO2@hypericin-FA displays outstanding performance in the induction of apoptosis of Hela cells and HepG2 cells under NIR light irradiation for a short time. At the same time, UCNPs@SiO2@hypericin-FA nanocomposites are proved to exhibit little cytotoxicity by cell viability experiments. By the use of confocal microscopy, cell uptake results show that folate receptor FR(+) cell lines such as Hela cells could internalize more UCNPs@SiO2@hypericin-FA than FR(-) cell lines, such as 293T cells, with highly selective cellular uptake. All the results indicate that UCNPs@SiO2@hypericin-FA nanocomposites have a promising potential in the application of PDT and other diseases in deep tissues.

Selective expression of S100A11 in lung cancer and its role in regulating proliferation of adenocarcinomas cells.

S100A11, one secreted protein, is overexpressed in certain cancers. We investigated S100A11 expression in various subtypes of lung cancer and explored its role in cell proliferation. S100A11 mRNA level was examined in 45 pairs of frozen lung cancer tissues by reverse transcriptase PCR (RT-PCR). The specific expression and subcellular distribution of S100A11 were examined in 78 paraffin-embedded lung cancers, 2 benign lung diseases as well as 22 healthy lung tissues by immunohistochemistry. S100A11 protein level was further analyzed in the sera of 86 lung cancer patients and 50 healthy individuals by enzyme-linked immunosorbent assay. We found that both mRNA and protein levels of S100A11 were overexpressed in adenocarcinomas (ADC) and squamous cell carcinomas (SCC) compared with paired non-cancerous lung tissues, while S100A11 was detected downregulated in small cell lung cancers (SCLC). Further immunohistochemistry staining was positive for S100A11 only in non-small cell lung cancer (NSCLC) (ADC, SCC, large cell carcinomas, et al.), but not SCLC. Conclusively, we found S100A11 protein level increased in the sera of NSCLC patients. Furthermore, when S100A11 expression was knocked down in lung adenocarcinoma cells A549 and LTEP-a-2, the cell proliferation was significantly inhibited in vitro and in vivo.

Triple labeling with three thymidine analogs reveals a well-orchestrated regulation of hepatocyte proliferation during liver regeneration.

AIM: After a two-thirds partial hepatectomy (PHx) in rodents, the remaining cells will proliferate and restore the lost liver mass within 7 days. Previous studies have proved that the residual hepatocytes proliferate in a synchronous manner. However, the existing data can not reflect the chronicle of individual hepatocytes proliferation during liver regeneration. METHODS: In this study, a combination of pulse and continuous labeling using three thymidine analogs, Bromodeoxyuridine (BrdU), Chlorodeoxyuridine (CldU) and Iododeoxyuridine (IdU), were used to analyze the cell proliferation of rat liver after PHx. This strategy allows us to follow an individual cell for more than one cell cycle and to define how many cells and which cells undergo multiple divisions. RESULTS: The residual hepatocytes clustered into three subpopulations to initiate the proliferation sequentially, and the corresponding percentage of each was 32%, 17%, and 36%. Meanwhile, the remaining 15% of hepatocytes never proliferated. In addition, the periportal hepatocytes were the first to respond to PHx stimulation and re-proliferated synchronously at 54 h. Furthermore, at least 11% of residual hepatocytes were identified to divide thrice or more. CONCLUSION: Based on the present analysis, we concluded a sequential model of the initial proliferation in residual hepatocytes, and for the first time, quantitatively elucidated the proliferation manner of three subpopulations during liver regeneration.