TGFß-induced EN1 promotes tumor budding of adenoid cystic carcinoma in patient-derived organoid model.

Adenoid cystic carcinoma (ACC) and basal cell adenoma (BCA) share many histological characteristics and often need a differential diagnosis in clinical pathology. Recently, we found homeobox protein engrailed-1 (EN1) was a potential diagnostic marker for ACC in an organoids library of salivary gland tumors (SGTs). Here we aim to confirm EN1 as a differential diagnostic marker for ACC, and further investigate the regulatory mechanism and biological function of EN1 in tumor progression. The transcriptional analysis, quantitative polymerase chain reaction, Western blot and immunohistochemistry staining were performed and revealed that EN1 was specifically and highly expressed in ACC, and accurately differentiated ACC from BCA. Furthermore, TGFß signaling pathway was found associated with ACC, and the regulation of EN1 through TGFß was detected in the human ACC cell lines and patient-derived organoids (PDOs). TGFß-induced EN1 was important in promoting tumor budding in the PDOs model. Interestingly, a high level of EN1 and TGFß1 in the budding tips was observed in ACC clinical samples, and the expression of EN1 and TGFß1 in ACC was significantly associated with the clinical stage. In summary, our study verified EN1 is a good diagnostic marker to differentiate ACC from BCA. TGFß-induced EN1 facilitates the tumor budding of ACC, which might be an important mechanism related to the malignant phenotype of ACC.

An organoid library of salivary gland tumors reveals subtype-specific characteristics and biomarkers.

BACKGROUND: Salivary gland tumors (SGTs) include a large group of rare neoplasms in the head and neck region, and the heterogeneous and overlapping features among the subtypes frequently make diagnostic difficulties. There is an urgent need to understand the cellular mechanisms underlying the heterogeneity and overlap among the subtypes, and explore the subtype-specific diagnostic biomarkers. METHODS: The tumor tissue and the adjacent normal tissue from the 6 most common types of SGTs were processed for organoid culture which only maintained tumor epithelial cells. Organoids were histologically evaluated based on phenotype markers, followed by transcriptional profiling using RNA-sequencing. The transcriptomic similarities and differences among the subtypes were analyzed by subtype consensus clustering and hierarchical clustering. Furthermore, by comparative transcriptional analysis for these 6 types of SGTs and the matched organoids, the potential diagnostic biomarkers from tumor epithelium were identified, in which two selected biomarkers were evaluated by qPCR and confirmed by immunohistochemistry staining using a tissue microarray. RESULTS: We generated a biobank of patient-derived organoids (PDOs) with 6 subtypes of SGTs, including 21 benign and 24 malignant SGTs. The PDOs recapitulated the morphological and transcriptional characteristics of the parental tumors. The overlap in the cell types and the heterogenous growth patterns were observed in the different subtypes of organoids. Comparing the bulk tissues, the cluster analysis of the PDOs remarkably revealed the epithelial characteristics, and visualized the intrinsic relationship among these subtypes. Finally, the exclusive biomarkers for the 6 most common types of SGTs were uncovered by comparative analysis, and PTP4A1 was demonstrated as a useful diagnostic biomarker for mucoepidermoid carcinoma. CONCLUSIONS: We established the first organoid biobank with multiple subtypes of SGTs. PDOs of SGTs recapitulate the morphological and transcriptional characteristics of the original tumors, which uncovers subtype-specific biomarkers and reveals the molecular distance among the subtype of SGTs.

PKCζ facilitates lymphatic metastatic spread of prostate cancer cells in a mice xenograft model.

Prostate cancer disseminates primarily into the adjacent lymph nodes, which is related to a poor outcome. Atypical protein kinase C ζ (PKCζ) is highly expressed in aggressive prostate cancer and correlates with Gleason score, clinical stage, and poor prognosis. Here, we report the molecular mechanisms of PKCζ in lymphatic metastasis during prostate cancer progression. Using zinc-finger nuclease technology or PKCζ shRNA lentiviral particles, and orthotopic mouse xenografts, we show that PKCζ-knockout or knockdown from aggressive prostate cancer (PC3 and PC3U) cells, decreasesd tumor growth and lymphatic metastasis in vivo. Intriguingly, PKCζ-knockout or knockdown impaired the activation of AKT, ERK, and NF-κB signaling in prostate cancer cells, thereby impairing the expression of lymphangiogenic factors and macrophage recruitment, resulting in aberrant lymphangiogenesis. Moreover, PKCζ regulated the expression of hyaluronan synthase enzymes, which is important for hyaluronan-mediated lymphatic drainage and tumor dissemination. Thus, PKCζ plays a crucial oncogenic role in the lymphatic metastasis of prostate cancer and is predicted to be a novel therapeutic target for prostate cancer.

APPL proteins promote TGFß-induced nuclear transport of the TGFß type I receptor intracellular domain.

The multifunctional cytokine transforming growth factor-ß (TGFß) is produced by several types of cancers, including prostate cancer, and promote tumour progression in autocrine and paracrine manners. In response to ligand binding, the TGFß type I receptor (TßRI) activates Smad and non-Smad signalling pathways. The ubiquitin-ligase tumour necrosis factor receptor-associated factor 6 (TRAF6) was recently linked to regulate intramembrane proteolytic cleavage of the TßRI in cancer cells. Subsequently, the intracellular domain (ICD) of TßRI enters in an unknown manner into the nucleus, where it promotes the transcription of pro-invasive genes, such as MMP2 and MMP9. Here we show that the endocytic adaptor molecules APPL1 and APPL2 are required for TGFß-induced nuclear translocation of TßRI-ICD and for cancer cell invasiveness of human prostate and breast cancer cell lines. Moreover, APPL proteins were found to be expressed at high levels in aggressive prostate cancer tissues, and to be associated with TßRI in a TRAF6-dependent manner. Our results suggest that the APPL-TßRI complex promotes prostate tumour progression, and may serve as a prognostic marker.

CIN85 modulates TGFß signaling by promoting the presentation of TGFß receptors on the cell surface.

Members of the transforming growth factor ß (TGFß) family initiate cellular responses by binding to TGFß receptor type II (TßRII) and type I (TßRI) serine/threonine kinases, whereby Smad2 and Smad3 are phosphorylated and activated, promoting their association with Smad4. We report here that TßRI interacts with the SH3 domains of the adaptor protein CIN85 in response to TGFß stimulation in a TRAF6-dependent manner. Small interfering RNA-mediated knockdown of CIN85 resulted in accumulation of TßRI in intracellular compartments and diminished TGFß-stimulated Smad2 phosphorylation. Overexpression of CIN85 instead increased the amount of TßRI at the cell surface. This effect was inhibited by a dominant-negative mutant of Rab11, suggesting that CIN85 promoted recycling of TGFß receptors. CIN85 enhanced TGFß-stimulated Smad2 phosphorylation, transcriptional responses, and cell migration. CIN85 expression correlated with the degree of malignancy of prostate cancers. Collectively, our results reveal that CIN85 promotes recycling of TGFß receptors and thereby positively regulates TGFß signaling.

TRAF6 stimulates the tumor-promoting effects of TGFß type I receptor through polyubiquitination and activation of presenilin 1.

Transforming growth factor-ß (TGFß) can be both a tumor promoter and suppressor, although the mechanisms behind the protumorigenic switch remain to be fully elucidated. The TGFß type I receptor (TßRI) is proteolytically cleaved in the ectodomain region. Cleavage requires the combined activities of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and TNF-α-converting enzyme (TACE). The cleavage event occurs selectively in cancer cells and generates an intracellular domain (ICD) of TßRI, which enters the nucleus to mediate gene transcription. Presenilin 1 (PS1), a γ-secretase catalytic core component, mediates intramembrane proteolysis of transmembrane receptors, such as Notch. We showed that TGFß increased both the abundance and activity of PS1. TRAF6 recruited PS1 to the TßRI complex and promoted lysine-63-linked polyubiquitination of PS1, which activated PS1. Furthermore, PS1 cleaved TßRI in the transmembrane domain between valine-129 and isoleucine-130, and ICD generation was inhibited when these residues were mutated to alanine. We also showed that, after entering the nucleus, TßRI-ICD bound to the promoter and increased the transcription of the gene encoding TßRI. The TRAF6- and PS1-induced intramembrane proteolysis of TßRI promoted TGFß-induced invasion of various cancer cells in vitro. Furthermore, when a mouse xenograft model of prostate cancer was treated with the γ-secretase inhibitor DBZ {(2S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-propionamide}, generation of TßRI-ICD was prevented, transcription of the gene encoding the proinvasive transcription factor Snail1 was reduced, and tumor growth was inhibited. These results suggest that γ-secretase inhibitors may be useful for treating aggressive prostate cancer.

APC and Smad7 link TGFß type I receptors to the microtubule system to promote cell migration.

Cell migration occurs by activation of complex regulatory pathways that are spatially and temporally integrated in response to extracellular cues. Binding of adenomatous polyposis coli (APC) to the microtubule plus ends in polarized cells is regulated by glycogen synthase kinase 3ß (GSK-3ß). This event is crucial for establishment of cell polarity during directional migration. However, the role of APC for cellular extension in response to extracellular signals is less clear. Smad7 is a direct target gene for transforming growth factor-ß (TGFß) and is known to inhibit various TGFß-induced responses. Here we report a new function for Smad7. We show that Smad7 and p38 mitogen-activated protein kinase together regulate the expression of APC and cell migration in prostate cancer cells in response to TGFß stimulation. In addition, Smad7 forms a complex with APC and acts as an adaptor protein for p38 and GSK-3ß kinases to facilitate local TGFß/p38-dependent inactivation of GSK-3ß, accumulation of ß-catenin, and recruitment of APC to the microtubule plus end in the leading edge of migrating prostate cancer cells. Moreover, the Smad7-APC complex links the TGFß type I receptor to the microtubule system to regulate directed cellular extension and migratory responses evoked by TGFß.

Non-Smad signaling pathways.

Transforming growth factor-beta (TGFß) is a key regulator of cell fate during embryogenesis and has also emerged as a potent driver of the epithelial-mesenchymal transition during tumor progression. TGFß signals are transduced by transmembrane type I and type II serine/threonine kinase receptors (TßRI and TßRII, respectively). The activated TßR complex phosphorylates Smad2 and Smad3, converting them into transcriptional regulators that complex with Smad4. TGFß also uses non-Smad signaling pathways such as the p38 and Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways to convey its signals. Ubiquitin ligase tumor necrosis factor (TNF)-receptor-associated factor 6 (TRAF6) and TGFß-associated kinase 1 (TAK1) have recently been shown to be crucial for the activation of the p38 and JNK MAPK pathways. Other TGFß-induced non-Smad signaling pathways include the phosphoinositide 3-kinase-Akt-mTOR pathway, the small GTPases Rho, Rac, and Cdc42, and the Ras-Erk-MAPK pathway. Signals induced by TGFß are tightly regulated and specified by post-translational modifications of the signaling components, since they dictate the subcellular localization, activity, and duration of the signal. In this review, we discuss recent findings in the field of TGFß-induced responses by non-Smad signaling pathways.

TRAF6 ubiquitinates TGFß type I receptor to promote its cleavage and nuclear translocation in cancer.

Transforming growth factor ß (TGFß) is a pluripotent cytokine promoting epithelial cell plasticity during morphogenesis and tumour progression. TGFß binding to type II and type I serine/threonine kinase receptors (TßRII and TßRI) causes activation of different intracellular signaling pathways. TßRI is associated with the ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here we show that TGFß, via TRAF6, causes Lys63-linked polyubiquitination of TßRI, promoting cleavage of TßRI by TNF-alpha converting enzyme (TACE), in a PKCζ-dependent manner. The liberated intracellular domain (ICD) of TßRI associates with the transcriptional regulator p300 to activate genes involved in tumour cell invasiveness, such as Snail and MMP2. Moreover, TGFß-induced invasion of cancer cells is TACE- and PKCζ- dependent and the TßRI ICD is localized in the nuclei of different kinds of tumour cells in tissue sections. Thus, our data reveal a specific role for TßRI in TGFß mediated tumour invasion.

Adenoviral mediated transduction of adenoid cystic carcinoma by human TRAIL gene driven with hTERT tumor specific promoter induces apoptosis.

Adenoid cystic carcinoma (ACC) is a common malignant tumor in salivary glands. Unfortunately, current treatment modalities which include surgery, radiation and chemotherapy have limited success rates. To develop new treatment strategies we hypothesized that a cancer-specific apoptotic ligand driven by a tumor specific promoter would specifically induce apoptosis in ACC. To test this concept, we selected tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the human telomerase reverse transcriptase (hTERT) promoter. The latter is highly active in 85% of human cancer cells while it is mostly inactive in somatic cells. Using immunohistochemistry we confirmed that ACC samples but not normal salivary cells were positive for hTERT. Similar results were also seen in an ACC cell line, SACC-83. We then constructed first generation Ad5 vectors which used the hTERT promoter to drive TRAIL (AdTERT-TRAIL). Transduction of SACC-83, but not of control human embryo-fibrocyte lung (HEL) cells, led to apoptosis as measured by MTT assay and flow cytomerty. We used the SACC-83 cells for a subcutaneous tumor model in vivo. Intratumoral injections of AdTERT-TRAIL (5 x 109 particles/tumor) but not of AdTERT-EGFP or PBS resulted in significant (p < 0.01) reduction in tumor sizes, which demonstrated that the human TRAIL driven by tumor-specific promoter could efficiently induce apoptosis in SACC-83 cells in vitro and in vivo. These results suggest that a novel gene therapy strategy can also be used in the treatment of ACC in the near future.

[Study of methods of decalcification for making united slices of tooth and affiliated periodontic tissues].

OBJECTIVE: To study the methods of decalcification for making united slices of tooth and affiliated periodontic tissues. METHODS: Twenty-one samples containing dog molars and affiliated periodontic tissues were divided into seven mean groups. The pH value of solution, time of decalcification, weight and volume of samples, and content of decalcified calcium were detected. The slices were observed by HE, specific, and immunohistochemical stain. RESULTS: The velocity of decalcification increased with decrease of solution pH. The weight of samples lightened by 37.61%, the volume reduced by 25.97% on average, and calcium decalcified was 174.49 mg per gram humid samples. The EDTA decalcification was slowest, but it was best. Decalcification was fast in Plank-Rycho solution while the section was worst, and faster in the formyl solution containing aluminium chloride than in EDTA, and the section was better. CONCLUSIONS: The 50% formyl solution containing aluminium chloride is an ideal decalcifying solution.

[Effect of nano-hydroxyapatite to glass ionomer cement].

OBJECTIVE: To investigate the mechanical character, microleakage and mineralizing potential of nano-hydroxyapatite (nano-HAP)-added glass ionomer cement(GIC). METHODS: 8% nano-HAP were incorporated into GIC as composite, and pure GIC as control. Both types of material were used to make 20 cylinders respectively in order to detect three-point flexural strength and compressive strength. Class V cavities were prepared in 120 molars extracted for orthodontic treatment, then were filled by two kinds of material. The microleakage at the composite-dentine interface was observed with confocal laser scanning microscope (CLSM) after stained with 1% rhodamin-B-isothiocyanate for 24 hours. Class V cavities were prepared in the molars of 4 healthy dogs, filled with composite, and the same molars in the other side were filled with GIC as control. The teeth were extracted to observe the mineralizing property with polarimetric microscope in 8 weeks after filling. RESULTS: Three-point flexural strength and compressive of nano-HAP-added GIC were increased compared with pure GIC (P < 0.001, P < 0.05). The nanoleakages and microleakages appeared at the material-dentine interface in the two groups, but there were more microleakages in control group than in experiment group (P = 0.004). New crystals of hydroxyapatite were formed into a new mineralizing zone at the interface of tooth and nano-HAP-added GIC, while there was no hydroxyapatite crystals formed at the interface of tooth and pure GIC. CONCLUSION: 8% nano-HAP-added GIC can tightly fill tooth and have mineralizing potential, and can be used as liner or filling material for prevention.

[Effect of TRAIL gene in human squamous cell carcinoma cell line induced by adenovirus].

OBJECTIVE: To study the apoptotic effect on the squamous cell carcinoma cell line TCa83 induced by recombined adenovirus vector containing TRAIL gene and CMV promoter. METHODS: The TCa83 cell line was firstly infected with different titre of AdCMV-EGFP containing enhanced green fluorescence protein gene (EGFP) as control, and investigated the transducing rate through fluorescence to obtain the definite titre. Then TCa83 cell line was infected with AdCMV-TRAIL in proper titre, and TRAIL gene was detected by means of RT-PCR. After TCa83 cell line was infected with AdCMV-TRAIL and AdCMV-EGFP at day 1, 3, 5, 7, the activity of TCa83 cell line were evaluated by MIT and the apoptosis were detected by flow cytometer. RESULTS: Proper titre was of 1,000 particles/cell, and TCa83 cell line could be infected 100% in this titre. TRAIL gene was detected by RT-PCR after infected with AdCMV-TRAIL. The activity of TCa83 decreased in both groups, but the AdCMV-TRAIL group decreased more sharply than AdCMV-EGFP group (P < 0.001). Both AdCMV-TRAIL and AdCMV-EGFP could lead to apoptosis of TCa83 cells, but the AdCMV-TRAIL, function stronger than AdCMV-EGFP. Especially there was remarkable statistic difference between two groups (P < 0.0001). CONCLUSION: AdCMV-TRAIL could effectively decrease the activity of TCa83 cell line and induce apoptosis.