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Background: Perineural invasion (PNI) frequently occurs in oral squamous cell carcinoma (OSCC) and predicts poor prognosis. Although PNI is increasingly recognised as a process driven by tumour-nerve crosstalk, the underlying molecular mechanisms remain unclear. We investigated the role of sympathetic nerve–derived neuropeptide Y (NPY) and its receptor NPY1R in OSCC PNI. Methods: NPY/NPY1R expression was assessed in human OSCC tissues by immunostaining, qPCR, and TCGA data analysis. Functional studies using Cal27 and SCC9 cells included migration, invasion, and sphere assays. The causal role of NPY1R was tested by lentiviral knockdown/overexpression, validated in tongue orthotopic xenografts, and further examined by NPY1R pharmacological inhibition in vivo. Results: NPY was enriched in the PNI microenvironment, and malignant OSCC expressed high NPY1R, particularly at invasive fronts. Mechanistically, NPY activated ERK and Smad2 via NPY1R, synergising with TGFβ signalling in tumour cells expressing TβRI. This crosstalk enhanced proliferation, invasion, and PNI in vivo. Importantly, NPY1R inhibition markedly reduced tumour growth, metastasis, and PNI. Conclusions: We identify NPY-NPY1R-TGFβ crosstalk as a novel mechanism enabling OSCC to exploit neural signals for PNI, highlighting a promising therapeutic target to block neural invasion and improve patient outcomes.

The repair and regeneration of maxillofacial bone defects are major clinical challenges. Titanium (Ti)-magnesium (Mg) composites are a new generation of revolutionary internal fixation materials encompassing the mechanical strength and bioactive advantages of Ti and Mg alloys, respectively. This study was aimed to construct a Ti-Mg composite internal plate/screw fixation system to fix and repair bone defects. Further, the effects of different internal fixation systems on bone repair were analyzed through radiological and histological analyses. Notably, Ti6Al4V with rolled Mg foil was used as the experimental group, and a bone defect model of transverse complete amputation of the ulna in rabbits similar to the clinical condition was established. The internal fixation system with the highest osteogenic efficiency was selected based on in vivo results, and the direct and indirect bone repair abilities of the selected materials were evaluated in vitro. Notably, the thin Mg foil-Ti6Al4V internal fixation system exhibited the best fixation effect in the bone defect model and promoted the formation of new bone and early healing of bone defect areas. In vitro, the thin Mg foil-Ti6Al4V composite enhanced the activity of MC3T3-E1 cells; promoted the proliferation, adhesion, extension, and osteogenic differentiation of MC3T3-E1 cells; and regulated new bone formation. Further, it also promoted the polarization of RAW264.7 cells to M2 macrophages, induced the osteogenic immune microenvironment, and indirectly regulated the bone repair process. Therefore, a internal fixation system holds a promising potential for the internal fixation of maxillofacial bone defects. Our findings provide a theoretical and scientific basis for the design and clinical application of Ti-Mg internal fixation systems.

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, TGFβ signaling pathway was found associated with ACC, and the regulation of EN1 through TGFβ was detected in the human ACC cell lines and patient-derived organoids (PDOs). TGFβ-induced EN1 was important in promoting tumor budding in the PDOs model. Interestingly, a high level of EN1 and TGFβ1 in the budding tips was observed in ACC clinical samples, and the expression of EN1 and TGFβ1 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. TGFβ-induced EN1 facilitates the tumor budding of ACC, which might be an important mechanism related to the malignant phenotype of ACC.

TGFβ potently modifies the extracellular matrix (ECM), which is thought to favor tumor cell invasion. However, the mechanism whereby the cancer cells employ the ECM proteins to facilitate their motility is largely unknown. In this study we used RNA-seq and proteomic analysis to examine the proteins secreted by castration-resistant prostate cancer (CRPC) cells upon TGFβ treatment and found that thrombospondin 1 (THBS1) was observed to be one of the predominant proteins. The CRISPR Cas9, or siRNA techniques was used to downregulate TGFβ type I receptor (TβRI) to interfere with TGFβ signaling in various cancer cells in vitro. The interaction of ECM proteins with the TβRI in the migratory prostate cancer cells in response to TGFβ1 was demonstrated by several different techniques to reveal that THBS1 mediates cell migration by interacting with integrin subunit alpha V (ITGAV) and TβRI. Deletion of TβRI or THBS1 in cancer cells prevented their migration and invasion. THBS1 belongs to a group of tumorigenic ECM proteins induced via TGFβ signaling in CRPC cells, and high expression of THBS1 in human prostate cancer tissues correlated with the degree of malignancy. TGFβ-induced production of THBS1 through TβRI facilitates the invasion and metastasis of CRPC cells as shown in vivo xenograft animal experiments.

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.

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.

The multifunctional cytokine transforming growth factor-beta (TGF beta) 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 TGF beta type I receptor (T beta RI) 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 T beta RI in cancer cells. Subsequently, the intracellular domain (ICD) of T beta RI 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 TGF beta-induced nuclear translocation of T beta RI-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 T beta RI in a TRAF6-dependent manner. Our results suggest that the APPL-T beta RI complex promotes prostate tumour progression, and may serve as a prognostic marker.

Members of the transforming growth factor beta (TGF beta) family initiate cellular responses by binding to TGF beta receptor type II (Tf3R11) and type I (TpRI) serine/threonine kinases, whereby Srnad2 and Smad3 are phosphorylated and activated, promoting their association with Smadzi. We report here that T beta RI interacts with the SH3 domains of the adaptor protein CIN85 in response to TGF beta stimulation in a TRAF6-dependent manner. Small interfering RNA mediated knockdown of CIN85 resulted in accumulation of T beta RI in intracellular compartments and diminished TGF beta-stimulated Sniad2 phosphorylation. Overexpression of CIN85 instead increased the amount of T beta RI at the cell surface. This effect was inhibited by a dominant-negative mutant of Rab11, suggesting that CIN85 promoted recycling of TGF beta receptors. CIN85 enhanced TGF beta-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 TGF beta receptors and thereby positively regulates TGF beta signaling.

Background:

The Par complex - comprising partition-defective 6 (Par6), Par3, and atypical protein kinase C (aPKC) - is crucial for cell polarisation, the loss of which contributes to cancer progression. Transforming growth factor beta (TGF beta)-induced phosphorylation of Par6 on the conserved serine 345 is implicated in epithelial-to-mesenchymal transition (EMT) in breast cancer. Here we investigated the importance of phosphorylated Par6 in prostate cancer.

Methods:

We generated a p-Par6(345)-specific antibody and verified its specificity in vitro. Endogenous p-Par6(345) was analysed by immunoblotting in normal human prostate RWPE1 and prostate cancer (PC-3U) cells. Subcellular localisation of p-Par6(345) in migrating TGF beta-treated PC-3U cells was analysed by confocal imaging. Invasion assays of TGF beta-treated PC-3U cells were performed. p-Par6 expression was immunohistochemically analysed in prostate cancer tissues.

Results:

TGF beta induced Par6 phosphorylation on Ser345 and its recruitment to the leading edge of the membrane ruffle in migrating PC-3U cells, where it colocalised with aPKC zeta. The p-Par6-aPKC zeta complex is important for cell migration and invasion, as interference with this complex prevented prostate cancer cell invasion. High levels of activated Par6 correlated with aggressive prostate cancer.

Conclusions: Increased p-Par6Ser(345) levels in aggressive prostate cancer tissues and cells suggest that it could be a useful novel biomarker for predicting prostate cancer progression.

Transforming growth factor-beta (TGF beta) can be both a tumor promoter and suppressor, although the mechanisms behind the protumorigenic switch remain to be fully elucidated. The TGF beta type I receptor (T beta RI) is proteolytically cleaved in the ectodomain region. Cleavage requires the combined activities of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and TNF-alpha-converting enzyme (TACE). The cleavage event occurs selectively in cancer cells and generates an intracellular domain (ICD) of T beta RI, which enters the nucleus to mediate gene transcription. Presenilin 1 (PS1), a gamma-secretase catalytic core component, mediates intramembrane proteolysis of transmembrane receptors, such as Notch. We showed that TGF beta increased both the abundance and activity of PS1. TRAF6 recruited PS1 to the T beta RI complex and promoted lysine-63-linked polyubiquitination of PS1, which activated PS1. Furthermore, PS1 cleaved T beta RI 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, T beta RI-ICD bound to the promoter and increased the transcription of the gene encoding T beta RI. The TRAF6- and PS1-induced intramembrane proteolysis of T beta RI promoted TGF beta-induced invasion of various cancer cells in vitro. Furthermore, when a mouse xenograft model of prostate cancer was treated with the gamma-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 T beta RI-ICD was prevented, transcription of the gene encoding the proinvasive transcription factor Snail1 was reduced, and tumor growth was inhibited. These results suggest that gamma-secretase inhibitors may be useful for treating aggressive prostate cancer.