Wnt pathway activation maintains the cancer stem cell (CSC) phenotype and promotes tumor progression, making it an attractive target for anti-cancer therapy. Wnt signaling at the tumor and tumor microenvironment (TME) front have not been investigated in depth in head and neck squamous cell carcinoma (HNSCC). In a cohort of 48 HNSCCs, increased Wnt signaling, including Wnt genes (AXIN2, LGR6, WISP1) and stem cell factors (RET, SOX5, KIT), were associated with a more advanced clinical stage. Key Wnt pathway proteins were most abundant at the cancer epithelial-stromal boundary. To investigate these observations, we generated three pairs of cancer-cancer associated fibroblast (CAF) cell lines derived from the same HNSCC patients. 3D co-culture of cancer spheres and CAFs mimicked these in vivo interactions, and using these we observed increased expression of Wnt genes (eg, WNT3A, WNT7A, WNT16) in both compartments. Of these Wnt ligands, we found Wnt3a, and less consistently Wnt16, activated Wnt signaling in both cancer cells and CAFs. Wnt activation increased CSC characteristics like sphere formation and invasiveness, which was further regulated by the presence of CAFs. Time lapse microscopy also revealed preferential Wnt activation of cancer cells. Wnt inhibitors, OMP-18R5 and OMP-54F28, significantly reduced growth of HNSCC patient-derived xenografts and suppressed Wnt activation at the tumor epithelial-stromal boundary. Taken together, our findings suggest that Wnt signaling is initiated in cancer cells which then activate CAFs, and in turn perpetuate a paracrine signaling loop. This suggests that targeting Wnt signaling in the TME is essential.

BACKGROUND: WNT1-inducible signaling pathway protein 1 (WISP1) is a member of CCN protein family and a downstream target of β-catenin. Aberrant WISP1 expression is associated with carcinogenesis. In the current study, we focused on examining WISP1 single nucleotide polymorphisms (SNPs) to elucidate hepatocellular carcinoma (HCC) clinicopathologic characteristics.
METHODOLOGY/PRINCIPAL FINDINGS: The WISP1 SNPs rs2977530, rs2977537, rs2929973, rs2929970, rs62514004, and rs16893344 were analyzed by real-time polymerase chain reaction in 332 patients with HCC and 664 cancer-free controls.
RESULTS: The patients with higher frequencies of WISP1 rs62514004 (AG + GG) and rs16893344 (CT + TT) variants revealed a lower risk to reach a later clinical stage compared with their wild-type carriers. Furthermore, individuals who carried WISP1 rs62514004 and rs16893344 haplotype G-T showed a greater synergistic effect combined with alcohol drinking on HCC development (AOR = 26.590, 95% CI = 9.780-72.295).
CONCLUSIONS: Our results demonstrated that the HCC patients with WISP1 SNPs are associated with HCC development, and WISP1 SNPs may serve as markers or therapeutic targets for HCC.

Recently, studies on transcriptome-proteome relationships have revealed mRNA/protein expression discordance for certain genes and speculated that protein posttranslational modification (PTM) may be involved. However, there is currently no evidence to support this hypothesis. Wnt-induced secreted protein-1 (WISP1) is the downstream target gene of β-catenin and plays an important role in tumorigenesis and progression, but the expression and role of WISP1 in different tumor types are controversial. Here, we first confirmed that WISP1 protein expression was significantly down-regulated in hepatocellular carcinoma (HCC) tissue and could be an independent predictor of poor prognosis for patients with HCC. In vivo and in vitro evidence was provided that WISP1 can suppress HCC cell proliferation. Further studies have found that low WISP1 protein expression was related to expression of human leukocyte antigen F locus adjacent transcript 10 (FAT10), a specific ubiquitin-like protein with both degradation and stabilization functions, which plays an important role in PTM. FAT10 overexpression facilitated WISP1 degradation by FAT10ylation to decrease WISP1 protein expression, thus promoting HCC proliferation. Interestingly, we found and demonstrated that FAT10 overexpression could result in WISP1 protein/mRNA expression discordance, with protein expression decreasing while mRNA expression increased. The underlying mechanism is that FAT10 exerts substrate stabilization and degradation functions simultaneously, while FAT10 overexpression promotes WISP1 mRNA expression by stabilizing β-catenin and directly degrades WISP1 protein. Conclusion: Our study demonstrated that overexpression of FAT10 results in expression discordance between WISP1 protein and mRNA, thereby promoting HCC progression by down-regulating WISP1 protein expression.

Toll-like receptor (TLR) 2 is a key regulator of innate immune responses and has been shown to play an important role in inflammation-associated cancers. In this study, we aimed to evaluate the role of TLR2 in colorectal cancer (CRC). We demonstrated that TLR2 mRNA and protein expression was significantly upregulated in tumors from CRC patients and indicated poor prognosis. Using the TLR2 agonist Pam3Cys (P3C) to activate TLR2 signaling in human CRC cell lines, we showed that TLR2 drives cellular proliferation, which was dependent upon PI3K/Akt and NF-κB signaling pathways and was associated with the upregulation of anti-apoptotic genes BCL2A1, WISP1 and BIRC3. Likewise, pharmacological blockade of PI3K/Akt and NF-κB pathways mitigated the CRC pro-survival effects of TLR2 stimulation. Furthermore, genetic ablation of TLR2 using CRISPR/Cas9 suppressed CRC cell proliferation, invasion and migration. Taken together, these findings demonstrate that TLR2 plays an important role in colorectal tumorigenesis and may represent a promising therapeutic target in CRC.

Long non-coding RNAs represent a fraction of the transcriptome that is being increasingly recognized. For most of them no function has been allocated so far. Here, we describe the nature and function of a novel non-protein-coding transcript, named WISP1-AS1, discovered in human renal proximal tubule cells exposed to the carcinogenic nephrotoxin ochratoxin A. WISP1-AS1 overlaps parts of the fourth intron and fifth exon of the Wnt1-inducible signaling pathway protein 1 (WISP1) gene. The transcript is 2922 nucleotides long, transcribed in antisense direction and predominantly localized in the nucleus. WISP1-AS1 is expressed in all 20 samples of a human tissue RNA panel with the highest expression levels detected in uterus, kidney and adrenal gland. Its expression was confirmed in primary tissues of human kidneys. In addition, WISP1-AS1 is expressed at higher levels in renal cell carcinoma (RCC) cell lines compared to primary proximal tubule cells as well as in RCC lesions than in the adjacent healthy control tissue from the same patient. Using specific gapmer antisense oligonucleotides to prevent its upregulation, we show that WISP1-AS1 (1) does not influence the mRNA expression of WISP1, (2) affects transcriptional regulation by Egr-1 and E2F as revealed by RNA-sequencing, enrichment analysis and reporter assays, and (3) modulates the apoptosis-necrosis balance. In summary, WISP1-AS1 is a novel lncRNA with modulatory transcriptional function and the potential to alter the cellular phenotype in situations of stress or oncogenic transformation. However, its precise mode of action and impact on cellular functions require further investigations.

Glioblastoma is the most common and aggressive primary brain tumor and has a high mortality in humans. However, mechanisms and factors involved in the progression of glioblastoma remain elusive. WISP1 (WNT1 inducible signaling pathway protein 1), has been suggested to be a critical regulator of cancer development. The aim of this study was to investigate the role of WISP1 in regulating the progression of glioblastoma. Clinicopathological characteristics of glioblastoma were assessed, and higher levels of WISP1 were positively associated with advanced clinical stage and a poor prognosis. Consistently, WISP1 expression was significantly upregulated in glioblastoma tissue and cell lines compared with normal tissue and cells. Additionally, inhibition of WISP1 greatly suppressed cell proliferation, migration, and invasion and promoted apoptosis and cell cycle arrest of glioblastoma cells. Further study indicated that downregulation of WISP1 suppressed cell proliferation associated with the gene expression of c‑myc and cyclin D1 and cellular signaling such as through the ERK pathway, while inhibiting epithelial-mesenchymal transition and MMP9. Finally, knockdown of WISP1 markedly suppressed in vivo tumor growth and sensitized glioblastoma cells to temozolomide. This study identified WISP1 as an oncogene in glioblastoma and suggests that WISP1 may serve as a potential molecular marker and treatment target for glioblastoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC), which is the most common head and neck cancer, accounts for 1%-2% of all human malignancies and is characterized by poor prognosis and reduced survival rates. WNT1-inducible signaling pathway protein 1 (WISP1), a cysteine-rich protein belonging to the Cyr61, CTGF, Nov (CCN) family of matricellular proteins, has many developmental functions and may be involved in carcinogenesis. This study investigated WISP1 single-nucleotide polymorphisms (SNPs) to elucidate OSCC susceptibility and clinicopathologic characteristics.
METHODOLOGY/PRINCIPAL FINDINGS: Real-time polymerase chain reaction was used to analyze 6 SNPs of WISP1 in 900 OSCC patients and 1200 cancer-free controls. The results showed that WISP1 rs2929970 polymorphism carriers with at least one G allele were susceptible to OSCC. Moreover, compared with smokers, non-smoker patients with higher frequencies of WISP1 rs2929970 (AG + GG) variants had a late stage (stages III and IV) and a large tumor size. In addition, OSCC patients who were betel quid chewers and carried WISP1 rs16893344 (CT + TT) variants had a low risk of lymph node metastasis.
CONCLUSION: Our results demonstrate that a joint effect of WISP1 rs2929970 with smoking as well as WISP1 rs16893344 with betel nut chewing causally contributes to the occurrence of OSCC. WISP1 polymorphism may serve as a marker or a therapeutic target in OSCC.

BACKGROUND: Previous work characterized variants of the EL4 murine lymphoma cell line. Some are non-metastatic, and others metastatic, in syngenic mice. In addition, metastatic EL4 cells were stably transfected with phospholipase D2 (PLD2), which further enhanced metastasis.
MATERIALS AND METHODS: Microarray analyses of mRNA expression was performed for non-metastatic, metastatic, and PLD2-expressing metastatic EL4 cells.
RESULTS: Many differences were observed between non-metastatic and metastatic cell lines. One of the most striking new findings was up-regulation of mRNA for the matricellular protein WNT1-inducible signaling pathway protein 1 (CCN4) in metastatic cells; increased protein expression was verified by immunoblotting and immunocytochemistry. Other differentially expressed genes included those for reproductive homeobox 5 (Rhox5; increased in metastatic) and cystatin 7 (Cst7; decreased in metastatic). Differences between PLD2-expressing and parental cell lines were limited but included the signaling proteins Ras guanyl releasing protein 1 (RGS18; increased with PLD2) and suppressor of cytokine signaling 2 (SOCS2; decreased with PLD2).
CONCLUSION: The results provide insights into signaling pathways potentially involved in conferring metastatic ability on lymphoma cells.

BACKGROUND/AIM: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) insufficiency is commonly found in breast cancer patients with metastasis. We investigated the mechanisms by which PTEN affects breast cancer metastatic behavior.
MATERIALS AND METHODS: Migration and invasion assay, western blot, immunofluorescent staining and zebrafish animal model were applied.
RESULTS: We showed that PTEN insufficiency induced an increase in MCF-7 cell migration and invasion through induction of epithelial-mesenchymal transition (EMT), which was triggered by up-regulation of the EMT-inducing transcriptional factors Zeb1, Zeb2, Snail, Slug and Twist. Simultaneously, E-cadherin expression was inhibited and P-cadherin was up-regulated. Further, WNT1 inducible signaling pathway protein 1 (WISP1) and lipocalin-2 (LCN2) expressions were increased after PTEN knockdown in MCF-7 cells, which also exhibited increased filamentous actin (F-actin) synthesis and extracellular matrix metalloproteinase-2 (MMP-2) and MMP-9 expression. We further showed that PTEN knockdown in MCF-7 cells could increase cell migration in the xenograft zebrafish model.
CONCLUSION: Our findings reveal new therapeutic targets for breast cancer patients with PTEN insufficiency.

Colon cancer (CC) likes many epithelial-derived cancers, resulting from a complex tumorigenic process. However, the exactly mechanisms of development and progression of CC are still unknown. In this study, integrated analysis in the GSE33113 and Fudan University Shanghai Cancer Center Hospital datasets revealed that WISP1 expression was significantly increased in CC cases, positivity correlated with the advanced pathologic stage and a poor prognosis was more likely in CC patients with higher levels of WISP1. Downregulation of WISP1 inhibited cell proliferation and invasion through increasing apoptosis and blocking cell cycle at G1 phase in CC LOVO and RKO cells. Besides, Gene set enrichment analysis (GSEA) revealed that relative genes involved in the Cell adhesion molecules and Cytokine-cytokine receptor interaction pathways were enriched in WISP1-higher expression patients. Western blot analysis showed that Cell adhesion molecules pathway associated genes (ICAM- 1, VCAM-1, SDC2 and CDH2) and Cytokine-cytokine receptor interaction pathway associated genes (VEGFC, CCL18, CXCR4 and TGFBR1) were also modulated by WISP1 downregulation. Then, we found that the protein β-catenin was identified as a binding partner of WISP1 and mediated the functions of WISP1 through promoting cell proliferation and invasion in LOVO and RKO cells. Further in vivo tumor formation study in nude mice indicated that inhibition of WISP1 delayed the progress of tumor formation and inhibited PCNA expression. These results indicate that WISP1 could act as an oncogene and may serve as a promising therapeutic strategy for colon cancer.

WISP1, a Wnt-induced secreted protein, has been found to have anticancer activity. ALL is a leading cause of death. Here we investigate the WISP1 effects on ALL Jurkat cells. Cell viability was assessed by CCK-8. Cell cycle and apoptosis were detected by flow cytometry. Mitochondrial membrane potential (MMP) was monitored using TMRM. Generation of reactive oxygen species (ROS) was quantified using DCFH-DA. Western blot was used to detect the expression of cell proliferation and apoptosis related genes. The results showed that knockdown of WISP1 significantly inhibited proliferation of Jurkat cells. Parallelly, cell cycle distribution was increased at G1 phase and apoptotic rate was induced after WISP1 knockdown. Furthermore, knockdown of WISP1 induced apoptosis of Jurkat cells was also associated with loss of MMP and generation of ROS. Western blot results showed that the protein expression p-AKT, PCNA, CDK1, P-ERK, CDK2, VEGF, VEGFR2 and Bcl2 were decreased, while the expression of Bax was up-regulated. In conclusion, WISP1 plays an important role in proliferation and apoptosis of Jurkat cells in mitochondria dependent pathway, the specific mechanisms need further study.

CONTEXT: Somatic mutations in the Med12 gene are known to activate Wnt/β-catenin signaling in human uterine fibroids (UFs).
OBJECTIVE: The objective of the study was to examine the role of vitamin D3 in the modulation of Wnt/β-catenin and mammalian target of rapamycin (mTOR) signaling in human UF cells.
DESIGN: Immortalized human UF cells (HuLM) and human primary UF (PUF) cells were treated with increasing concentrations of vitamin D3 and thereafter analyzed using Western blots and immunocytochemistry.
MAIN OUTCOME MEASURES: Wnt/β-catenin and mTOR signaling proteins in cultured HuLM and PUF cells were measured.
RESULTS: UF tumors with Med12 somatic mutations showed an up-regulation of Wnt4 and β-catenin as compared with adjacent myometrium. Vitamin D3 administration reduced the levels of Wnt4 and β-catenin in both HuLM and PUF cells. Vitamin D3 also reduced the expression/activation of mTOR signaling in both cell types. In contrast, vitamin D3 induced the expression of DNA damaged-induced transcription 4 (an inhibitor of mTOR) and tuberous sclerosis genes (TSC1/2) in a concentration-dependent manner in HuLM cells. Furthermore, we observed a concentration-dependent reduction of Wisp1 (Wnt induced signaling protein 1) and flap endonuclease 1 proteins in HuLM cells. Additionally, abrogation of vitamin D receptor expression (by silencing) in normal myometrial cells induces Wnt4/β-catenin as well as prompts a fibrotic process including an increase in cell proliferation and increased extracellular matrix production. Together these results suggest that vitamin D3 functions as an inhibitor of Wnt4/β-catenin and mTOR signaling pathways, which may play major roles in fibroid pathogenesis.
CONCLUSIONS: Vitamin D3 may have utility as a novel long-term therapeutic and/or preventive option for uterine fibroids.

PURPOSE: To correlate the differentially expressed miRNAs with clinico-pathological features in uveal melanoma (UM) tumors harbouring chromosomal 3 aberrations among South Asian Indian cohort.
METHODS: Based on chromosomal 3 aberration, UM (n = 86) were grouped into monosomy 3 (M3; n = 51) and disomy 3 (D3; n = 35) by chromogenic in-situ hybridisation (CISH). The clinico-pathological features were recorded. miRNA profiling was performed in formalin fixed paraffin embedded (FFPE) UM samples (n = 6) using Agilent, Human miRNA microarray, 8x15KV3 arrays. The association between miRNAs and clinico-pathological features were studied using univariate and multivariate analysis. miRNA-gene targets were predicted using Target-scan and MiRanda database. Significantly dys-regulated miRNAs were validated in FFPE UM (n = 86) and mRNAs were validated in frozen UM (n = 10) by qRT-PCR. Metastasis free-survival and miRNA expressions were analysed by Kaplen-Meier analysis in UM tissues (n = 52).
RESULTS: Unsupervised analysis revealed 585 differentially expressed miRNAs while supervised analysis demonstrated 82 miRNAs (FDR; Q = 0.0). Differential expression of 8 miRNAs: miR-214, miR-149*, miR-143, miR-146b, miR-199a, let7b, miR-1238 and miR-134 were studied. Gene target prediction revealed SMAD4, WISP1, HIPK1, HDAC8 and C-KIT as the post-transcriptional regulators of miR-146b, miR-199a, miR-1238 and miR-134. Five miRNAs (miR-214, miR146b, miR-143, miR-199a and miR-134) were found to be differentially expressed in M3/ D3 UM tumors. In UM patients with liver metastasis, miR-149* and miR-134 expressions were strongly correlated.
CONCLUSION: UM can be stratified using miRNAs from FFPE sections. miRNAs predicting liver metastasis and survival have been identified. Mechanistic linkage of de-regulated miRNA/mRNA expressions provide new insights on their role in UM progression and aggressiveness.

Forward genetic screens using Sleeping Beauty (SB)-mobilized T2/Onc transposons have been used to identify common insertion sites (CISs) associated with tumor formation. Recurrent sites of transposon insertion are commonly identified using ligation-mediated PCR (LM-PCR). Here, we use RNA sequencing (RNA-seq) data to directly identify transcriptional events mediated by T2/Onc. Surprisingly, the majority (∼80%) of LM-PCR identified junction fragments do not lead to observable changes in RNA transcripts. However, in CIS regions, direct transcriptional effects of transposon insertions are observed. We developed an automated method to systematically identify T2/Onc-genome RNA fusion sequences in RNA-seq data. RNA fusion-based CISs were identified corresponding to both DNA-based CISs (Cdkn2a, Mycl1, Nf2, Pten, Sema6d, and Rere) and additional regions strongly associated with cancer that were not observed by LM-PCR (Myc, Akt1, Pth, Csf1r, Fgfr2, Wisp1, Map3k5, and Map4k3). In addition to calculating recurrent CISs, we also present complementary methods to identify potential driver events via determination of strongly supported fusions and fusions with large transcript level changes in the absence of multitumor recurrence. These methods independently identify CIS regions and also point to cancer-associated genes like Braf. We anticipate RNA-seq analyses of tumors from forward genetic screens will become an efficient tool to identify causal events.

OBJECTIVE: The aim of the present study was to investigate the effect of inhibition of endoplasmic reticulum stress signaling mediated by IRE1/ERN1 (inositol-requiring enzyme 1/endoplasmic reticulum to nucleus signaling 1) on the expression of genes encoding different groups of insulin-like growth binding proteins (IGFBP6 and IGFBP7) and CCN family (IGFBP8/CTGF/CCN2, IGFBP9/NOV/CCN3, IGFBP10/CYR61/CCN1, WISP1/CCN4, and WISP2/CCN5) and its sensitivity to glucose deprivation in U87 glioma cells.
METHODS: The expression of IGFBP6, IGFBP7, IGFBP8, IGFBP9, IGFBP10, WISP1, and WISP2 genes was studied by qPCR in control U87 glioma cells (wild-type) and its subline with IRE1 signaling enzyme loss of function upon glucose deprivation.
RESULTS: The expression of IGFBP8, IGFBP9, and WISP2 genes was up-regulated in control glioma cells upon glucose deprivation with most significant changes for IGFBP9 gene. At the same time, the expression of IGFBP6, IGFBP10, and WISP1 genes was resistant to glucose deprivation in these glioma cells, but the IGFBP7 gene expression was down-regulated. The inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in glioma cells modified the sensitivity of most studied gene expressions to glucose deprivation condition: introduced sensitivity of IGFBP10 and WISP1 genes to glucose deprivation, enhanced the effect of this deprivation on IGFBP7 and IGFBP9 gene expressions, and reduced this effect on WISP2 gene and induced suppressive effect of glucose deprivation on the expression of IGFBP8 gene. Furthermore, the inhibition of IRE1 strongly affected the expression of all studied genes in glioma cells upon regular growing condition in gene specific manner: up-regulated the expression levels of IGFBP7, IGFBP8, IGFBP10, WISP1, and WISP2 genes and down-regulated the IGFBP6 and IGFBP9 genes.
CONCLUSIONS: The data of this investigation demonstrate that the expression of IGFBP7, IGFBP8, IGFBP9, and WISP2 genes are sensitive to glucose deprivation in U87 glioma cells and that inhibition of IRE1 signaling enzyme function may significantly affect the expression of all studied genes in the presence of glucose as well as modify the effect of glucose deprivation on the expression of most studied genes. These data also show that proteins encoded by these genes may participate in the regulation of metabolic and proliferative processes via IGF/INS receptors and possibly other signaling pathways as well, via IRE1 signaling, which is a central mediator of the unfolded protein response and an important component of the tumor growth and metabolic diseases.

A role of Wnt signaling in Dupuytren disease, a fibroproliferative disease of the hand and fingers, has not been fully elucidated. We examined a large set of Wnt pathway components and signaling targets and found significant dysregulation of 41 Wnt-related genes in tissue from the Dupuytren nodules compared with patient-matched control tissue. A large proportion of genes coding for Wnt proteins themselves was downregulated. However, both canonical Wnt targets and components of the noncanonical signaling pathway were upregulated. Immunohistochemical analysis revealed that protein expression of Wnt1-inducible secreted protein 1 (WISP1), a known Wnt target, was increased in nodules compared with control tissue, but knockdown of WISP1 using small interfering RNA (siRNA) in the Dupuytren myofibroblasts did not confirm a functional role. The protein expression of noncanonical pathway components Wnt5A and VANGL2 as well as noncanonical coreceptors Ror2 and Ryk was increased in nodules. On the contrary, the strongest downregulated genes in this study were 4 antagonists of Wnt signaling (DKK1, FRZB, SFRP1, and WIF1). Downregulation of these genes in the Dupuytren tissue was mimicked in vitro by treating normal fibroblasts with transforming growth factor β1 (TGF-β1), suggesting cross talk between different profibrotic pathways. Furthermore, siRNA-mediated knockdown of these antagonists in normal fibroblasts led to increased nuclear translocation of Wnt target β-catenin in response to TGF-β1 treatment. In conclusion, we have shown extensive dysregulation of Wnt signaling in affected tissue from Dupuytren disease patients. Components of both the canonical and the noncanonical pathways are upregulated, whereas endogenous antagonists are downregulated, possibly via interaction with other profibrotic pathways.

Lymph node (LN) metastasis is the most important prognostic factor in oral squamous cell carcinoma (OSCC) patients. However, in approximately one third of OSCC patients nodal metastases remain undetected, and thus are not adequately treated. Therefore, clinical assessment of LN metastasis needs to be improved. The purpose of this study was to identify DNA methylation biomarkers to predict LN metastases in OSCC. Genome wide methylation assessment was performed on six OSCC with (N+) and six without LN metastases (N0). Differentially methylated sequences were selected based on the likelihood of differential methylation and validated using an independent OSCC cohort as well as OSCC from The Cancer Genome Atlas (TCGA). Expression of WISP1 using immunohistochemistry was analyzed on a large OSCC cohort (n = 204). MethylCap-Seq analysis revealed 268 differentially methylated markers. WISP1 was the highest ranking annotated gene that showed hypomethylation in the N+ group. Bisulfite pyrosequencing confirmed significant hypomethylation within the WISP1 promoter region in N+ OSCC (P = 0.03) and showed an association between WISP1 hypomethylation and high WISP1 expression (P = 0.01). Both these results were confirmed using 148 OSCC retrieved from the TCGA database. In a large OSCC cohort, high WISP1 expression was associated with LN metastasis (P = 0.05), disease-specific survival (P = 0.022), and regional disease-free survival (P = 0.027). These data suggest that WISP1 expression is regulated by methylation and WISP1 hypomethylation contributes to LN metastasis in OSCC. WISP1 is a potential biomarker to predict the presence of LN metastases.

The matricellular protein WISP1 is a member of the CCN protein family. It is induced by WNT1 and is a downstream target of β-catenin. WISP1 is expressed during embryonic development, wound healing and tissue repair. Aberrant WISP1 expression is associated with various pathologies including osteoarthritis, fibrosis and cancer. Its role in tumor progression and clinical outcome makes WISP1 an emerging candidate for the detection and treatment of tumors.

Our previous work demonstrates that DHX32 is upregulated in colorectal cancer (CRC) compared to its adjacent normal tissues. However, how overexpressed DHX32 contributes to CRC remains largely unknown. In this study, we reported that DHX32 was overexpressed in human colon cancer cells. Overexpressed DHX32 promoted SW480 cancer cells proliferation, migration, and invasion, as well as decreased the susceptibility to chemotherapy agent 5-Fluorouracil. Furthermore, PCR array analyses revealed that depleting DHX32 in SW480 colon cancer cells suppressed expression of WISP1, MMP7 and VEGFA in the Wnt pathway, and anti-apoptotic gene BCL2 and CA9, however, elevated expression of pro-apoptotic gene ACSL5. The findings suggested that overexpressed DHX32 played an important role in CRC progression and metastasis and that DHX32 has the potential to serve as a biomarker and a novel therapeutic target for CRC.

Radiotherapy is a primary treatment modality for esophageal squamous cell carcinoma (ESCC). However, most of patients benefited little from radiotherapy due to refractory radioresistance. We found that WISP1, a downstream target gene of Wnt/β-catenin pathway, was re-expressed in 67.3% of ESCC patients as an oncofetal gene. Expression of WISP1 predicted prognosis of ESCC patients treated with radiotherapy. Overall survival in WISP1-positive patients was significantly poorer than in WISP1-negative patients. Serum concentration of WISP1 after radiotherapy reversely correlated with relapse-free survival. Gain and loss of function studies confirmed that WISP1 mediated radioresistance both in esophageal squamous cancer cells and in xenograft tumor models. Further studies revealed that WISP1 contributed to radioresistance primarily by repressing irradiation-induced DNA damage and activating PI3K kinase. LncRNA BOKAS was up-regulated following radiation and promoted WISP1 expression and resultant radioresistance. Furthermore, WISP1 facilitated its own expression in response to radiation, creating a positive feedback loop and increased radioresistance. Our study revealed WISP1 as a potential target to overcome radioresistance in ESCC.

WNT1 inducible signaling pathway protein 1 (WISP1) plays a key role in many cellular functions in a highly tissue-specific manner; however the role of WISP1 in breast cancer is still poorly understood. Here, we demonstrate that WISP1 acts as an oncogene in human breast cancer. We demonstrated that human breast cancer tissues had higher WISP1 mRNA expression than normal breast tissues and that treatment of recombinant WISP1 enhanced breast cancer cell proliferation. Further, ectopic expression of WISP1 increased the growth of breast cancer cells in vitro and in vivo. WISP1 transfection also induced epithelial-mesenchymal-transition (EMT) in MCF-7 cells, leading to higher migration and invasion. During this EMT-inducing process, E-cadherin was repressed and N-cadherin, snail, and β-catenin were upregulated. Filamentous actin (F-actin) remodeling and polarization were also observed after WISP1 transfection into MCF-7 cells. Moreover, forced overexpression of WISP1 blocked the expression of NDRG1, a breast cancer tumor suppressor gene. Our study provides novel evidence that WISP1-modulated NDRG1 gene expression is dependent on a DNA fragment (-128 to +46) located within the human NDRG1 promoter. Thus, we concluded that WISP1 is a human breast cancer oncogene and is a potential therapeutic target.

The current study was undertaken with a view to identify differential biomarkers in chewing-tobacco-associated oral cancer tissues in patients of Indian ethnicity. The gene expression profile was analyzed in oral cancer tissues as compared to clinically normal oral buccal mucosa. We examined 30 oral cancer tissues and 27 normal oral tissues with 16 paired samples from contralateral site of the patient and 14 unpaired samples from different oral cancer patients, for whole genome expression using high-throughput IlluminaSentrix Human Ref-8 v2 Expression BeadChip array. The cDNA microarray analysis identified 425 differentially expressed genes with >1.5-fold expression in the oral cancer tissues as compared to normal tissues in the oral cancer patients. Overexpression of 255 genes and downregulation of 170 genes (p < 0.01) were observed. Further, a minimum twofold overexpression was observed in 32 genes and downregulation in 12 genes, in 30-83% of oral cancer patients. Biological pathway analysis using Kyoto Encyclopedia of Genes and Genome Pathway database revealed that the differentially regulated genes were associated with critical biological functions. The biological functions and representative deregulated genes include cell proliferation (AIM2, FAP, TNFSF13B, TMPRSS11A); signal transduction (FOLR2, MME, HTR3B); invasion and metastasis (SPP1, TNFAIP6, EPHB6); differentiation (CLEC4A, ELF5); angiogenesis (CXCL1); apoptosis (GLIPR1, WISP1, DAPL1); and immune responses (CD300A, IFIT2, TREM2); and metabolism (NNMT; ALDH3A1). Besides, several of the genes have been differentially expressed in human cancers including oral cancer. Our data indicated differentially expressed genes in oral cancer tissues and may identify prognostic and therapeutic biomarkers in oral cancers, postvalidation in larger numbers and varied population samples.

BACKGROUND: Platinum-based chemotherapy is the main treatment method for lung cancer patients. The genetic polymorphisms of Wnt-inducible signaling pathway protein 1 (WISP1) were reported to be associated with the development of diverse lung diseases. In this study, we aimed to investigate the relationship of WISP1 genetic polymorphisms with lung cancer susceptibility and platinum-based chemotherapy response in Chinese lung cancer patients.
MATERIALS AND METHODS: A total of 556 lung cancer patients and 254 healthy controls were enrolled onto this study. The 28 polymorphisms of the WISP1 gene were genotyped by the Sequenom MassARRAY system.
RESULTS: We found that WISP1 rs16893344, rs2977530, rs2977537, and rs62514004 (P = .009, .033, .049, and .036, respectively) polymorphisms were related to susceptibility of lung cancer; and WISP1 rs11778573 (P = .023, nonsmokers), rs16893344 (P = .013, ≥ 50 years old), rs2977536 (P = .039, ≥ 50 years old; P = .044, nonsmokers; P = .047, non-small-cell lung cancer, respectively), rs2977549 (P = .013, smokers), and rs62514004 (P = .033, ≥ 50 years old) polymorphisms were significantly associated with platinum-based chemotherapy response in lung cancer patients.
CONCLUSION: Genotypes of WISP1 may be novel and useful biomarkers for diagnosis of lung cancer and evaluation of platinum-based chemotherapy response in lung cancer patients.

Studies have reported that the CCN family of proteins plays an important role in stimulating tumorigenesis. However, the relationship between the CCN protein family members and the features of hepatocellular carcinoma (HCC) remains unclear. The objective of this study was to determine the relationship between the expression levels of CCN protein family members and the features of HCC. Expression levels of the CCN family of proteins in 80-paired primary HCC samples and 11 normal liver samples were determined by a quantitative real-time PCR assay. Enhanced expression of nephroblastoma overexpressed protein (NOV) and decreased expression of Wnt-induced secreted protein 1 (WISP1), cysteine-rich protein 61 (CYR61) and connective tissue growth factor (CTGF) were found in HCC samples when compared to levels in matched non-cancerous tissues. No significant difference in WISP2 was found between matched-pair samples; only a few samples showed WISP3 expression. Furthermore, the expression levels of NOV, WISP1 and CYR61 were closely correlated with certain clinical features, including venous invasion, cellular differentiation, pTNM stage, disease-free survival and overall survival. Our results suggest that HCC progression may be enhanced by NOV and suppressed by WISP1 and CYR61. Our statistical analysis suggests that these proteins may be valuable in determining the prognosis of this deadly disease and directs attention to modulating the levels of these proteins as a potential mode of therapy.

Platinum-based chemotherapy toxicity is always one of the serious problems from which lung cancer patients suffer. The genetic polymorphism of WISP1 was revealed to be associated with susceptibility and platinum-based chemotherapy response in our previous studies. In this study, we aimed to investigate the relationship of WISP1 genetic polymorphisms with platinum-based chemotherapy toxicity in lung cancer patients. A total of 412 lung cancer patients were enrolled in this study, and 28 polymorphisms of the WISP1 gene were genotyped by SequenomMassARRAY. We found that WISP1 polymorphisms (rs2929965, rs2929969, rs2929970, rs2929973 and rs754958) were related to the overall chemotherapy toxicity of lung cancer in subgroup analyses. Rs16904853, rs2929970, rs2977549 and rs2977551 (p = 0.021, 0.028, 0.024, 0.048, respectively) polymorphisms were significantly associated with hematologic toxicity. Rs2929946, rs2929970, rs2977519, rs2977536, rs3739262 and rs754958 (p = 0.031, 0.046, 0.029, 0.016, 0.042, 0.035, respectively) polymorphisms were significantly associated with the gastrointestinal toxicity of lung cancer. Genotypes of WISP1 may be novel and useful biomarkers for predicting platinum-based chemotherapy toxicity in lung cancer patients.

To explore the universal law of the abnormal gene expression and the structural variation of genes related to lung adenocarcinoma, the gene expression profile of GSE37765 were downloaded from Gene Expression Omnibus database. The differentially expressed genes (DEGs) were analyzed with t-test and NOISeq tool, and the core DEGs were screened out by combining with another RNA-seq data containing totally 77 pairs of samples in 77 patients with lung adenocarcinoma. Moreover, the functional annotation of the core DEGs was performed by using the Database for Annotation Visualization and Integrated Discovery following selection of oncogene and tumor suppressor by combining with tumor suppressor genes and Cancer Genes database, and motif-finding of core DEGs was performed with motif-finding algorithm Seqpos. We also used Tophat-fusion tool to further explore the fusion genes. In total, 850 downregulated DEGs and 206 upregulated DEGs were screened out in lung adenocarcinoma tissues. Next, we selected 543 core DEGs, including 401 downregulated and 142 upregulated genes, and vasculature development (P=1.89E-06) was significantly enriched among downregulated core genes, as well as mitosis (P=6.26E-04) enriched among upregulated core genes. On the basis of the cellular localization analysis of core genes, wnt-1-induced secreted protein 1 (WISP1) and receptor (G protein-coupled) activity modifying protein 1 (RAMP1) identified mainly located in extracellular region and extracellular space. We also screened one oncogene, v-myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2). Moreover, transcription factor GATA2 was mined by motif-finding analysis. Finally, four fusion genes belonged to the human leukocyte antigen (HLA) family. WISP1, RAMP1, MYBL2 and GATA2 could be potential targets of treatment for lung adenocarcinoma and the fusion of HLA family genes might have important roles in lung adenocarcinoma.

Oral tumors are a growing health problem worldwide; thus, it is mandatory to establish genetic markers in order to improve diagnosis and early detection of tumors, control relapses and, ultimately, delineate individualized therapies. This study was the first to evaluate and discuss the clinical applicability of a multiplex ligation-dependent probe amplification (MLPA) probe panel directed to head and neck cancer. Thirty primary oral squamous cell tumors were analyzed using the P428 MLPA probe panel. We detected genetic imbalances in 26 patients and observed a consistent pattern of distribution of genetic alterations in terms of losses and gains for some chromosomes, particularly for chromosomes 3, 8, and 11. Regarding the latter, some specific genes were highlighted due to frequent losses of genetic material--RARB, FHIT, CSMD1, GATA4, and MTUS1--and others due to gains--MCCC1, MYC, WISP1, PTK2, CCND1, FGF4, FADD, and CTTN. We also verified that the gains of MYC and WISP1 genes seem to suggest higher propensity of tumors localized in the floor of the mouth. This study proved the value of this MLPA probe panel for a first-tier analysis of oral tumors. The probemix was developed to include target regions that have been already shown to be of diagnostic/prognostic relevance for oral tumors. Furthermore, this study emphasized several of those specific genetic targets, suggesting its importance to oral tumor development, to predict patients' outcomes, and also to guide the development of novel molecular therapies.

Innate and type 1 cell-mediated cytotoxic immunity function as important extracellular control mechanisms that maintain cellular homeostasis. Interleukin-12 (IL12) is an important cytokine that links innate immunity with type 1 cell-mediated cytotoxic immunity. We recently observed in vitro that tumor-derived Wnt-inducible signaling protein-1 (WISP1) exerts paracrine action to suppress IL12 signaling. The objective of this retrospective study was three fold: 1) to determine whether a gene signature associated with type 1 cell-mediated cytotoxic immunity was correlated with overall survival, 2) to determine whether WISP1 expression is increased in invasive breast cancer, and 3) to determine whether a gene signature consistent with inhibition of IL12 signaling correlates with WISP1 expression. Clinical information and mRNA expression for genes associated with anti-tumor immunity were obtained from the invasive breast cancer arm of the Cancer Genome Atlas study. Patient cohorts were identified using hierarchical clustering. The immune signatures associated with the patient cohorts were interpreted using model-based inference of immune polarization. Reverse phase protein array, tissue microarray, and quantitative flow cytometry in breast cancer cell lines were used to validate observed differences in gene expression. We found that type 1 cell-mediated cytotoxic immunity was correlated with increased survival in patients with invasive breast cancer, especially in patients with invasive triple negative breast cancer. Oncogenic transformation in invasive breast cancer was associated with an increase in WISP1. The gene expression signature in invasive breast cancer was consistent with WISP1 as a paracrine inhibitor of type 1 cell-mediated immunity through inhibiting IL12 signaling and promoting type 2 immunity. Moreover, model-based inference helped identify appropriate immune signatures that can be used as design constraints in genetically engineering better pre-clinical models of breast cancer.

Aggressive fibromatosis (AF) represents a group of tumors with a variable and unpredictable clinical course, characterized by a monoclonal proliferation of myofibroblastic cells. The optimal treatment for AF remains unclear. Identification and validation of genes whose expression patterns are associated with AF may elucidate biological mechanisms in AF, and aid treatment selection. This study was designed to examine the protein expression by immunohistochemistry (IHC) of four genes, ADAM12, FAP, SOX11, and WISP1, that were found in an earlier study to be uniquely overexpressed in AF compared with normal tissues. Digital image analysis was performed to evaluate inter- and intratumor heterogeneity, and correlate protein expression with histologic features, including a histopathologic assessment of tumor activity, defined by nuclear chromatin density ratio (CDR). AF tumors exhibited marked inter- and intratumor histologic heterogeneity. Pathologic assessment of tumor activity and digital assessment of average nuclear size and CDR were all significantly correlated. IHC revealed protein expression of all four genes. IHC staining for ADAM12, FAP, and WISP1 correlated with CDR and was higher, whereas SOX11 staining was lower in tumors with earlier recurrence following excision. All four proteins were expressed, and the regional variation in tumor activity within and among AF cases was demonstrated. A spatial correlation between protein expression and nuclear morphology was observed. IHC also correlated with the probability of recurrence following excision. These proteins may be involved in AF pathogenesis and the corresponding pathways could serve as potential targets of therapy.

Ablation of tetraspanin protein TSPAN12 from human MDA-MB-231 cells significantly decreased primary tumor xenograft growth, while increasing tumor apoptosis. Furthermore, TSPAN12 removal markedly enhanced tumor-endothelial interactions and increased metastasis to mouse lungs. TSPAN12 removal from human MDA-MB-231 cells also caused diminished association between FZD4 (a key canonical Wnt pathway receptor) and its co-receptor LRP5. The result likely explains substantially enhanced proteosomal degradation of β-catenin, a key effecter of canonical Wnt signaling. Consistent with disrupted canonical Wnt signaling, TSPAN12 ablation altered expression of LRP5, Naked 1 and 2, DVL2, DVL3, Axin 1, and GSKβ3 proteins. TSPAN12 ablation also altered expression of several genes regulated by β-catenin (e.g. CCNA1, CCNE2, WISP1, ID4, SFN, ME1) that may help to explain altered tumor growth and metastasis. In conclusion, these results provide the first evidence for TSPAN12 playing a role in supporting primary tumor growth and suppressing metastasis. TSPAN12 appears to function by stabilizing FZD4-LRP5 association, in support of canonical Wnt-pathway signaling, leading to enhanced β-catenin expression and function.