KITENIN promotes aerobic glycolysis through PKM2 induction by upregulating the c-Myc/hnRNPs axis in colorectal cancer.

PURPOSE: The oncoprotein KAI1 C-terminal interacting tetraspanin (KITENIN; vang-like 1) promotes cell metastasis, invasion, and angiogenesis, resulting in shorter survival times in cancer patients. Here, we aimed to determine the effects of KITENIN on the energy metabolism of human colorectal cancer cells. EXPERIMENTAL DESIGN: The effects of KITENIN on energy metabolism were evaluated using in vitro assays. The GEPIA web tool was used to extrapolate the clinical relevance of KITENIN in cancer cell metabolism. The bioavailability and effect of the disintegrator of KITENIN complex compounds were evaluated by LC-MS, in vivo animal assay. RESULTS: KITENIN markedly upregulated the glycolytic proton efflux rate and aerobic glycolysis by increasing the expression of GLUT1, HK2, PKM2, and LDHA. ß-catenin, CD44, CyclinD1 and HIF-1A, including c-Myc, were upregulated by KITENIN expression. In addition, KITENIN promoted nuclear PKM2 and PKM2-induced transactivation, which in turn, increased the expression of downstream mediators. This was found to be mediated through an effect of c-Myc on the transcription of hnRNP isoforms and a switch to the M2 isoform of pyruvate kinase, which increased aerobic glycolysis. The disintegration of KITENIN complex by silencing the KITENIN or MYO1D downregulated aerobic glycolysis. The disintegrator of KITENIN complex compound DKC1125 and its optimized form, DKC-C14S, exhibited the inhibition activity of KITENIN-mediated aerobic glycolysis in vitro and in vivo. CONCLUSIONS: The oncoprotein KITENIN induces PKM2-mediated aerobic glycolysis by upregulating the c-Myc/hnRNPs axis.

MicroRNA-122 targets δ-catenin to suppress the tumorigenic potential of prostate cancer cells.

δ-Catenin is expressed abundantly in various human cancers, including prostate, brain, breast, and lung carcinomas, and is recognized as an oncogene that promotes cancer cell growth and tumorigenesis. Although several transcriptional and post-translational pathways for δ-catenin regulation have been identified in cancer cells, the potential effects of microRNA-mediated regulation remain elusive. Here, we used a δ-catenin 3'-UTR luciferase reporter assay to identify regulatory microRNAs. Subsequent bioinformatics analyses and molecular studies revealed that overexpression of miR-122 downregulated δ-catenin expression significantly via targeted binding to a seed sequence in the 3'-UTR region of δ-catenin, and suppressed the invasion, migration, and proliferation of prostate cancer cells in vitro. In a TRAMP-C2 mouse syngeneic prostate tumor model, stable expression of miR-122 decreased both δ-catenin expression and tumor growth. Mechanistically, overexpression of miR-122 inhibited the expression of δ-catenin-mediated downstream factors significantly in prostate cancer cells, including c-myc and cyclin D1. In cells overexpressing miR-122, there was no additive or synergistic effect of siRNA-mediated knockdown of δ-catenin on cell invasiveness, and overexpression of miR-122 alone had a more pronounced suppressive effect on cell invasion than knockdown of δ-catenin alone. These results suggest that miR-122 acts as tumor suppressor in prostate cancer, mainly by downregulating δ-catenin expression, but also by targeting other factors. Indeed, subsequent experiments showed that overexpression of miR-122 reduced the levels of the mRNAs encoding myc, snail, and VEGF in prostate cancer cells. Overall, our findings demonstrate that targeting of δ-catenin by miR-122 represses the motility and tumorigenesis of prostate cancer cells, indicating a tumor suppressive effect of this miRNA in prostate cancer.

A peptide interfering with the dimerization of oncogenic KITENIN protein and its stability suppresses colorectal tumour progression.

The stability of a protein, as well as its function and versatility, can be enhanced through oligomerization. KITENIN (KAI1 C-terminal interacting tetraspanin) is known to promote the malignant progression of colorectal cancer (CRC). How KITENIN maintains its structural integrity and stability are largely unknown, however. Here we investigated the mechanisms regulating the stability of KITENIN with the aim of developing therapeutics blocking its oncogenic functions. We found that KITENIN formed a homo-oligomeric complex and that the intracellular C-terminal domain (KITENIN-CTD) was needed for this oligomerization. Expression of the KITENIN-CTD alone interfered with the formation of the KITENIN homodimer, and the amino acid sequence from 463 to 471 within the KITENIN-CTD was the most effective. This sequence coupled with a cell-penetrating peptide was named a KITENIN dimerization-interfering peptide (KDIP). We next studied the mechanisms by which KDIP affected the stability of KITENIN. The KITENIN-interacting protein myosin-X (Myo10), which has oncogenic activity in several cancers, functioned as an effector to stabilize the KITENIN homodimer in the cis formation. Treatment with KDIP resulted in the disintegration of the homodimer via downregulation of Myo10, which led to increased binding of RACK1 to the exposed RACK1-interacting motif (463-471 aa), and subsequent autophagy-dependent degradation of KITENIN and reduced CRC cell invasion. Intravenous injection of KDIP significantly reduced the tumour burden in a syngeneic mouse tumour model and colorectal liver metastasis in an intrasplenic hepatic metastasis model. Collectively, our present results provide a new cancer therapeutic peptide for blocking colorectal liver metastasis, which acts by inducing the downregulation of Myo10 and specifically targeting the stability of the oncogenic KITENIN protein.

Bifunctional Role of CrkL during Bone Remodeling.

Coupled signaling between bone-forming osteoblasts and bone-resorbing osteoclasts is crucial to the maintenance of bone homeostasis. We previously reported that v-crk avian sarcoma virus CT10 oncogene homolog-like (CrkL), which belongs to the Crk family of adaptors, inhibits bone morphogenetic protein 2 (BMP2)-mediated osteoblast differentiation, while enhancing receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated whether CrkL can also regulate the coupling signals between osteoblasts and osteoclasts, facilitating bone homeostasis. Osteoblastic CrkL strongly decreased RANKL expression through its inhibition of runt-related transcription factor 2 (Runx2) transcription. Reduction in RANKL expression by CrkL in osteoblasts resulted in the inhibition of not only osteoblast-dependent osteoclast differentiation but also osteoclast-dependent osteoblast differentiation, suggesting that CrkL participates in the coupling signals between osteoblasts and osteoclasts via its regulation of RANKL expression. Therefore, CrkL bifunctionally regulates osteoclast differentiation through both a direct and indirect mechanism while it inhibits osteoblast differentiation through its blockade of both BMP2 and RANKL reverse signaling pathways. Collectively, these data suggest that CrkL is involved in bone homeostasis, where it helps to regulate the complex interactions of the osteoblasts, osteoclasts, and their coupling signals.

A new KSRP-binding compound suppresses distant metastasis of colorectal cancer by targeting the oncogenic KITENIN complex.

BACKGROUND: Distant metastasis is the major cause of death in patients with colorectal cancer (CRC). Previously, we identified KITENIN as a metastasis-enhancing gene and suggested that the oncogenic KITENIN complex is involved in metastatic dissemination of KITENIN-overexpressing CRC cells. Here, we attempted to find substances targeting the KITENIN complex and test their ability to suppress distant metastasis of CRC. METHODS: We screened a small-molecule compound library to find candidate substances suppressing the KITENIN complex in CRC cells. We selected a candidate compound and examined its effects on the KITENIN complex and distant metastasis through in vitro assays, a molecular docking model, and in vivo tumor models. RESULTS: Among several compounds, we identified DKC1125 (Disintegrator of KITENIN Complex #1125) as the best candidate. DKC1125 specifically suppressed KITENIN gain of function. After binding KH-type splicing regulatory protein (KSRP), DKC1125 degraded KITENIN and Dvl2 by recruiting RACK1 and miRNA-124, leading to the disintegration of the functional KITENIN-KSRP-RACK1-Dvl2 complex. A computer docking model suggested that DKC1125 specifically interacted with the binding pocket of the fourth KH-domain of KSRP. KITENIN-overexpressing CRC cells deregulated certain microRNAs and were resistant to 5-fluorouracil, oxaliplatin, and cetuximab. DKC1125 restored sensitivity to these drugs by normalizing expression of the deregulated microRNAs, including miRNA-124. DKC1125 effectively suppressed colorectal liver metastasis in a mouse model. Interestingly, the combination of DKC1125 with 5-fluorouracil suppressed metastasis more effectively than either drug alone. CONCLUSION: DKC1125 targets the KITENIN complex and could therefore be used as a novel therapeutic to suppress liver metastasis in CRC expressing high levels of KITENIN.

Solitary Fibrous Tumor/Hemangiopericytoma Metastasizes Extracranially, Associated with Altered Expression of WNT5A and MMP9.

Solitary fibrous tumor/hemangiopericytoma (SFT/HPC) is a mesenchymal tumor originating from various soft tissues and meninges, which carries the NAB2-STAT6 fusion gene. Meningeal/intracranial SFT/HPCs (icSFT/HPC) have a poor clinical outcome with metastatic behavior compared to soft tissue/extracranial SFT/HPCs (exSFT/HPC), but the underlying genetic factors are unclear. Differentially expressed genes (DEGs) were analyzed by NanoString nCounter assay using RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue samples. Additionally, immunohistochemistry (IHC) was performed on 32 cases of exSFT/HPC, 18 cases of icSFT/HPC, and additional recurrent or metastatic cases to verify the findings. Pathway analysis revealed that the WNT signaling pathway was enriched in exSFT/HPC. Analysis of DEGs showed that expression of WNT5A was lower and that of MMP9 was higher in icSFT/HPC than in exSFT/HPC (p = 0.008 and p = 0.035, respectively). IHC showed that WNT5A and CD34 expression was high in exSFT/HPC (p < 0.001, both), while that of MMP9 was high in icSFT/HPC (p = 0.001). Expression of CLDN5 in tumoral vessels was locally decreased in icSFT/HPC (p < 0.001). The results suggested that decreased WNT5A expression, together with increased MMP9 expression, in icSFT/HPC, may affect vascular tightness and prompt tumor cells to metastasize extracranially.

Fibroblast growth factor receptor 4 increases epidermal growth factor receptor (EGFR) signaling by inducing amphiregulin expression and attenuates response to EGFR inhibitors in colon cancer.

Fibroblast growth factor receptor 4 (FGFR4) is known to induce cancer cell proliferation, invasion, and antiapoptosis through activation of RAS/RAF/ERK and PI3K/AKT pathways, which are also known as major molecular bases of colon cancer carcinogenesis related with epidermal growth factor receptor (EGFR) signaling. However, the interaction between FGFR4 and EGFR signaling in regard to colon cancer progression is unclear. Here, we investigated a potential cross-talk between FGFR4 and EGFR, and the effect of anti-EGFR therapy in colon cancer treatment. To explore the biological roles of FGFR4 in cancer progression, RNA sequencing was carried out using FGFR4 transfected colon cell lines. Gene ontology data showed the upregulation of genes related to EGFR signaling, and we identified that FGFR4 overexpression secretes EGFR ligands such as amphiregulin (AREG) with consequent activation of EGFR and ErbB3. This result was also shown in in vivo study and the cooperative interaction between EGFR and FGFR4 promoted tumor growth. In addition, FGFR4 overexpression reduced cetuximab-induced cytotoxicity and the combination of FGFR4 inhibitor (BLU9931) and cetuximab showed profound antitumor effect compared to cetuximab alone. Clinically, we found the positive correlation between FGFR4 and AREG expression in tumor tissue, but not in normal tissue, from colon cancer patients and these expressions were significantly correlated with poor overall survival in patients treated with cetuximab. Therefore, our results provide the novel mechanism of FGFR4 in connection with EGFR activation and the combination of FGFR4 inhibitor and cetuximab could be a promising therapeutic option to achieve the optimal response to anti-EGFR therapy in colon cancer.

Adaptor protein CrkII negatively regulates osteoblast differentiation and function through JNK phosphorylation.

The adaptor protein CrkII is involved in several biological activities, including mitogenesis, phagocytosis, and cytoskeleton reorganization. Previously, we demonstrated that CrkII plays an important role in osteoclast differentiation and function through Rac1 activation both in vitro and in vivo. In this study, we investigated whether CrkII also regulates the differentiation and function of another type of bone cells, osteoblasts. Overexpression of CrkII in primary osteoblasts inhibited bone morphogenetic protein (BMP) 2-induced osteoblast differentiation and function, whereas knockdown of CrkII expression exerted the opposite effect. Importantly, CrkII strongly enhanced c-Jun-N-terminal kinase (JNK) phosphorylation, and the CrkII overexpression-mediated attenuation of osteoblast differentiation and function was recovered by JNK inhibitor treatment. Furthermore, transgenic mice overexpressing CrkII under control of the alpha-1 type I collagen promoter exhibited a reduced bone mass phenotype. Together, these results indicate that CrkII negatively regulates osteoblast differentiation and function through JNK phosphorylation. Given that CrkII acts as a negative and positive regulator of osteoblast and osteoclast differentiation, respectively, the regulation of CrkII expression in bone cells may help to develop new strategies to enhance bone formation and inhibit bone resorption.

Recurrent Glioma With Lineage Conversion From Oligodendroglioma to Astrocytoma in Two Cases.

Following the introduction of the molecular classification of gliomas by the WHO in 2016, molecularly-proven lineage conversion during glioma recurrence has never been reported. The reported two cases were initially diagnosed as oligodendroglioma with 1p/19q-codeletion and mutation of isocitrate dehydrogenase 1 (IDH1)-R132H. The recurrent tumors showed loss of alpha-thalassemia/mental retardation X-linked (ATRX) expression, strong P53 positivity, and 1p/19q-nondeletion. Next generation sequencing analysis performed on the first case confirmed the transition of molecular traits from oligodendroglioma to astrocytoma. An IDH mutation of R132H was preserved in the episodes of recurrence, but ATRX and TP53 mutations were newly acquired and TERT promoter mutation C228T was lost at the most recent recurrence. The issue in question for the presented cases is whether the original tumors were pure oligodendrogliomas that then transdifferentiated into astrocytomas, or whether the original tumor was an oligoastrocytoma having oligodendroglioma cells that outnumbered the astrocytoma cells and where the astrocytoma cells becoming more dominant over the episodes of recurrence. With the recognition of the possibility of lineage conversion, our study suggests that molecular examination should be performed to adjust therapeutic strategies in recurrent gliomas. Indeed, our observation of lineage conversion in glioma recurrence calls into question the current distinction drawn between oligodendroglioma, astrocytoma and oligoastrocytoma, rather than simply bidding "farewell to oligoastrocytoma."

MYO1D binds with kinase domain of the EGFR family to anchor them to plasma membrane before their activation and contributes carcinogenesis.

The cell surface receptor tyrosine kinase (RTK) exists in a dynamic state, however, it remains unknown how single membrane-spanning RTK proteins are retained in the plasma membrane before their activation. This study was undertaken to investigate how RTK proteins are anchored in the plasma membrane before they bind with their respective extracellular ligands for activation through protein-protein interaction, co-localization, and functional phenotype studies. Here we show that unconventional myosin-I MYO1D functions to hold members of the EGFR family (except ErbB3) at the plasma membrane. MYO1D binds only with unphosphorylated EGFRs and anchors them to underlying actin cytoskeleton at the plasma membrane. The C-terminal end region of the MYO1D tail domain containing a ß-meander motif is critical for direct binding with kinase domain of the EGFR family, and expression of the tail domain alone suppresses the oncogenic action of full-length MYO1D. Overexpressed MYO1D increases colorectal and breast cancer cell motility and viability through upregulating EGFR level, and thereby promotes colorectal tumor progression in a syngeneic mouse model. MYO1D is upregulated in human colorectal cancer tissues from advanced stages. Collectively, molecular motor MYO1D plays a distinct role in the dynamic regulation of EGFR family levels by holding them at the plasma membrane before their activation. Overexpressed MYO1D contributes to colorectal carcinogenesis possibly as a novel oncogene and thus may serve as an additional target for suppression of RTK signaling in cancer treatment.

Caveolin-1 enhances brain metastasis of non-small cell lung cancer, potentially in association with the epithelial-mesenchymal transition marker SNAIL.

BACKGROUND: Caveolin-1 (Cav-1) plays an important role in the development of various human cancers. We investigated the relationship between Cav-1 expression and non-small cell lung cancer (NSCLC) progression in the context of brain metastasis (BM). METHODS: Cav-1 expression was investigated in a series of 102 BM samples and 49 paired primary NSCLC samples, as well as 162 unpaired primary NSCLC samples with (63 cases) or without (99 cases) metastasis to distant organs. Human lung cancer cell lines were used for in vitro functional analysis. RESULTS: High Cav-1 expression in tumor cells was observed in 52% (38/73) of squamous cell carcinomas (SQCs) and 33% (45/138) of non-SQCs. In SQC, high Cav-1 expression was increased after BM in both paired and unpaired samples of lung primary tumors and BM (53% vs. 84% in paired samples, P = 0.034; 52% vs. 78% in unpaired samples, P = 0.020). Although the difference in median overall survival in patients NSCLC was not statistically significant, high Cav-1 expression in tumor cells (P = 0.005, hazard ratio 1.715, 95% confidence index 1.175-2.502) was independent prognostic factors of overall survival on multivariate Cox regression analyses, in addition to the presence of BM and non-SQC type. In vitro assays revealed that Cav-1 knockdown inhibited the invasion and migration of lung cancer cells. Genetic modulation of Cav-1 was consistently associated with SNAIL up- and down-regulation. These findings were supported by increased SNAIL and Cav-1 expression in BM samples of SQC. CONCLUSIONS: Cav-1 plays an important role in the BM of NSCLC, especially in SQC. The mechanism may be linked to SNAIL regulation.

Deoxypodophyllotoxin Exerts Anti-Cancer Effects on Colorectal Cancer Cells Through Induction of Apoptosis and Suppression of Tumorigenesis.

Deoxypodophyllotoxin (DPT) is a cyclolignan compound that exerts anti-cancer effects against various types of cancers. DPT induces apoptosis and inhibits the growth of breast, brain, prostate, gastric, lung, and cervical tumors. In this study, we sought to determine the effect of DPT on cell proliferation, apoptosis, motility, and tumorigenesis of three colorectal cancer (CRC) cell lines: HT29, DLD1, and Caco2. DPT inhibited the proliferation of these cells. Specifically, the compound-induced mitotic arrest in CRC cells by destabilizing microtubules and activating the mitochondrial apoptotic pathway via regulation of B-cell lymphoma 2 (Bcl-2) family proteins (increasing Bcl-2 associated X (BAX) and decreasing B-cell lymphoma-extra-large (Bcl-xL)) ultimately led to caspase-mediated apoptosis. In addition, DPT inhibited tumorigenesis in vitro, and in vivo skin xenograft experiments revealed that DPT significantly decreased tumor size and tumor weight. Taken together, our results suggest DPT to be a potent compound that is suitable for further exploration as a novel chemotherapeutic for human CRC.

The Role Played by SLUG, an Epithelial-Mesenchymal Transition Factor, in Invasion and Therapeutic Resistance of Malignant Glioma.

In malignant gliomas, invasive phenotype and cancer stemness promoting resurgence of residual tumor cells render treatment very difficult. Hence, identification of epithelial-mesenchymal transition (EMT) factors associated with invasion and stemness of glioma cells is critical. To address the issue, we investigated several EMT factors in hypermotile U87MG and U251 cells, orthotopic mouse glioma model, and human glioma samples. Of several EMT markers, SLUG expression was notably increased at the invasive fronts of gliomas, both in mouse tumor grafts and human glioma samples. The biological role played by SLUG was investigated using a colony-forming assay after chemotherapy and irradiation, and by employing a neurosphere culture assay. The effect of SLUG on glioma progression was examined in our patient cohort and samples, and compared to large public data from the REMBRANDT and TCGA. Genetic upregulation of SLUG was associated with increased levels of stemness factors and enhanced resistance to radiation and temozolomide. In our cohort, patients exhibiting lower-level SLUG expression evidenced longer progression-free survival (P = 0.042). Also, in the REMBRANDT dataset, a group in which SLUG was downregulated exhibited a significant survival benefit (P < 0.001). Although paired glioblastoma samples from our patients did not show a significant increase of SLUG expression, increased mRNA levels of SLUG were found in recurrent glioblastoma from TCGA (P = 0.052), and in temozolomide-treated glioma cells and mouse tumor grafts. SLUG may contribute to glioma progression by controlling invasion at infiltrating margins, associated with increased stemness and therapeutic resistance.

p300/CBP-associated factor promotes autophagic degradation of δ-catenin through acetylation and decreases prostate cancer tumorigenicity.

δ-Catenin shares common binding partners with ß-catenin. As acetylation and deacetylation regulate ß-catenin stability, we searched for histone acetyltransferases (HATs) or histone deacetylases (HDACs) affecting δ-catenin acetylation status and protein levels. We showed that p300/CBP-associated factor (PCAF) directly bound to and acetylated δ-catenin, whereas several class I and class II HDACs reversed this effect. Unlike ß-catenin, δ-catenin was downregulated by PCAF-mediated acetylation and upregulated by HDAC-mediated deacetylation. The HDAC inhibitor trichostatin A attenuated HDAC1-mediated δ-catenin upregulation, whereas HAT or autophagy inhibitors, but not proteasome inhibitors, abolished PCAF-mediated δ-catenin downregulation. The results suggested that PCAF-mediated δ-catenin acetylation promotes its autophagic degradation in an Atg5/LC3-dependent manner. Deletions or point mutations identified several lysine residues in different δ-catenin domains involved in PCAF-mediated δ-catenin downregulation. PCAF overexpression in prostate cancer cells markedly reduced δ-catenin levels and suppressed cell growth and motility. PCAF-mediated δ-catenin downregulation inhibited E-cadherin processing and decreased the nuclear distribution of ß-catenin, resulting in the suppression of ß-catenin/LEF-1-mediated downstream effectors. These data demonstrate that PCAF downregulates δ-catenin by promoting its autophagic degradation and suppresses δ-catenin-mediated oncogenic signals.

Author Correction: PP2A negatively regulates the hypertrophic response by dephosphorylating HDAC2 S394 in the heart.

After the publication of this article, the authors noticed an error in one of the grant numbers (2015R1A2A1A05001708) in the Acknowledgements section.