Hypoxia-activated ADCC-enhanced humanized anti-CD147 antibody for liver cancer imaging and targeted therapy with improved selectivity.

Therapeutic antibodies (Abs) improve the clinical outcome of cancer patients. However, on-target off-tumor toxicity limits Ab-based therapeutics. Cluster of differentiation 147 (CD147) is a tumor-associated membrane antigen overexpressed in cancer cells. Ab-based drugs targeting CD147 have achieved inadequate clinical benefits for liver cancer due to side effects. Here, by using glycoengineering and hypoxia-activation strategies, we developed a conditional Ab-dependent cellular cytotoxicity (ADCC)-enhanced humanized anti-CD147 Ab, HcHAb18-azo-PEG5000 (HAP18). Afucosylated ADCC-enhanced HcHAb18 Ab was produced by a fed-batch cell culture system. Azobenzene (Azo)-linked PEG5000 conjugation endowed HAP18 Ab with features of hypoxia-responsive delivery and selective targeting. HAP18 Ab potently inhibits the migration, invasion, and matrix metalloproteinase secretion, triggers the cytotoxicity and apoptosis of cancer cells, and induces ADCC, complement-dependent cytotoxicity, and Ab-dependent cellular phagocytosis under hypoxia. In xenograft mouse models, HAP18 Ab selectively targets hypoxic liver cancer tissues but not normal organs or tissues, and has potent tumor-inhibiting effects. HAP18 Ab caused negligible side effects and exhibited superior pharmacokinetics compared to those of parent HcHAb18 Ab. The hypoxia-activated ADCC-enhanced humanized HAP18 Ab safely confers therapeutic efficacy against liver cancer with improved selectivity. This study highlights that hypoxia activation is a promising strategy for improving the tumor targeting potential of anti-CD147 Ab drugs.

Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

Morphine is a frequently used analgesic that activates the mu-opioid receptor (MOR), which has prominent side effects of tolerance. Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance, currently, there is no effective therapy to treat morphine tolerance. In the current study, we aimed to develop a monoclonal antibody (mAb) precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms. We successfully prepared a mAb targeting MOR, named 3A5C7, by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization, and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation. Treatment of two cell lines, HEK293T and SH-SY5Y, with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2 (GRK2)/ß-arrestin2-dependent mechanism, as demonstrated by immunofluorescence staining, flow cytometry, Western blotting, coimmunoprecipitation, and small interfering ribonucleic acid (siRNA)-based knockdown. This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR. We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid. Western blot, enzyme-linked immunosorbent assays, and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase, the in vitro biomarker of morphine tolerance, via the GRK2/ß-arrestin2 pathway. Furthermore, in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice, and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence. Finally, intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/ß-arrestin2 pathway. Collectively, our study provided a therapeutic mAb, 3A5C7, targeting MOR to treat morphine tolerance, mediated by enhancing morphine-induced MOR endocytosis. The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

Targeting the up-regulated CNOT3 reverses therapeutic resistance and metastatic progression of EGFR-mutant non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related mortality worldwide. CNOT3, a subunit of the CCR4-NOT complex, has recently been suggested to be overexpressed in lung cancer and involved in tumor malignancy. However, its precise role and the underlying mechanisms still need to be fully revealed. In the present study, we found in lung cancer cells the expression of CNOT3 could be regulated by EGFR signaling pathway and c-Jun, a transcription factor downstream of EGFR, transcriptionally regulated its expression. Interestingly, CNOT3 could inversely regulate the expression of c-Jun via modulating its translation. Thus, a feedback loop existed between c-Jun and CNOT3. CNOT3 reduction post EGFR blockade facilitated the drug-induced cell death, and simultaneously inhibited cell proliferation via impacting TSC1/mTOR axis. Whereas, further up-regulation of the CNOT3 expression was observed in gefitinib-resistant cells, which dampened gefitinib sensitivity. Mechanically, the elevation of CNOT3 was induced by the bypass activation of HER2/c-Jun signaling. Depleting CNOT3 in vitro and in vivo sensitized the drug-resistant cells to gefitinib treatment and inhibited metastatic progression. These results give novel insights into the role of CNOT3 in lung cancer malignancy and provide a theoretical basis for the development of therapeutic strategies to solve acquired resistance to EGFR-TKIs.

PDGFA-associated protein 1 is a novel target of c-Myc and contributes to colorectal cancer initiation and progression.

BACKGROUND: The mechanism underlying colorectal cancer (CRC) initiation and progression remains elusive, and overall survival is far from satisfactory. Previous studies have shown that PDGFA-associated protein 1 (PDAP1) is upregulated in several cancers including CRC. Here, we aimed to identify the cause and consequence of PDAP1 dysregulation in CRC and evaluate its role as a potential therapeutic target. METHODS: Multi-omics data analysis was performed to identify potential key players in CRC initiation and progression. Immunohistochemistry (IHC) staining was applied to determine the expression pattern of PDAP1 in CRC tissues. Pdap1 conditional knockout mice were used to establish colitis and CRC mouse models. RNA sequencing, a phosphoprotein antibody array, western blotting, histological analysis, 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, and interactome analysis were applied to identify the underlying mechanisms of PDAP1. A human patient-derived xenograft (PDX) model was used to assess the potential of PDAP1 as a therapeutic target. RESULTS: PDAP1 was identified as a potential key player in CRC development using multi-omics data analysis. PDAP1 was overexpressed in CRC cells and correlated with reduced overall survival. Further investigation showed that PDAP1 was critical for the regulation of cell proliferation, migration, invasion, and metastasis. Significantly, depletion of Pdap1 in intestinal epithelial cells impaired mucosal restitution in dextran sulfate sodium salt-induced colitis and inhibited tumor initiation and growth in colitis-associated cancers. Mechanistic studies showed that c-Myc directly transactivated PDAP1, which contributed to the high PDAP1 expression in CRC cells. PDAP1 interacted with the juxtamembrane domain of epidermal growth factor receptor (EGFR) and facilitated EGFR-mitogen-activated protein kinase (MAPK) signaling activation, which resulted in FOS-related antigen 1 (FRA-1) expression, thereby facilitating CRC progression. Notably, silencing of PDAP1 could hinder the growth of patient-derived xenografts that sustain high PDAP1 levels. CONCLUSIONS: PDAP1 facilitates mucosal restitution and carcinogenesis in colitis-associated cancer. c-Myc-driven upregulation of PDAP1 promotes proliferation, migration, invasion, and metastasis of CRC cells via the EGFR-MAPK-FRA-1 signaling axis. These findings indicated that PDAP1 inhibition is warranted for CRC patients with PDAP1 overexpression.

CD147 receptor is essential for TFF3-mediated signaling regulating colorectal cancer progression.

Major gaps in understanding the molecular mechanisms of colorectal cancer (CRC) progression and intestinal mucosal repair have hampered therapeutic development for gastrointestinal disorders. Trefoil factor 3 (TFF3) has been reported to be involved in CRC progression and intestinal mucosal repair; however, how TFF3 drives tumors to become more aggressive or metastatic and how TFF3 promotes intestinal mucosal repair are still poorly understood. Here, we found that the upregulated TFF3 in CRC predicted a worse overall survival rate. TFF3 deficiency impaired mucosal restitution and adenocarcinogenesis. CD147, a membrane protein, was identified as a binding partner for TFF3. Via binding to CD147, TFF3 enhanced CD147-CD44s interaction, resulting in signal transducer and activator of transcription 3 (STAT3) activation and prostaglandin G/H synthase 2 (PTGS2) expression, which were indispensable for TFF3-induced migration, proliferation, and invasion. PTGS2-derived PGE2 bound to prostaglandin E2 receptor EP4 subtype (PTGER4) and contributed to TFF3-stimulated CRC progression. Solution NMR studies of the TFF3-CD147 interaction revealed the key residues critical for TFF3 binding and the induction of PTGS2 expression. The ability of TFF3 to enhance mucosal restitution was weakened by a PTGS2 inhibitor. Blockade of TFF3-CD147 signaling using competitive inhibitory antibodies or a PTGS2 inhibitor reduced CRC lung metastasis in mice. Our findings bring strong evidence that CD147 is a novel receptor for TFF3 and PTGS2 signaling is critical for TFF3-induced mucosal restitution and CRC progression, which widens and deepens the understanding of the molecular function of trefoil factors.

Endothelial genetic deletion of CD147 induces changes in the dual function of the blood-brain barrier and is implicated in Alzheimer's disease.

AIMS: The blood-brain barrier (BBB) is a specialized and indispensable structure in brain blood vessels that is damaged during Alzheimer's disease (AD). CD147 is expressed on the BBB and deeply engaged in the AD pathological process. In this study, we aimed to provide a better understanding of the roles of CD147 in BBB function in health and neurodegenerative disease. METHODS AND RESULTS: We measured CD147 expression in mouse brains and demonstrated that CD147 is exclusively expressed in brain endothelial cells (BECs), and its expression decreases with age. After constructing endothelial-specific CD147 knockout mice, we performed RNA-sequencing on BECs isolated from mice of different ages as well as a range of database analyses. We found that endothelial CD147 is essential for the dual functions of the BBB, including barrier maintenance and transporter regulation. This study also shows that CD147 plays a pivotal role in neurodegenerative diseases, particularly in AD. CONCLUSIONS: Our findings suggested that targeting CD147 in BECs may represent a novel therapeutic strategy, which promoted the design of future experimental investigations and the mechanistic understanding of neurodegenerative diseases.

Prognostic Landscape of Tumor-Infiltrating T and B Cells in Human Cancer.

Recently, immunotherapy targeting tumor-infiltrating lymphocytes (TILs) has emerged as a critical and promising treatment in several types of cancer. However, not all cancer types have been tested in immunotherapeutic trials, and different patients and cancer types may have unpredictable clinical outcomes. This situation has created a particular exigency for analyzing the prognostic significance of tumor-infiltrating T cells (TIL-T) and B cells (TIL-B) across different cancer types. To address the critical role of TILs, the abundances of TIL-T and TIL-B cells, as determined by the protein levels of LCK and CD20, were analyzed across heterogeneous human malignancies. TIL-T and TIL-B cells showed varying prognostic significances across heterogeneous cancer types. Additionally, distinct distributions of TIL-T and TIL-B cells were observed in different cancer and tumor microenvironment (TME) subtypes. Next, we analyzed the cellular context for the TME communication network involving the well-acknowledgeable chemokine receptors of TIL-T and TIL-B cells, implying the functional interactions with TME. Additionally, these chemokine receptors, expressed by TIL-T and TIL-B cells, were remarkably correlated with the levels of TIL-T or TIL-B cell infiltrations across nearly all the cancer types, indicating these chemokine receptors as universal targets for up- and down-regulating the TIL-T and TIL-B cells. Lastly, we provide the prognostic landscape of TIL-T and TIL-B cells across 30 cancer types and the subgroups defined by gender, histopathology, histological grade, therapeutic approach, drug, and TME subtype, which are intended to be a resource to fuel the investigations of TILs, with important implications for cancer immunotherapy.

CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells.

In face of the everlasting battle toward COVID-19 and the rapid evolution of SARS-CoV-2, no specific and effective drugs for treating this disease have been reported until today. Angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, mediates the virus infection by binding to spike protein. Although ACE2 is expressed in the lung, kidney, and intestine, its expressing levels are rather low, especially in the lung. Considering the great infectivity of COVID-19, we speculate that SARS-CoV-2 may depend on other routes to facilitate its infection. Here, we first discover an interaction between host cell receptor CD147 and SARS-CoV-2 spike protein. The loss of CD147 or blocking CD147 in Vero E6 and BEAS-2B cell lines by anti-CD147 antibody, Meplazumab, inhibits SARS-CoV-2 amplification. Expression of human CD147 allows virus entry into non-susceptible BHK-21 cells, which can be neutralized by CD147 extracellular fragment. Viral loads are detectable in the lungs of human CD147 (hCD147) mice infected with SARS-CoV-2, but not in those of virus-infected wild type mice. Interestingly, virions are observed in lymphocytes of lung tissue from a COVID-19 patient. Human T cells with a property of ACE2 natural deficiency can be infected with SARS-CoV-2 pseudovirus in a dose-dependent manner, which is specifically inhibited by Meplazumab. Furthermore, CD147 mediates virus entering host cells by endocytosis. Together, our study reveals a novel virus entry route, CD147-spike protein, which provides an important target for developing specific and effective drug against COVID-19.

Detachment Activated CyPA/CD147 Induces Cancer Stem Cell Potential in Non-stem Breast Cancer Cells.

BACKGROUND: Cancer stem cells (CSCs), responsible for cancer metastasis and recurrence, are generated from non-CSCs after chemo-radiation therapy. This study investigated the induction of CSC potential in non-stem breast cancer cells and the underlying molecular mechanisms in detachment culture. METHODS: Bulk breast cancer cells, or sorted non-CSCs and CSCs were cultured under an attached or detached condition to assess CSC numbers, ability to form tumor spheres, expression of stemness markers, and chemoresistance. Lentivirus carrying CD147 shRNA or cDNA was used to manipulate CD147 expression, while CD147 ligand recombinant cyclophilin A (CyPA) or its inhibitor was used to activate or inhibit CD147 signaling. RESULTS: Detachment promoted anoikis resistance, chemoresistance, sphere formation, self-renewal, and expression of stemness markers in breast cancer cells. Detachment increased functional ALDH+ or CD44highCD24-/low CSCs, and induced CSC potential in ALDH- or CD44 low CD24high non-CSCs. Upon detachment, both CD147 expression and CyPA secretion were enhanced, and CyPA-CD147 activation mediated detachment induced CSC potential in non-CSCs via STAT3 signaling. Clinically, CD147 and pSTAT3 were highly co-expressed and correlated with poor overall survival and tumor recurrence in breast cancer patients. CONCLUSION: This study demonstrates that detachment induces the generation of CSCs from non-stem breast cancer cells via CyPA-CD147 signaling, indicating that targeting CD147 may serve as a potential novel therapeutic strategy for lethal metastatic breast cancer by eliminating induced CSCs.

CD147 promotes collective invasion through cathepsin B in hepatocellular carcinoma.

BACKGROUND: Mounting evidence suggests that solid tumors display the features of collective invasion, however, the molecular mechanisms are far from clear. This study aims to verify the role and the underlying mechanisms of CD147 in collective invasion in hepatocellular carcinoma. METHODS: Immunostaining was used to analyze human hepatocellular carcinoma specimens and three-dimensional cultures. Three-dimensional invasion model was established to mimic in vivo invasion. RNA-sequencing was used to identify downstream effectors. RESULTS: Human hepatocellular carcinoma underwent collective invasion and CD147 was observed to be upregulated at the invasive front of tumor cell groups. CD147 was demonstrated to promote collective invasion using the modified three-dimensional invasion model, which recapitulated the main features of collective invasion. Through transcriptome analysis and enzyme activity assay, we found that CD147 enhanced cathepsin B expression and activity. Upregulated cathepsin B in hepatocellular carcinoma cells facilitated migration and invasion, which mediated CD147-induced invasive phenotype in hepatocellular carcinoma. In terms of mechanism, we found that CD147 promoted cathepsin B transcription by activating ß-catenin signaling as a result of reduced GSK-3ß expression. Furthermore, we found that elevated expression of CD147 as well as cathepsin B were correlated with poor prognosis in patients with hepatocellular carcinoma. CONCLUSIONS: CD147 promotes hepatocellular carcinoma cells collective invasion via upregulating cathepsin B expression and targeting CD147 would be valuable for the development of novel therapeutic modalities against invasion and metastasis of cancer.

CD147 promotes DNA damage response and gemcitabine resistance via targeting ATM/ATR/p53 and affects prognosis in pancreatic cancer.

The acquisition of chemoresistance is a major clinical challenge for pancreatic cancer (PC) treatment. Chemoresistance is largely attributed to aberrant DNA damage repair. However, the underlying mechanisms of chemoresistance in pancreatic cancer remain unclear. Here, we showed that CD147 was strongly correlated to DNA damage response (DDR) indices and poor prognosis in pancreatic ductal adenocarcinoma (PDAC) patients. CD147 knockdown or monoclonal antibodies improved the killing effects of gemcitabine in gemcitabine resistant cells, exhibiting reduced activation of ATM/p53. Moreover, we found the interaction of CD147 with ATM, ATR and p53, which was augmented in gemcitabine resistant cells. High CD147/p-ATM/p-ATR/p-p53 cytoplasmic expression associated with poor survival of PC patients. Our studies thus identify CD147 as a critical player in DDR programing that affects gemcitabine therapeutic outcomes of pancreatic cancer patients.

Synthesis and Evaluation of a Novel Small-molecule Compound as an Anticancer Inhibitor of CD147.

OBJECTIVE: Cancer is a serious threat to human health. Despite extensive research on cancer treatment, there is a growing demand for new therapies. CD147 is widely involved in tumor development, but it is unclear whether cancer cell malignancy is affected by CD147 expression level. The first compound (AC-73) targeting CD147 could only act on advanced tumors and inhibit metastasis. Therefore, new compounds with better anticancer activity should be explored. METHODS: Wst-1 assays were used to confirm the effect of novel compounds on proliferation. Apoptosis tests were used to evaluate their proapoptotic capacity. A nude mouse model was used to demonstrate in vivo anticancer activity and safety of the compounds. Western blots were used to suggest a molecule mechanism. RESULTS: There is a positive correlation between CD147 expression and tumor cell proliferation. A new compound, HA-08, was synthesized and proved to be more active than AC-73. HA-08 could inhibit cancer cell viability and promote cancer cell apoptosis both in vitro and in vivo. HA-08 induces cancer apoptosis, mainly by disrupting the CD147-CD44 interaction and then down-regulating the JAK/STAT3/Bcl-2 signaling pathway. CONCLUSION: Our results have clarified the tumor specificity of CD147 and its drug target characteristics. The biological profile of HA-08 suggests that this compound could be developed as a potential anticancer agent.

Therapeutic anti-CD147 antibody sensitizes cells to chemoradiotherapy via targeting pancreatic cancer stem cells.

We have previously demonstrated that anti-CD44s H4C4 or liposomal-delivered STAT3 inhibitor FLLL32 sensitized pancreatic cancer cells to radiotherapy through the elimination or inhibition of cancer stem cells (CSCs) and that HAb18G/CD147 promoted STAT3-mediated pancreatic tumor development by forming a signaling complex with CD44s. In this paper, we therefore explored whether anti-CD147 HAb18IgG sensitized pancreatic cancer cells to chemoradiotherapy via the targeting of CSCs. We tested the influence of HAb18IgG on the sensitivity of pancreatic cancer cells to chemoradiotherapy by clonogenic and MTT assays and on pancreatic CSCs by colony and sphere formation assays, flow cytometry, quantitative real-time RT-PCR (qRT-PCR) and stem cell transcription factors PCR array analysis. Changes in CD147 signaling were examined by immunoblot and reporter assays. We found that HAb18IgG sensitized pancreatic cancer cells to chemoradiotherapy by dose-dependently decreasing colony and sphere formation. Furthermore, HAb18IgG reduced the pancreatic CSC subpopulation and the expression of stem cell transcription factors OCT4, SOX2 and NANOG. Mechanistically, HAb18IgG inhibited CSCs by blocking CD44s-pSTAT3 signaling. The present findings indicated the promising therapeutic role of anti-CD147 HAb18IgG in suppressing pancreatic tumor initiation and overcoming post-chemoradiotherapy recurrence through the direct targeting of CSCs.

RIP3 promotes colitis-associated colorectal cancer by controlling tumor cell proliferation and CXCL1-induced immune suppression.

Rationale: Necroptosis is a programmed form of non-apoptotic cell death that requires receptor-interacting protein 3 (RIP3). RIP3 has been shown to be relevant in multiple tumor types and has differential impact on tumor progression. We investigated whether RIP3 is involved in the progression of colitis-associated cancer (CAC) in mice. Methods: Tissues from colorectal cancer patients were examined for RIP3 expression. CAC was induced using azoxymethane (AOM) injection followed by dextran sodium sulfate (DSS) treatment in RIP3-deficient or wild-type mice. Colon tissues were collected and analyzed by Western blotting and gene expression profile analyses. Immune cell infiltration and CXCL1 expression were examined by flow cytometry and Real-time PCR, respectively. Results: RIP3 expression was upregulated in mouse CAC and human colon cancer. RIP3-deficient mice showed significantly attenuated colitis-associated tumorigenesis. Bone marrow transplantation experiments suggested that RIP3's function in hematopoietic cells primarily contributes to the phenotype. RIP3 supported epithelial proliferation and tumor growth via JNK signaling but had no effect on apoptosis. RIP3 deletion increased T cell accumulation and reduced infiltration by immunosuppressive subsets of myeloid cells during acute colitis and CAC. The immune-suppressive tumor microenvironment was dependent on RIP3-induced expression of the chemokine attractant CXCL1, and administration of recombinant CXCL1 during CAC restored tumorigenesis in Rip3-/- mice. Conclusion: Our results reveal an unexpected function of RIP3 in enhancing the proliferation of premalignant intestinal epithelial cells (IECs) and promoting myeloid cell-induced adaptive immune suppression. These two distinct mechanisms of RIP3-induced JNK and CXCL1 signalling contribute to CAC progression.

CNOT3 contributes to cisplatin resistance in lung cancer through inhibiting RIPK3 expression.

Chemotherapeutic resistance always results in poor clinical outcomes of cancer patients and its intricate mechanisms are large obstacles in overcoming drug resistance. CCR4-NOT transcription complex subunit 3 (CNOT3), a post-translational regulator, is suggested to be involved in cancer development and progression. However, its role in chemotherapeutic resistance is not well understood. In this study, after screening the CNOT3 mRNA in a cancer microarray database called Oncomine and examining the expression levels of CNOT3 mRNA in normal tissues and lung cancer tissues, we found that CNOT3 was up-regulated in lung cancer tissues. Besides, its high-expression was associated with poor prognosis of lung cancer patients. We also found higher expression level of CNOT3 and lower expression level of receptor-interacting protein kinase 3 (RIPK3) in cisplatin-resistant A549 (A549/DDP) cells, and knocking down CNOT3 expression could sensitize A549/DDP cells to cisplatin-induced apoptosis. We demonstrated that CNOT3 depletion up-regulated the expression level of RIPK3 and the enhanced apoptosis was mediated by the elevated RIPK3 to further trigger Caspase 8 activation. Taken together, our results reveal a role of CNOT3 in cisplatin resistance of lung cancer and provide a potential target for lung cancer therapy.

Hypo-phosphorylated CD147 promotes migration and invasion of hepatocellular carcinoma cells and predicts a poor prognosis.

PURPOSE: CD147 is a tumor-associated antigen that plays a key regulatory role in tumor invasion and distant metastasis. However, the exact role of CD147 phosphorylation, which is deregulated during cancer progression, is unknown. Here, the effects of CD147 phosphorylation on the malignant behavior of hepatocellular carcinoma (HCC) cells and its possible underlying mechanisms are explored. METHODS: An in situ Duolink-proximity ligation assay (PLA) was used to detect CD147 phosphorylation. Tandem mass spectrometry was employed to identify the phosphorylation sites of CD147. The effects of CD147 phosphorylation on the malignant behavior of HCC cells were evaluated using scratch wound healing assays, transwell invasion assays and cell cycle assays. The genes regulated by CD147 phosphorylation were detected by RNA sequencing. RESULTS: We identified phosphorylated serine-246 in the C terminus of CD147 in primary HCC tissues, whereas serine to alanine substitution mutation analysis suggested that CD147 is phosphorylated mainly at serine-252 in HCC-derived Huh-7 cells. Recovery expression of S246A/S252A mutants in CD147 knockout cells revealed significantly increased migration and invasion capacities compared to wildtype CD147 expressing cells. Cyclophilin A (CyPA) treatment decreased the phosphorylation level of CD147, whereas NIMA-related kinase 6 (NEK6) increased the CD147 phosphorylation level. Moreover, the CD147 phosphorylation level was found to be dramatically decreased in HCC tissues in patients with distant metastases, and a low phosphorylation level of CD147 was found to be associated with a high serum AFP level, recurrence and a poor overall survival. CONCLUSIONS: From our data we conclude that hypo-phosphorylated CD147 promotes the migration and invasion of HCC cells and correlates with an unfavorable prognosis in HCC patients, indicating that targeting the aberrantly hypo-phosphorylated form of CD147 may be instrumental for the development of novel therapeutic modalities directed against HCC metastasis.

N-glycosylation by N-acetylglucosaminyltransferase V enhances the interaction of CD147/basigin with integrin ß1 and promotes HCC metastasis.

While the importance of protein N-glycosylation in cancer cell migration is well appreciated, the precise mechanisms by which N-acetylglucosaminyltransferase V (GnT-V) regulates cancer processes remain largely unknown. In the current study, we report that GnT-V-mediated N-glycosylation of CD147/basigin, a tumor-associated glycoprotein that carries ß1,6-N-acetylglucosamine (ß1,6-GlcNAc) glycans, is upregulated during TGF-ß1-induced epithelial-to-mesenchymal transition (EMT), which correlates with tumor metastasis in patients with hepatocellular carcinoma (HCC). Interruption of ß1,6-GlcNAc glycan modification of CD147/basigin decreased matrix metalloproteinase (MMP) expression in HCC cell lines and affected the interaction of CD147/basigin with integrin ß1. These results reveal that ß1,6-branched glycans modulate the biological function of CD147/basigin in HCC metastasis. Moreover, we showed that the PI3K/Akt pathway regulates GnT-V expression and that inhibition of GnT-V-mediated N-glycosylation suppressed PI3K signaling. In summary, ß1,6-branched N-glycosylation affects the biological function of CD147/basigin and these findings provide a novel approach for the development of therapeutic strategies targeting metastasis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cytoplasmic fragment of CD147 generated by regulated intramembrane proteolysis contributes to HCC by promoting autophagy.

Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers worldwide. CD147 (EMMPRIN or basigin) is a leading gene relating to hepatocarcinogenesis and metastasis, and is detected in transmembrane, exosome or circulating forms in HCC patients. The endosome recycling of CD147 further enhances the function of this oncoprotein from a dynamic perspective. However, previous studies about CD147 mainly focused on one separate form, and little attention has been paid to how the different forms of tumor-derived CD147 changes. Moreover, uncovering the roles of the residual C-terminal portion of CD147 after shedding is inevitable to fully understand CD147 promoting tumor progression. In this study, we discovered that under low-cholesterol condition, CD147 endocytosis is inhibited but its shedding mediated by ADAM10 is enhanced. Further procession of residual CD147 in the lysosome produces nuclear-localized CD147-ICD (intracellular domain of CD147), which contributes to autophagy through NF-κB-TRAIL-caspase8-ATG3 axis. As autophagy endows cancer cells with increased adaptability to chemotherapy, and HAb 18 (a specific antibody targeting CD147) inhibits CD147 shedding and sequential CD147-ICD enhances autophagy, we found the combination of HAb 18 and cisplatin exhibited marked antitumor efficiency.

Rab22a enhances CD147 recycling and is required for lung cancer cell migration and invasion.

Rab22a is a member of the Ras-related small GTPase family, which plays a key role in regulating the recycling of cargo proteins entering cells through clathrin-independent endocytosis (CIE). Rab22a is overexpressed in different cancer types, including liver cancer, malignant melanoma, ovarian cancer and osteosarcoma. However, its oncogenic role remains unknown. In this study, we found that silencing of Rab22a suppressed the migration and invasion of lung cancer cells. Furthermore, Rab22a interacts with CD147, and knockdown of Rab22a blocks CD147 recycling and promotes CD147 degradation. Taken together, our findings indicate that Rab22a enhances recycling of CD147, which is required for lung cancer cell migration and invasion,and targeting CD147 recycling may be a rational strategy for lung cancer therapy.

F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells.

Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells.