ICAM2 initiates trans-blood-CSF barrier migration and stemness properties in leptomeningeal metastasis of triple-negative breast cancer.

Leptomeningeal metastasis (LM) occurs when tumor cells spread to the leptomeningeal space surrounding the brain and the spinal cord, thereby causing poor clinical outcomes. The triple-negative breast cancer (TNBC) has been associated with symptoms of LM and mechanism remained unclear. Through proteomic analysis, we identified high expression of ICAM2 in leptomeningeal metastatic TNBC cells, which promoted the colonization of the spinal cord and resulted in poor survival in vivo. Two-way demonstration indicated that high levels of ICAM2 promoted blood-cerebrospinal fluid barrier (BCB) adhesion, trans-BCB migration, and stemness abilities and determined the specificity of LM in vivo. Furthermore, pull-down and antibody neutralizing assay revealed that ICAM2 determined the specificity of LM through interactions with ICAM1 in the choroid plexus epithelial cells. Therefore, neutralizing ICAM2 can attenuate the progression of LM and prolong survival in vivo. The results suggested that targeting ICAM2 is a potential therapeutic strategy for LM in TNBC.

Carboxyl-terminal modulator protein facilitates tumor metastasis in triple-negative breast cancer.

Currently, the survival rate for breast cancer is more than 90%, but once the cancer cells metastasize to distal organs, the survival rate is dramatically reduced, to less than 30%. Triple-negative breast cancer accounts for 15-20% of all breast cancers. Triple-negative breast cancer (TNBC) is associated with poor prognostic and diagnostic outcomes due to the limiting therapeutic strategies, relative to non-TNBC breast cancers. Therefore, the development of targeted therapy for TNBC metastasis remains an urgent issue. In this study, high Carboxyl-terminal modulator protein (CTMP) is significantly associated with recurrence and disease-free survival rate in TNBC patients. Overexpression of CTMP promotes migration and invasion abilities in BT549 cells. Down-regulating of CTMP expression inhibits migration and invasion abilities in MDA-MB-231 cells. In vivo inoculation of high-CTMP cells enhances distant metastasis in mice. The metastasis incidence rate is decreased in mice injected with CTMP-downregulating MDA-MB-231 cells. Gene expression microarray analysis indicates the Akt-dependent pathway is significantly enhanced in CTMP overexpressing cells compared to the parental cells. Blocking Akt activation via Akt inhibitor treatment or co-expression of the dominant-negative form of Akt proteins successfully abolishes the CTMP mediating invasion in TNBC cells. Our findings suggest that CTMP is a potential diagnostic marker for recurrence and poor disease-free survival in TNBC patients. CTMP promotes TNBC metastasis via the Akt-activation-dependent pathway.

Concurrent Chemoradiotherapy-Driven Cell Plasticity by miR-200 Family Implicates the Therapeutic Response of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a common and fatal malignancy with an increasing incidence worldwide. Over the past decade, concurrent chemoradiotherapy (CCRT) with or without surgery is an emerging therapeutic approach for locally advanced ESCC. Unfortunately, many patients exhibit poor response or develop acquired resistance to CCRT. Once resistance occurs, the overall survival rate drops down rapidly and without proper further treatment options, poses a critical clinical challenge for ESCC therapy. Here, we utilized lab-created CCRT-resistant cells as a preclinical study model to investigate the association of chemoradioresistantresistance with miRNA-mediated cell plasticity alteration, and to determine whether reversing EMT status can re-sensitize refractory cancer cells to CCRT response. During the CCRT treatment course, refractory cancer cells adopted the conversion of epithelial to mesenchymal phenotype; additionally, miR-200 family members were found significantly down-regulated in CCRT resistance cells by miRNA microarray screening. Down-regulated miR-200 family in CCRT resistance cells suppressed E-cadherin expression through snail and slug, and accompany with an increase in N-cadherin. Rescuing expressions of miR-200 family members in CCRT resistance cells, particularly in miR-200b and miR-200c, could convert cells to epithelial phenotype by increasing E-cadherin expression and sensitize cells to CCRT treatment. Conversely, the suppression of miR-200b and miR-200c in ESCC cells attenuated E-cadherin, and that converted cells to mesenchymal type by elevating N-cadherin expression, and impaired cell sensitivity to CCRT treatment. Moreover, the results of ESCC specimens staining established the clinical relevance that higher N-cadherin expression levels associate with the poor CCRT response outcome in ESCC patients. Conclusively, miR-200b and miR-200c can modulate the conversion of epithelial-mesenchymal phenotype in ESCC, and thereby altering the response of cells to CCRT treatment. Targeting epithelial-mesenchymal conversion in acquired CCRT resistance may be a potential therapeutic option for ESCC patients.

Sialylation of CD55 by ST3GAL1 Facilitates Immune Evasion in Cancer.

Altered glycosylations, which are associated with expression and activities of glycosyltransferases, can dramatically affect the function of glycoproteins and modify the behavior of tumor cells. ST3GAL1 is a sialyltransferase that adds sialic acid to core 1 glycans, thereby terminating glycan chain extension. In breast carcinomas, overexpression of ST3GAL1 promotes tumorigenesis and correlates with increased tumor grade. In pursuing the role of ST3GAL1 in breast cancer using ST3GAL1-siRNA to knockdown ST3GAL1, we identified CD55 to be one of the potential target proteins of ST3GAL1. CD55 is an important complement regulatory protein, preventing cells from complement-mediated cytotoxicity. CD55 had one N-linked glycosylation site in addition to a Ser/Thr-rich domain, which was expected to be heavily O-glycosylated. Detailed analyses of N- and O-linked oligosaccharides of CD55 released from scramble or ST3GAL1 siRNA-treated breast cancer cells by tandem mass spectrometry revealed that the N-glycan profile was not affected by ST3GAL1 silencing. The O-glycan profile of CD55 demonstrated a shift in abundance to nonsialylated core 1 and monosialylated core 2 at the expense of the disialylated core 2 structure after ST3GAL1 silencing. We also demonstrated that O-linked desialylation of CD55 by ST3GAL1 silencing resulted in increased C3 deposition and complement-mediated lysis of breast cancer cells and enhanced sensitivity to antibody-dependent cell-mediated cytotoxicity. These data demonstrated that ST3GAL1-mediated O-linked sialylation of CD55 acts like an immune checkpoint molecule for cancer cells to evade immune attack and that inhibition of ST3GAL1 is a potential strategy to block CD55-mediated immune evasion.

Sialylation of vasorin by ST3Gal1 facilitates TGF-ß1-mediated tumor angiogenesis and progression.

ST3Gal1 is a key sialyltransferase which adds α2,3-linked sialic acid to substrates and generates core 1 O-glycan structure. Upregulation of ST3Gal1 has been associated with worse prognosis of breast cancer patients. However, the protein substrates of ST3Gal1 implicated in tumor progression remain elusive. In our study, we demonstrated that ST3GAL1-silencing significantly reduced tumor growth along with a notable decrease in vascularity of MCF7 xenograft tumors. We identified vasorin (VASN) which was shown to bind TGF-ß1, as a potential candidate that links ST3Gal1 to angiogenesis. LC-MS/MS analysis of VASN secreted from MCF7, revealed that more than 80% of its O-glycans are sialyl-3T and disialyl-T. ST3GAL1-silencing or desialylation of VASN by neuraminidase enhanced its binding to TGF-ß1 by 2- to 3-fold and thereby dampening TGF-ß1 signaling and angiogenesis, as indicated by impaired tube formation of HUVECs, suppressed angiogenesis gene expression and reduced activation of Smad2 and Smad3 in HUVEC cells. Examination of 114 fresh primary breast cancer and their adjacent normal tissues showed that the expression levels of ST3Gal1 and TGFB1 were high in tumor part and the expression of two genes was positively correlated. Kaplan Meier survival analysis showed a significantly shorter relapse-free survival for those with lower expression VASN, notably, the combination of low VASN with high ST3GAL1 yielded even higher risk of recurrence (p = 0.025, HR = 2.967, 95% CI = 1.14-7.67). Since TGF-ß1 is known to transcriptionally activate ST3Gal1, our findings illustrated a feedback regulatory loop in which TGF-ß1 upregulates ST3Gal1 to circumvent the negative impact of VASN.

Reciprocal feedback regulation of ST3GAL1 and GFRA1 signaling in breast cancer cells.

GFRA1 and RET are overexpressed in estrogen receptor (ER)-positive breast cancers. Binding of GDNF to GFRA1 triggers RET signaling leading to ER phosphorylation and estrogen-independent transcriptional activation of ER-dependent genes. Both GFRA1 and RET are membrane proteins which are N-glycosylated but no O-linked sialylation site on GFRA1 or RET has been reported. We found GFRA1 to be a substrate of ST3GAL1-mediated O-linked sialylation, which is crucial to GDNF-induced signaling in ER-positive breast cancer cells. Silencing ST3GAL1 in breast cancer cells reduced GDNF-induced phosphorylation of RET, AKT and ERα, as well as GDNF-mediated cell proliferation. Moreover, GDNF induced transcription of ST3GAL1, revealing a positive feedback loop regulating ST3GAL1 and GDNF/GFRA1/RET signaling in breast cancers. Finally, we demonstrated ST3GAL1 knockdown augments anti-cancer efficacy of inhibitors of RET and/or ER. Moreover, high expression of ST3GAL1 was associated with poor clinical outcome in patients with late stage breast cancer and high expression of both ST3GAL1 and GFRA1 adversely impacted outcome in those with high grade tumors.

Potent adjuvant effects of novel NKT stimulatory glycolipids on hemagglutinin based DNA vaccine for H5N1 influenza virus.

H5N1 influenza virus is a highly pathogenic virus, posing a pandemic threat. Previously, we showed that phenyl analogs of α-galactosylceramide (α-GalCer) displayed greater NKT stimulation than α-GalCer. Here, we examined the adjuvant effects of one of the most potent analogs, C34, on consensus hemagglutinin based DNA vaccine (pCHA5) for H5N1 virus. Upon intramuscular electroporation of mice with pCHA5 with/without various α-GalCer analogs, C34-adjuvanted group developed the highest titer against consensus H5 and more HA-specific IFN-γ secreting CD8 cells (203±13.5) than pCHA5 alone (152.6±13.7, p<0.05). Upon lethal challenge of NIBRG-14 virus, C34-adjuvanted group (84.6%) displayed higher survival rate than pCHA5 only group (46.1%). In the presence of C34 as adjuvant, the antisera displayed broader and greater neutralizing activities against virions pseudotyped with HA of clade 1, and 2.2 than pCHA5 only group. Moreover, to simulate an emergency response to a sudden H5N1 outbreak, we injected mice intramuscularly with single dose of a new consensus H5 (pCHA5-II) based on 1192 full-length H5 sequences, with C34 as adjuvant. The latter not only enhanced the humoral immune response and protection against virus challenge, but also broadened the spectrum of neutralization against pseudotyped HA viruses. Our vaccine strategy can be easily implemented for any H5N1 virus outbreak by single IM injection of a consensus H5 DNA vaccine based on updated HA sequences using C34 as an adjuvant.

Chimeras of p14ARF and p16: functional hybrids with the ability to arrest growth.

The INK4A locus codes for two independent tumor suppressors, p14ARF and p16/CDKN2A, and is frequently mutated in many cancers. Here we report a novel deletion/substitution from CC to T in the shared exon 2 of p14ARF/p16 in a melanoma cell line. This mutation aligns the reading frames of p14ARF and p16 mid-transcript, producing one protein which is half p14ARF and half p16, chimera ARF (chARF), and another which is half p16 and half non-p14ARF/non-p16 amino acids, p16-Alternate Carboxyl Terminal (p16-ACT). In an effort to understand the cellular impact of this novel mutation and others like it, we expressed the two protein products in a tumor cell line and analyzed common p14ARF and p16 pathways, including the p53/p21 and CDK4/cyclin D1 pathways, as well as the influence of the two proteins on growth and the cell cycle. We report that chARF mimicked wild-type p14ARF by inducing the p53/p21 pathway, inhibiting cell growth through G2/M arrest and maintaining a certain percentage of cells in G1 during nocodazole-induced G2 arrest. chARF also demonstrated p16 activity by binding CDK4. However, rather than preventing cyclin D1 from binding CDK4, chARF stabilized this interaction through p21 which bound CDK4. p16-ACT had no p16-related function as it was unable to inhibit cyclin D1/CDK4 complex formation and was unable to arrest the cell cycle, though it did inhibit colony formation. We conclude that these novel chimeric proteins, which are very similar to predicted p16/p14ARF chimeric proteins found in other primary cancers, result in maintained p14ARF-p53-p21 signaling while p16-dependent CDK4 inhibition is lost.

Effect of a C/EBP gene replacement on mitochondrial biogenesis in fat cells.

CCAAT/enhancer-binding proteins, C/EBPalpha and C/EBPbeta, are required for fat cell differentiation and maturation. Previous studies showed that replacement of C/EBPalpha with C/EBPbeta, generating the beta/beta alleles in the mouse genome, prevents lipid accumulation in white adipose tissue (WAT). In this study, beta/beta mice lived longer and had higher energy expenditure than their control littermates due to increased WAT energy oxidation. The WAT of beta/beta mice was enriched with metabolically active, thermogenic mitochondria known for energy burning. The beta/beta allele exerted its effect through the elevated expression of the G protein alpha stimulatory subunit (Galphas) in WAT. Galphas, when overexpressed in fat-laden 3T3-L1 cells, stimulated mitochondrial biogenesis similar to that seen in the WAT of beta/beta mice, and effectively diminished the stored lipid pool.

Anti-4-1BB-based immunotherapy for autoimmune diabetes: lessons from a transgenic non-obese diabetic (NOD) model.

Various therapeutic strategies have been developed to tolerize autoreactive T cells and prevent autoimmune pathology in type 1 diabetes. 4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a costimulatory receptor primarily expressed on activated T cells. The administration of an agonistic anti-4-1BB antibody (2A) dramatically reduced the incidence and severity of experimental autoimmune encephalomyelitis (EAE). Treatment with the same antibody in Fas-deficient MRL/lpr mice blocked lymphadenopathy and lupus-like autoimmune processes. Paradoxically, transgenic non-obese diabetic (NOD) mice overexpressing membrane-bound agonistic single-chain anti-4-1BB Fv in pancreatic beta cells developed more severe diabetes than their non-transgenic littermates, with earlier onset, faster diabetic processes, and higher mortality. Forty percent of transgenic mice developed diabetes by 4 weeks of age, compared with their control littermates, which first exhibited diabetes at 14 weeks. The frequency of diabetes in female transgenics reached 70% by 8 weeks of age. Most female transgenic mice died around 12 weeks. Consistent with this, transgenic mice developed earlier and more severe insulitis and showed stronger GAD-specific T-cell responses, compared with age-matched control littermates. Our results indicate an adverse effect of transgenic anti-4-1BB scFv in NOD mice and suggest a potential risk of this anti-4-1BB-based immunotherapy for autoimmune diseases.