Activated Leukocyte Cell Adhesion Molecule Expression Research Articles

Activated Leukocyte Cell Adhesion Molecule (ALCAM) Regulate Myeloma Cancer Stem Cell Mediated Myelomagenesis and Chemoresistance

IntroductionMultiple myeloma (MM) is an incurable plasma cell cancer. People introduced the concept of cancer stem cells (CSCs) in MM, known as MM-CSCs, to characterize a small population of malignant cells with distinguishable activities of self-renewal and differentiation into regular MM cells. In this study, we showed that activated leukocyte cell adhesion molecule (ALCAM) interacted with epidermal growth factor receptor (EGFR). ALCAM-EGFR axis regulated MM-CSC mediated myelomagenesis and chemoresistance.MethodsMM-CSCs in MM cell lines and primary MM cells were identified by Hoechst staining or ALDEFLUOR dye, followed by flow cytometry analyses. MM-CSC clonalization activity was examined by soft agar colony formation assay. The protein-protein interaction was identified by co-immunoprecipitation, proximity ligation assay, and proteins' structure simulation. MM-CSC mediated myelomagenesis in vivo was examined in human MM xenograft mouse model and murine adoptive MM model. MM-CSC mediated chemoresistance in vivo was examined using 5T MM model. The clinical significance of ALCAM expression in MM was analyzed using patients' gene expression profiles.ResultsBoth MM cell lines and primary MM cells expressed ALCAM. When cultured with bone marrow stromal cells (BMSCs) or in BMSC-conditioned medium, ALCAM knocked down (AL-KD) MM cells exhibited increased colony formation activity. Hoechst staining showed that under such culture conditions, AL-KD MM cells had increased stemness side population (SP), which was MM-CSC. Depletion of MM-CSC reduced MM colony formation activity. In human MM xenograft model, subcutaneous co-injection of AL-KD MM cells and BMSCs had large amount of MM-CSC and fast tumorigenesis. In murine adoptive MM model, which had tumor derived from viral transformed plasma cells with mandatory expression of MYC, KRAS genes and a shRNA sequence, ALCAM shRNA expression resulted significant more rapid tumor development than non-target shRNA.Mechanistic studies showed that ALCAM and EGFR co-immunoprecipitated in MM cells. Proximity ligation assay showed interaction of ALCAM and EGFR in primary MM cells. Immunofluorescent staining showed that ALCAM colocalized with unphosphorylated EGFR in MM. Epidermal growth factor (EGF), the canonical ligand of EGFR, stimulated EGFR phosphorylation and abolished ALCAM-EGFR colocalization. Protein structure simulation suggested that EGF and ALCAM mutual exclusively bound to the same region of EGFR. EGF addition stimulated MM-CSC in cultured MM. Inhibitors of EGFR, ERK, or hedgehog pathway repressed MM-CSC. EGFR activation led to hedgehog pathway activation via RAS/MEK/ERK cascade, while ALCAM inhibited such pro-MM-CSC cell signaling by binding to EGFR.Chemotherapeutics, such as bortezomib and melphalan, induced MM apoptosis, but also stimulated MM-CSCs, which were resistant to chemotherapeutics. Although CTR-KD and AL-KD MM had similar death rate after the drug treatment, AL-KD MM cells had more MM-CSC generated. MM-CSCs derived in AL-KD MM cells had more active cell proliferation even in the presence of drugs. Further study showed that chemotherapy stimulated EGFR expression and repressed ALCAM expression, therefore switched the balance of ALCAM-EGFR axis to pro-MM-CSC direction. EGFR inhibitors, such as gefitinib, did not have anti-MM activity, but repressed MM-CSCs. In 5T MM model, Gefitinib/melphalan combination therapy resulted prolonged remission period, and significant better mouse survival.We identified MM-CSC in patients BM. ALCAM expression correlated with MM overall survival (OS): ALCAMhigh patients had better OS. In ALCAM-EGFR axis, only ALCAM expression, but not EGFR expression, correlated with MM OS. ALCAM expression varied in different patients, while EGFR expression was more even. Therefore we believe in ALCAM-EGFR axis, it was ALCAM, but not the EGFR, that played the regulatory role. Furthermore, pathway enrichment analysis showed that MM-CSC, identified by Hoechst staining, and MM minimum residual disease (MRD) shared similarities in signaling pathways, as well as pattern of ALCAM expressions.Conclusion and DiscussionALCAM-EGFR axis regulates MM-CSC mediated myelomagenesis and chemoresistance. EGFR inhibitors repress chemoresistant MM-CSC. Combination therapy with conventional chemotherapeutics and an EGFR inhibitor might be promising in MM treatment. DisclosuresNo relevant conflicts of interest to declare.

Abstract 30: Effect of Wnt5a on aggressiveness of estrogen receptor-positive breast cancer and cancer cell migration through ALCAM pathway

Abstract Purpose: Wnt signaling occurs in β-catenin-dependent pathways, through β-catenin regulates the expression of many genes, and in the β-catenin-independent planar cell polarity (PCP) and Wnt/Ca2+ pathways. Wnt5a is a key ligand in activation of β-catenin-independent pathways, involved in cell motility and polarity through downstream signaling (e.g., JNK phosphorylation). Wnt5a expression correlates significantly with malignancy and stage of progression in malignant melanoma, gastric cancer, prostate cancer, lung cancer, and pancreatic cancer. In contrast, Wnt5a is a tumor suppressor in colorectal cancer, thyroid cancer, liver cancer, and malignant lymphoma. Thus, the effect of Wnt5a expression differs between organs. The role of Wnt5a expression in breast cancer remains elusive. In this study, we examined significance of Wnt5a expression in breast cancer. Experimental design: The relationships between Wnt5a expression and clinicopathologic factors were assessed in 178 consecutive cases of invasive breast cancer resected in Hiroshima University Hospital between January 2011 and February 2014. In addition, altered gene expression following Wnt5a addition was mapped as possible Wnt5a-induced transformation pathways. Results: Wnt5a was positively expressed in 69 of 178 cases (39%) of invasive breast cancer. Wnt5a expression was strongly correlated with estrogen receptor (ER)-positive (P < 0.001). Analysis of the relationship between Wnt5a expression and malignancy in 153 cases of ER-positive breast cancer revealed significant correlations with lymph node metastasis, nuclear grade, and lymphatic invasion. Relapse-free survival was shorter in cases of Wnt5a-positive breast cancer compared to Wnt5a-negative breast cancer cases (P = 0.024). Constitutive expression of Wnt5a in MCF7 cells enhanced migratory capacity, whereas knockdown of Wnt5a reduced this capacity. DNA microarray analysis identified activated leukocyte cell adhesion molecule (ALCAM) as the molecule which is primarily induced by Wnt5a. ALCAM was expressed in 69% of Wnt5a-positive breast cancers but only 27% of Wnt5a-negative breast cancers in 153 cases of ER-positive breast cancer, showing a statistical correlation between Wnt5a and ALCAM expressions. Inhibition of ALCAM in MCF7 constitutively expressing Wnt5a cells reverted the effect of Wnt5a, confirming the important of this molecule in migration of ER-positive breast cancer cells. Conclusions: In ER-positive breast cancer, Wnt5a expression is related with high malignancy and poor prognosis. We speculate that Wnt5a expression increases malignancy in breast cancer by enhancing migratory capacity of cancer cells through induction of ALCAM expression. Wnt5a may be useful as a predictor of malignancy, a therapeutic target, and a prognostic indicator in ER-positive breast cancer. Citation Format: Yoshie Kobayashi, Takayuki Kadoya, Ai Amioka, Hideaki Hanaki, Shinsuke Sasada, Norio Masumoto, Koji Arihiro, Akira Kikuchi, Morihito Okada. Effect of Wnt5a on aggressiveness of estrogen receptor-positive breast cancer and cancer cell migration through ALCAM pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 30.

Wnt5a-induced cell migration is associated with the aggressiveness of estrogen receptor-positive breast cancer.

Elevated expression of Wnt5a is associated with malignancy, cell invasion, and metastasis. The role of Wnt5a expression in breast cancer remains elusive. We investigated the significance of Wnt5a expression in breast cancer. The relationship between Wnt5a expression and clinicopathologic factors was assessed in invasive breast cancer (n = 178) resected at Hiroshima University Hospital between January 2011 and February 2014. Wnt5a was expressed in 69 of 178 cases (39%) of invasive breast cancer and correlated strongly with estrogen receptor (ER) expression (P < 0.001). Wnt5a expression in ER-positive breast cancer correlated significantly with lymph node metastasis, nuclear grade, and lymphatic invasion. The recurrence-free survival was shorter in breast cancer patients with Wnt5a expression than in those without (P = 0.024). The migratory capacity of ER-positive breast cancer cells increased with constitutive expression of Wnt5a and decreased with Wnt5a knockdown. DNA microarray analysis identified activated leukocyte cell adhesion molecule (ALCAM) as the primary gene induced by Wnt5a. ALCAM was expressed in 69% of Wnt5a-positive but only 27% of Wnt5a-negative cancers (κ = 0.444; P < 0.001). The inhibition of ALCAM reversed the enhanced migratory effect of Wnt5a, confirming the importance of this protein in the migration of ER-positive breast cancer cells. Wnt5a expression is related to high malignancy and a poor prognosis in ER-positive breast cancer. We suspect that Wnt5a expression increases the malignancy of breast cancer by increasing the migratory capacity of cancer cells through the induction of ALCAM expression.

Sonic Hedgehog Signalling Activation Contributes to ALCAM Over-Expression and Poor Clinical Outcome in Patients with Oral Squamous Cell Carcinoma.

To investigate the Sonic Hedgehog (SHH) signalling molecules and activated leukocyte cell adhesion molecule (ALCAM) expression in the mechanisms regulating invasion and metastasis in oral squamous cell carcinoma (OSCC). The expressions of SHH signalling molecules Gli family zinc finger 1/2 (Gli1/Gli2), as well as ALCAM expression, was analysed in 101 OSCC patients by immunohistochemistry. The potential relationship between Gli1/Gli2 and ALCAM in regard to invasion and metastasis were studied by western blot, invasion and wound-healing assays. Gli1, Gli2 and ALCAM were expressed in 54.5%, 49.5% and 47.5% of the 101 OSCC specimens, respectively. High expression of ALCAM was associated with shorter survival in the patient population (P = 0.018), which was independent of other clinical parameters. Notably, when both ALCAM expression and positive nodal status were considered, an enhanced prediction of clinical outcomes was achieved (P = 0.001). In OSCC cell lines, down-regulation of ALCAM resulted in reduced cell invasion and metastasis. Importantly, SHH activation increased the half-life of ALCAM leading to ALCAM accumulation and increased cell invasion and migration. ALCAM over-expression in OSCC is an independent prognostic factor for OSCC patients. Its over-expression may be the result of the activation of the SHH signalling pathway and contributes to OSCC progression.

Predicting Value of ALCAM as a Target Gene of microRNA-483-5p in Patients with Early Recurrence in Hepatocellular Carcinoma.

The long-term survival rate of hepatocellular carcinoma (HCC) is poor. One of the reasons for the poor rate of survival is the high rate of recurrence caused by intrahepatic metastas is that adversely affects long-term outcome. Many studies have indicated that microRNAs play an important role in HCC, but there has been no research of clonal origins on recurrent HCC (RHCC) by analzing microRNAs. In the present study, we found that miR-483-5p was significantly upregulated in RHCC tissues of short-term recurrence (≤ 2 years) by miRNA microarray screening, and can significantly promote migration and invasion of HCC cells in vitro and increase intrahepatic metastasis in nude mice in vivo. Furthermore, we demonstrated that activated leukocyte cell adhesion molecule (ALCAM), which significantly suppressed migration and invasion of HCC cells, was a direct target of miR-483-5p, and the re-introduction of ALCAM expression could antagonize the promoting effects of miR-483-5p on the capacity of HCC cells for migration and invasion. In addition, expression level of ALCAM was negatively correlated with microvascular invasion and tumor size recognized as prognostic factors. The cases which were negative for ALCAM expression had shorter time to recurrence than positive cases, and univariate and multivariate survival analyses showed that ALCAM was an independent risk factor of HCC recurrence. qRT-PCR and Western blotting showed that the expression of EMT related genes (MMP-2, MMP-9, E-caherin and vimentin) significantly changed as a result of interfering or overexpression of ALCAM, and ALCAM was significantly associated with EMT in HCC. These results suggest that the miR-483-5p/ALCAM axis is an important regulator in invasion and metastasis and biomarker for recurrence risk assessment of HCC.

Prognostic Significance of Activated Leukocyte Cell Adhesion Molecule (ALCAM) in Association with Promoter Methylation of the ALCAM Gene in Breast Cancer.

Activated leukocyte cell adhesion molecule (ALCAM) has been implicated in tumorigenesis. In this study, we studied DNA methylation status of the ALCAM gene using pyrosequencing in breast cancer tissues. We analyzed the association between the methylation status of the ALCAM gene and its expression. Also, the effects of inflammation on the ALCAM gene methylation and its expression were investigated. The ALCAM gene methylation was associated with the ALCAM transcripts in tumor tissues. The methylation status of the ALCAM gene was not significantly different between tumor and normal tissues. The level of ALCAM transcripts was associated with the expression of TNFα, NF-κB p50, IL-4, and intratumoral inflammation. The IHC expression of ALCAM was associated with histologic grade, HER2 overexpression and molecular subtype. The expression of TNFα, NF-κB p50, and IL-4 showed significant association with the clinicopathologic characteristics. In conclusion, the ALCAM gene methylation was related to the level of ALCAM transcripts. Also, the level of ALCAM transcripts was associated with the inflammatory markers in breast cancer. Our results suggest that the methylation of the ALCAM gene contributes to the decreased expression of ALCAM. Also, ALCAM is linked to the inflammatory response in breast cancer.

Co-detection of ALDH1A1, ABCG2, ALCAM and CD133 in three A549 subpopulations at the single cell level by one-step digital RT-PCR.

Cancer stem-like cells (CSCs) displaying the properties of normal stem cells have become the main culprit associated with cancer transportation and recurrence. As of now, various CSC functions and marker genes have been identified due to the heterogeneity of cancer, such as aldehyde dehydrogenase (ALDH), the second member of the ABC transporter G-subfamily (ABCG2), activated leukocyte cell adhesion molecule (ALCAM) and CD133. To investigate these markers, most conventional approaches are bulk-based strategies, which may veil the disparity of single cells' gene expression. In this study, one-step digital RT-PCR at the single cell level was developed to co-determine the expression of ALDH1A1, ABCG2, ALCAM and CD133 genes in A549 cancer stem cells that perform high ALDH activities (ALDH+ A549 cells), as well as in ALDH- A549 cells and A549 cells, with 36, 20 and 20 cell samples each. The results demonstrated that, when compared to single ALDH- or A549 cells, the majority of single ALDH+ A549 cells displayed a 1.5- and 2.0-fold increase in the gene expression of ALDH1A1 and ALCAM (P < 0.001), respectively. However, for ABCG2 and CD133, there was no significant difference (P > 0.05), which means that they are not appropriate as co-indicated markers to identify ALDH+ A549 cells. Conclusively, as a single cell level approach, one-step digital RT-PCR has potential in exploring efficient co-detection markers for the classification and identification of CSCs.

Histopathological and genotypic characterization of metastatic colorectal carcinoma with PD-L1 (CD274)-expression: Possible roles of tumour micro environmental factors for CD274 expression.

Aberrant PD‐L1 (CD274) expression has been described in different types of tumour and linked to tumour aggressiveness and a poor prognosis. In primary colorectal carcinomas (CRCs), CD274 expression was reported to be associated with mismatch repair (MMR)‐deficiency, BRAF mutation, and “stem‐like” immunophenotype defined by down‐regulation of homeobox protein CDX2 and membranous expression of activated leukocyte cell adhesion molecule (ALCAM). However, the immunophenotype and genotype of CD274‐positive metastatic CRC have not been extensively analysed. In this study, 189 CRC metastases were evaluated immunohistochemically for CD274, MMR proteins, CDX2, and ALCAM expression. Immunostaining for CD4, CD8, and FOXP3 was also performed to characterize tumour‐associated immune cells. In addition, 34 arbitrarily selected lesions were genotyped using Sanger‐ and next‐generation sequencing. Univariate analyses showed no clear association between CD274 expression and clinicopathological parameters including MMR‐deficiency or “stem‐like” immunophenotype after adjustment for multiple testing. Comparison of the clinicopathological profiles of CD274‐positive primary and metastatic tumours revealed in the latter younger age of occurrence (60.9 ± 13.3 versus 72.6 ± 13.1 years, p = 0.001), cytoplasm‐dominant CD274 expression (p < 0.001), infrequent MMR‐deficiency (p < 0.001), and common KRAS mutations (54%, p < 0.001). In five cultured colon cancer cell lines, CD274 was expressed and modulated after exogenous exposure to IFNγ and TGF‐β1. Thus, CD274 regulation mechanisms might include tumour micro environmental factors. Based on significantly different characteristics in CD274‐positive metastatic and primary CRCs, evaluation of metastases should also be considered when planning immune checkpoint inhibitor therapy.

Stromal Expression of Activated Leukocyte Cell Adhesion Molecule Promotes Lung Tumor Growth and Metastasis

Activated leukocyte cell adhesion molecule (ALCAM) is expressed on various cell types, including leukocytes, endothelial cells, and certain tumor cells. Although ALCAM expression on tumor cells has been linked to tumor invasion and metastatic spread, the contribution of ALCAM expressed in cells forming the tumor stroma to cancer progression has not been investigated. In this study, ALCAM-deficient (ALCAM-/-) mice were used to evaluate the role of ALCAM in lung tumor growth and metastasis. ALCAM-/- mice displayed an altered blood vascular network in the lung and the diaphragm, indicative of an angiogenetic defect. The absence of ALCAM expression in cells forming the stromal tumor microenvironment profoundly affected lung tumor growth in three different i.v. metastasis models. In the case of Lewis lung carcinoma (LLC), an additional defect in tumor cell homing to the lungs and a resulting reduction in the number of lung tumor nodules were observed. Similarly, when LLC cells were implanted subcutaneously for the study of spontaneous tumor cell metastasis, the rate of LLC metastasis to the lungs was profoundly reduced in ALCAM-/- mice. Taken together, our work demonstrates for the first time the invivo contribution of ALCAM to angiogenesis and reveals a novel role of stromally expressed ALCAM in supporting tumor growth and metastatic spread.

Activated leukocyte cell adhesion molecule is involved in excitatory synaptic transmission and plasticity in the rat spinal dorsal horn.

Activated leukocyte cell adhesion molecule (ALCAM), a member of type I transmembrane immunoglobulin superfamily of cell adhesion molecule, is expressed in the surface membrane of various cell types including neurons. In the spinal cord dorsal horn (DH), the first gate for the sensory and pain transmission to the brain, the expression and function of ALCAM have not been known yet. Therefore, we here investigate the synaptic function of ALCAM in the substantia gelatinosa (lamina II) of the spinal DH, as well as its expression in the DH. Bath-application of ALCAM/Fc or CD6/Fc, the recombinant human IgG1-Fc chimeric proteins, specifically potentiated C-fiber-mediated excitatory synaptic transmission and predominantly increased spontaneous release of glutamate. In addition, the development of long-term potentiation, a form of synaptic plasticity, at excitatory synapses was significantly inhibited in the presence of the recombinant proteins. The functional roles of ALCAM in the spinal DH were further supported by immunohistochemical analysis; it showed that ALCAM intensely expressed through laminae I/II with the exception of lateral portion of the dorsal part of inner lamina II and distinctly co-localized with molecular markers of C-fibers, such as peptidergic calcitonin gene-related protein and transient receptor potential vanilloid type 1 and non-peptidergic isolectin B4. This study, for the first time, suggests the modulatory roles of ALCAM in the excitatory synaptic transmission and plasticity in the rat spinal DH.

Abstract 5442: Down-regulation of miRNA-214 expression by bioactive molecules mitigates invasive potential of melanoma cells through ALCAM/TFAP2 signaling

Abstract Melanoma is responsible for 80% of skin cancer related deaths in the United States and represents fifth most common neoplasia in humans. Over the past decades, the incidence of melanoma has increased by 3% to 8% per year in western countries. Therefore, it is essential to understand the molecular regulatory pathways that contribute to melanoma aggressiveness and metastatic dissemination. MicroRNAs (miRNAs) are small noncoding regulatory RNAs that contribute to tumor formation and progression, for their ability to regulate gene expression post-transcriptionally by either inhibiting protein translation or degrading target mRNAs. miR-214 is overexpressed in various types of cancers and is associated with the regulation of numerous carcinogenic processes including cell differentiation, senescence, angiogenesis and cell migration/invasion, etc. In the present study, we demonstrated that miR-214 shows distinct over expression in melanoma cell lines (A375, Hs294t, SK-Mel119, and SK-Mel28) as compared to its expression in normal human epidermal melanocytes (NHEM), and this overexpression of miR-214 in melanoma cells was associated with the invasive potential than NHEM. Treatment of melanoma cells with an inhibitor of miR-214 (50 nM) resulted in inhibition of cell invasion ability. We also determined the effects of bioactive phytochemical grape seed proanthocyanidins (GSPs) on the expression levels of miRNA-214 in melanoma cell lines (A375 and Hs294t) and its underlying molecular targets. Treatment of A375 and Hs294t cells with GSPs resulted in suppression of the levels of miRNA-214, which leads to decrease in migration potential of melanoma cell lines. By using 3D melanoma progression model, we also verified this suppressive effect of GPSs on melanoma invasion. The transition from the non-invasive to the invasive and metastatic stage is accompanied by the loss of AP-2 transcription factors (TFAP-2) and expression of activated leukocyte cell adhesion molecule (ALCAM) and those are associated with miRNA-214 overexpression. Here, we observed that the treatment of GSPs decreases ALCAM expression and restore TFAP-2 levels in melanoma cell lines. Further, dietary administration of GSPs significantly reduce the metastasis potential of A375 cells in different vital organs such as lung, liver, kidney, and spleen, and this suppressive effect of GSPs on invasive potential of A375 cells was associated with the reduction in the levels of miRNA-214 and ALCAM. Our results indicate that modulation of miR-214 influences in vitro cell movement and extravasation from blood vessels in vivo. Together, these studies suggest that an identification of metastasis related miRNAs may provide potential diagnostic biomarkers and better understanding of the complex functions of miRNAs, as well as therapeutic targets for clinical application. Note: This abstract was not presented at the meeting. Citation Format: Ram Prasad, Santosh K. Katiyar. Down-regulation of miRNA-214 expression by bioactive molecules mitigates invasive potential of melanoma cells through ALCAM/TFAP2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5442. doi:10.1158/1538-7445.AM2017-5442

Meta-analysis indicating that high ALCAM expression predicts poor prognosis in colorectal cancer.

Activated leukocyte cell adhesion molecule (ALCAM) has been linked to the development and progress of colorectal cancer (CRC). In this meta-analysis, we examined whether ALCAM expression is predictive of survival outcomes in CRC patients. We included 7 studies with 2048 patients in our meta-analysis after searching the PubMed, Cochrane Library, EMBASE, OVID and Web of Science databases. High ALCAM expression was associated with poor overall survival among CRC patients (HR = 1.94, 95%CI = 1.05–3.58, P = 0.03). High ALCAM expression was also associated with aggressive clinicopathological features such as tumor stage (T3,T4/T1,T2; HR = 2.66, 95%CI = 2.01–3.51, P < 0.00001), nodal status (Positive/Negative, HR = 2.12, 95%CI = 1.61–2.82, P < 0.00001), distant metastasis (M1/M0, HR = 3.30, 95%CI = 2,21–4.91, P < 0.00001), tumor grade (grade3/grade1,2, HR = 1,28, 95% CI = 1.00–1.62, P = 0.05), and patient age (> 60/< 60, HR = 1.29, 95%CI = 1.01–1.66, P = 0.05). These findings indicate that high ALCAM expression is associated with poor prognosis and advanced clinicopathological characteristics in CRC patients.

MiR-148a and miR-152 reduce tamoxifen resistance in ER+ breast cancer via downregulating ALCAM

Activated leukocyte cell adhesion molecule (ALCAM), also called CD166 is a 105-kDa transmembrane glycoprotein of the immunoglobin superfamily. In this study, we studied the association between ALCAM expression and tamoxifen resistance in ER + breast cancer and further investigated how ALCAM is regulated in the cancer cells. IHC staining data showed that the tumor tissues from non-responders (N = 20) generally had significantly stronger ALCAM staining than that from tamoxifen responders (N = 16). In vitro cell assay also confirmed ALCAM upregulation in tamoxifen resistant (TamR) MCF-7 cells than in tamoxifen sensitive (TamS) MCF-7 cells. ALCAM overexpression significantly alleviated 4-Hydroxytestosterone (4-OHT) induced cell viability inhibition and cell apoptosis in TamS MCF-7 cells, while ALCAM knockdown remarkably enhanced 4-OHT induced cell viability inhibition and cell apoptosis in TamR MCF-7 cells. Demethylation reagent treatment significantly restored miR-148a and miR-152 expression in TamR MCF-7 cells. MiR-148a and miR-152 can directly target ALCAM 3′UTR and decrease ALCAM expression. MiR-148a overexpression had similar effect as ALCAM siRNA on enhancing 4-OHT induced cell viability inhibition and cell apoptosis in TamR MCF-7 cells. MiR-152 overexpression alone caused growth inhibition and increased cell apoptosis in TamR MCF-7 cells. It also enhanced the effect of 4-OHT. Simultaneous inhibition of miR-148a and miR-152 significantly protected TamS MCF-7 cells from 4-OHT induced cell viability inhibition and cell apoptosis. Based on these findings, we infer that MiR-148a and miR-152 can sensitize TamR MCF-7 cells to tamoxifen at least via downregulating ALCAM.

Shed urinary ALCAM is an independent prognostic biomarker of three-year overall survival after cystectomy in patients with bladder cancer.

Proteins involved in tumor cell migration can potentially serve as markers of invasive disease. Activated Leukocyte Cell Adhesion Molecule (ALCAM) promotes adhesion, while shedding of its extracellular domain is associated with migration. We hypothesized that shed ALCAM in biofluids could be predictive of progressive disease. ALCAM expression in tumor (n = 198) and shedding in biofluids (n = 120) were measured in two separate VUMC bladder cancer cystectomy cohorts by immunofluorescence and enzyme-linked immunosorbent assay, respectively. The primary outcome measure was accuracy of predicting 3-year overall survival (OS) with shed ALCAM compared to standard clinical indicators alone, assessed by multivariable Cox regression and concordance-indices. Validation was performed by internal bootstrap, a cohort from a second institution (n = 64), and treatment of missing data with multiple-imputation. While ALCAM mRNA expression was unchanged, histological detection of ALCAM decreased with increasing stage (P = 0.004). Importantly, urine ALCAM was elevated 17.0-fold (P < 0.0001) above non-cancer controls, correlated positively with tumor stage (P = 0.018), was an independent predictor of OS after adjusting for age, tumor stage, lymph-node status, and hematuria (HR, 1.46; 95% CI, 1.03–2.06; P = 0.002), and improved prediction of OS by 3.3% (concordance-index, 78.5% vs. 75.2%). Urine ALCAM remained an independent predictor of OS after accounting for treatment with Bacillus Calmette-Guerin, carcinoma in situ, lymph-node dissection, lymphovascular invasion, urine creatinine, and adjuvant chemotherapy (HR, 1.10; 95% CI, 1.02–1.19; P = 0.011). In conclusion, shed ALCAM may be a novel prognostic biomarker in bladder cancer, although prospective validation studies are warranted. These findings demonstrate that markers reporting on cell motility can act as prognostic indicators.

ALCAM (CD166) is involved in extravasation of monocytes rather than T cells across the blood–brain barrier

Activated leukocyte cell adhesion molecule (ALCAM) has been proposed to mediate leukocyte migration across the blood–brain barrier (BBB) in multiple sclerosis or experimental autoimmune encephalomyelitis (EAE). Here, we confirmed vascular ALCAM expression in human brain tissue samples in situ and on two different human in vitro BBB models. Antibody-mediated inhibition of ALCAM reduced diapedesis of human CD4+ Th1 but not of Th17 cells across the human BBB in vitro. In accordance to human Th1 cells, mouse Th1 cells showed reduced diapedesis across an ALCAM−/− in vitro BBB model under static but no longer under flow conditions. In contrast to the limited role of ALCAM in T cell extravasation across the BBB, we found a contribution of ALCAM to rolling, adhesion, and diapedesis of human CD14+ monocytes across the human BBB under flow and static conditions. Taken together, our study highlights the potential differences in the CNS expression of ALCAM in mouse and human and supports a prominent role for ALCAM in the multi-step extravasation of monocytes across the BBB.

Human T-Lymphotropic Virus Type 1-Induced Overexpression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) Facilitates Trafficking of Infected Lymphocytes through the Blood-Brain Barrier.

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this study, we investigated the role of activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically infected cell lines and in primary infected CD4(+) T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-κB pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients showed that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis. Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The CNS is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into the CNS is unclear. Here, we show that the viral transactivator Tax increases activated leukocyte cell adhesion molecule (ALCAM/CD166) expression. This molecule facilitates the migration of lymphocytes across the BBB endothelium. Targeting this molecule could be of interest in preventing or reducing the development of HAM/TSP.

Activated leukocyte cell adhesion molecule (ALCAM) facilitates trafficking of HTLV-1 infected lymphocytes through the blood brain barrier

HAM/TSP is a neurodegenerative disease that develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS). Physiologically, the CNS is protected and isolated from the immune system by a structure called blood-brain barrier (BBB). Thus HTLV-1-infected lymphocytes have the capacity to cross the blood-brain barrier (BBB). The mechanisms of such crossing are still poorly understood. In the context of multiple sclerosis and neuro-AIDS, the Activated Leukocyte Cell Adhesion Molecule (ALCAM) was shown to amplify leukocyte extravasation. In the earlier, ALCAM is over expressed on the BBB endothelium, whereas in the latter ALCAM expression is increased on HIV-1-infected monocytes. We therefore studied the possible role of ALCAM in extravasation of HTLV-1 infected lymphocytes. We demonstrated by FACS that ALCAM is over expressed both on HTLV-1-infected lymphocytes cell lines and on primary cells from HTLV-1 asymptomatic carriers or HAM/TSP patients. Via transduction with a Tax-encoding lentiviral vectors, we showed that ALCAM over expression is the consequence of Tax-1-induced NF-κB pathway activation. We finally demonstrated that inhibiting ALCAM with a monoclonal blocking antibody reduces significantly the extravasation of HTLV-1 chronically infected cells through a monolayer of BBB endothelial cells (the hCMEC/D3 model). This study reports a potential role of HTLV-1-induced ALCAM overpression in HAM/TSP pathogenesis.

Overexpression of activated leukocute cell adhesion molecule in gastric cancer is associated with advanced stages and poor prognosis and miR-9 deregulation.

Activated leukocyte cell adhesion molecule (ALCAM) has been identified as a novel potential molecular marker of human tumors. The present study aimed to assess ALCAM as a prognostic marker for gastric cancer (GC), and to explore the mRNA deregulation underlying the abnormal expression of ALCAM. The mRNA and protein expression of ALCAM in GC and adjacent non‑tumor tissues from 66 patients with GC were analyzed. The association between miR‑9 and ALCAM mRNA expression was determined by quantitative polymerase chain reaction. Serum soluble ALCAM (sALCAM) was analyzed by ELISA in 72 patients with GC, 82 patients with gastric precancerous lesions and 73 controls. ALCAM and sALCAM levels were associated with certain clinicopathological variables, including overall survival. Compared with the non‑tumor tissues, the expression of ALCAM mRNA in the GC tissues was significantly upregulated (P=0.013). The expression of miR‑9 was reduced and inversely correlated with ALCAM mRNA levels in GC tissues and cell lines. The ALCAM mRNA level was reduced following ectopic overexpression of miR‑9 in SGC‑7901 human gastric cancer cells. The rates of membranous and cytoplasmic expression of ALCAM in GC tissues were 59.1 and 48.48%, respectively, and the serum sALCAM levels were significantly elevated in patients with GC. Elevated ALCAM mRNA, membranous ALCAM expression in GC tissues and high sALCAM levels are associated with advanced tumor stage, lymphatic invasion and shorter overall survival duration. The results of the current study indicated that membranous ALCAM expression and high serum sALCAM levels are independent prognostic markers of poor survival for patients with GC, and that the overexpression of ALCAM may be due to the downregulation of miR‑9.

Loss of ALCAM expression is linked to adverse phenotype and poor prognosis in breast cancer: a TMA-based immunohistochemical study on 2,197 breast cancer patients.

Activated leukocyte cell adhesion molecule (ALCAM) is a membranous cell adhesion protein that is often expressed in breast cancer. Data on the prognostic impact of ALCAM expression is highly controversial in this cancer. To evaluate the clinical impact of ALCAM expression in a sufficiently large patient cohort, we utilized a tissue microarray (TMA) containing more than 2,100 primary breast cancers with clinical follow-up data by immunohistochemistry. TMA spots containing normal breast epithelium showed moderate to strong membranous ALCAM staining. ALCAM staining was strong in 66.2%, moderate in 10.9%, weak in 11.1% and absent in 11.8% of 1,778 (80.9%) interpretable breast cancer tissue spots. Decreased ALCAM expression was significantly associated with advanced tumor size (p=0.0017), unfavorable tumor grade (p<0.0001), negative ER and PR status (p<0.0001 each) as well as high Ki67 labeling index (p<0.0001). Cancers with ACLAM expression loss had a significantly poorer overall (p<0.0001) and disease-specific survival (p=0.0088). This association also held true in the subset of nodal positive cancers (p<0.0001). In conclusion, these data demonstrate that ALCAM is generally expressed in normal and cancerous breast epithelium and that a marked reduction of ALCAM expression characterizes a subset of breast cancer patients with adverse tumor characteristics and unfavorable clinical outcome.

Enhanced down-regulation of ALCAM/CD166 in African-American Breast Cancer.

BackgroundVariation in tumor biology in African-American (AA) and Caucasian (CAU) women with breast cancer is poorly defined. Activated leukocyte cell adhesion molecule (ALCAM) is a bad prognostic factor of breast cancer yet it has never being studied in the AA population. We tested the hypothesis that ALCAM expression would be markedly lower in cases of AA breast cancer when compared to CAU.MethodsCases of breast cancer among AA (n = 78) and CAU (n = 95) women were studied. Immunohistochemical staining was used to semi-quantitatively score ALCAM expression in tumor and adjacent non-tumor breast tissues. Clinico-pathological characteristics including histological type, histological grade, tumor size, lymph node metastasis, estrogen receptor (ER), progesterone receptor (PR), and HER2-neu status were abstracted, and their association with ALCAM expression tested.ResultsUnivariate analysis revealed that the level of ALCAM expression at intercellular junctions of primary tumors correlates with histological grade (AA; p = 0.04, CUA; p = 0.02), ER status (AA; p = 0.0004, CAU; p = 0.0015), PR status (AA; p = 0.002, CUA p = 0.034) and triple-negative tumor status (AA; p = 0.0002, CAU; p = 0.0006,) in both ethnic groups. Multivariate analysis demonstrated that ethnicity contribute significantly to ALCAM expression after accounting for basal-like subtype, age, histological grade, tumor size, and lymph node status. Compared to CAU tumors, the AA are 4 times more likely to have low ALCAM expression (p = 0.003).ConclusionsMarkedly low expression of ALCAM at sites of cell-cell contact in primary breast cancer tumors regardless of differentiation, size and lymph node involvement may contribute to the more aggressive phenotype of breast cancer among AA women.