Journal: Cancers

Article Title: MARCH8 Suppresses Tumor Metastasis and Mediates Degradation of STAT3 and CD44 in Breast Cancer Cells

doi: 10.3390/cancers13112550

Figure Lengend Snippet: MARCH8 interacts with and degrades CD44 through the lysosome pathway. ( A ) Immunoblots to detect decreased CD44 and increased BAX, BID, and MARCH8 expression levels in MARCH8-overexpressing cells compared to GFP control cells. ( B ) Immunofluorescence staining with anti-CD44 antibody showing decreased expression of membrane protein CD44 (blue) in MARCH8-GFP expressing cells. ( C ) Flow cytometry histogram overlay (left panel) and dot plots (right panels) indicating MARCH8-decreased CD44 expression levels in negative association with MARCH8-GFP signals. ( D ) Immunoblots of endogenous CD44 and exogenous MARCH8-GFP after transient transfection of MARCH-GFP and treatment with MG-132 or chloroquine (CLQ) to block the proteasomal or lysosomal degradation pathways, respectively. ( E ) Immunoblots of MARCH8 and FLAG-tagged CD44 after anti-FLAG mediated immunoprecipitation (IP) of the lysates of HEK-293 cells after transfections with MARCH8-GFP and CD44-FLAG (standard isoform CD44s and full-length CD44f) and treatment with CLQ, indicating the interactions between MARCH8 and CD44 (CD44s or CD44f). ( F ) Immunoblots of BAX, BID, CD44, and MARCH8 in the MDA-MB-231 cells with stable expression of GFP or MARCH8-GFP with transient transfection of a FLAG vector control or restoration of CD44 expression via CD44s-FLAG. CD44 overexpression slightly inhibited the expression of proapoptotic BID and BAX in MARCH8-GFP-overexpressing cells.

Article Snippet: Plasmids that were used for overexpression include human CD44 standard form (NM_001001391), FLAG-tagged ORF Clone pCMV6-Flag-CD44s (OriGene Technologies, Rockville, MD, USA, Cat RC221820), CD44 full-length pCMV3-CD44f-HA ((Sino Biological, Beijing, China, Cat HG12211-CY), Lenti ORF clone of human MARCH8-GFP (OriGene Technologies, Rockville, MD, USA, Cat RC209891L2), GFP control (OriGene Technologies, Rockville, MD, USA, Cat PS10007), STAT3 (Addgene, Watertown, MA, USA, Cat 71450), and STAT3 Y705F (Addgene, Watertown, MA, USA, Cat 71445) mutant.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, Transfection, Blocking Assay, Immunoprecipitation, Plasmid Preparation, Over Expression

Journal: Cancers

Article Title: MARCH8 Suppresses Tumor Metastasis and Mediates Degradation of STAT3 and CD44 in Breast Cancer Cells

doi: 10.3390/cancers13112550

Figure Lengend Snippet: MARCH8 interacts with and degrades STAT3 through the proteasome pathway. ( A ) Immunoblots of AKT, pAKT, ERK, and pERK in MDA-MB-231 cells with stable expression of GFP and MARCH8-GFP, both adherent and in suspension. ( B ) Immunoblots of STAT3, pSTAT3 (Y705), CD44, and MARCH8 in GFP- and MARCH8-GFP-expressing cells in the absence or presence of MG-132 and CLQ, both adherent and in suspension. ( C ) Immunoblots of MARCH8, STAT3, pSTAT3 (Y705), and ubiquitin in lysates of cells co-transfected with STAT3 (wildtype or Y705F) and MARCH8-GFP (or GFP control) and immunoprecipitated by anti-STAT3 and anti-pSTAT3 (Y705). ( D ) Immunoblots of CD44, STAT3, BID, and BAX in MDA-MB-231 cells with stable expression of GFP and MARCH8-GFP, with transient transfection with CD44, STAT3, or mutant Y705F as indicated, both adherent and in suspension.

Article Snippet: Plasmids that were used for overexpression include human CD44 standard form (NM_001001391), FLAG-tagged ORF Clone pCMV6-Flag-CD44s (OriGene Technologies, Rockville, MD, USA, Cat RC221820), CD44 full-length pCMV3-CD44f-HA ((Sino Biological, Beijing, China, Cat HG12211-CY), Lenti ORF clone of human MARCH8-GFP (OriGene Technologies, Rockville, MD, USA, Cat RC209891L2), GFP control (OriGene Technologies, Rockville, MD, USA, Cat PS10007), STAT3 (Addgene, Watertown, MA, USA, Cat 71450), and STAT3 Y705F (Addgene, Watertown, MA, USA, Cat 71445) mutant.

Techniques: Western Blot, Expressing, Transfection, Immunoprecipitation, Mutagenesis

Journal: Leukemia

Article Title: Evidence of a role for CD44 and cell adhesion in mediating resistance to lenalidomide in multiple myeloma: therapeutic implications.

doi: 10.1038/leu.2013.174

Figure Lengend Snippet: Figure 1. Lenalidomide resistance and CD44 expression in myeloma cells (A) Lenalidomide-sensitive wild-type (wt) and lenalidomide-resistant (R10R) cells were subjected to qPCR, and CD44 mRNA content was analyzed using the comparative CT method and normalized to GAPDH internal control. CD44 expression in drug-naïve wt cells was arbitrarily set at 1.0, and representative data are shown from one of three independent experiments along with the S.D. (B) CD44 protein levels were evaluated in drug-naïve and lenalidomide-resistant cells by Western blotting. Densitometric quantitation of the total CD44 levels is shown from one of two independent experiments, and was first normalized to the β-actin loading control, and then compared to wild-type levels, which were arbitrarily set at 1.0. (C) Lenalidomide-sensitive (dotted line) and -resistant (solid line) cells were subjected to FACS analysis to determine the levels of cell surface CD44 using an Alexa Fluor® 488 conjugated CD44 antibody. On the left, a representative FACS profile is shown from one of three independent experiments, each of which collected 20,000 events. Quantitative analysis of the mean fluorescent intensity of these data was then used to calculate the fold-increase of surface CD44 levels in the lenalidomide-resistant cells following normalization to the isotype control, which is displayed on the right.

Article Snippet: In an attempt to show the reverse effect, we tried to overexpress the full length CD44 (Origene– NM_000610), however this overexpression appeared to be lethal by both transient transfection in HEK293 cells, and by Lentiviral transduction in lenalidomide-sensitive U266 or ANBL-6 cells (data not shown).

Techniques: Expressing, Control, Western Blot, Quantitation Assay

Journal: Leukemia

Article Title: Evidence of a role for CD44 and cell adhesion in mediating resistance to lenalidomide in multiple myeloma: therapeutic implications.

doi: 10.1038/leu.2013.174

Figure Lengend Snippet: Figure 2. Adhesive properties of drug-resistant and-na ïve cells. (A) HS5-GFP bone marrow stromal cells (BMSCs) were plated and allowed to adhere overnight, and Calcein Blue AM+, lenalidomide-sensitive wt or lenalidomide-resistant ANBL-6 or KAS-6 cells were then added. Non-adherent cells were subsequently washed off and the GFP−/Calcein Blue AM+ signal was measured. The percentage of adhesion was calculated as described in the “Materials and Methods,” and normalized to the adhesion of wt cells, which were arbitrarily set at 1.0. (B) Lenalidomide-sensitive and -resistant ANBL-6 and KAS-6/1 cells were evaluated for their ability to adhere to increasing concentrations of hyaluranon (HA) immobilized in culture plates, and evaluated as above. (C) Free hyaluronan or a CD44 blocking antibody were pre-incubated with the indicated myeloma cell lines, which were then allowed to adhere to BMSCs and analyzed as above. Relative adhesion was determined and compared to an isotype control, with data displayed as the mean ± S.D. All results are representative of three independent experiments.

Article Snippet: In an attempt to show the reverse effect, we tried to overexpress the full length CD44 (Origene– NM_000610), however this overexpression appeared to be lethal by both transient transfection in HEK293 cells, and by Lentiviral transduction in lenalidomide-sensitive U266 or ANBL-6 cells (data not shown).

Techniques: Adhesive, Blocking Assay, Incubation, Control

Journal: Leukemia

Article Title: Evidence of a role for CD44 and cell adhesion in mediating resistance to lenalidomide in multiple myeloma: therapeutic implications.

doi: 10.1038/leu.2013.174

Figure Lengend Snippet: Figure 3. Impact of CD44 blockade on the effectiveness of lenalidomide (A) Utilizing immobilized HA to represent the marrow microenvironment, lenalidomide- resistant ANBL-6 and U266 cells were pre-incubated with increasing concentrations of free HA (25–100 μg/mL). They were then exposed either to vehicle or lenalidomide for 72 hours, and viability was determined with the tetrazolium reagent WST-1. Data are from three independent experiments, and are presented as the mean ± S.D., with “*” representing statistical significance at a level of p<0.05 using the student’s paired t-test. (B) The experiment described in panel A was performed with lenalidomide-naïve ANBL-6 and U266 cells to study the impact of CD44 blockade on the acute effects of lenalidomide. (C) FACS analysis was used to determine the proportion of apoptotic (Annexin V+) cells in the U266 model system after the above-described experiments. (D) Again utilizing immobilized HA to represent the marrow microenvironment, lenalidomide-resistant ANBL-6 and U266 cells were pre-incubated with increasing concentrations of a humanized anti-CD44 monoclonal antibody (RO5429083). They were then exposed either to vehicle or lenalidomide for 72 hours, and viability was determined with the tetrazolium reagent WST-1. Data are from three independent experiments, and are presented as the mean ± S.D., with “*” representing statistical significance at a level of p<0.05 using the student’s paired t-test. (E) The experiment described in panel D was performed with lenalidomide-naïve ANBL-6 and U266 cells to study the impact of CD44 blockade on the acute effects of lenalidomide. (F) FACS

Article Snippet: In an attempt to show the reverse effect, we tried to overexpress the full length CD44 (Origene– NM_000610), however this overexpression appeared to be lethal by both transient transfection in HEK293 cells, and by Lentiviral transduction in lenalidomide-sensitive U266 or ANBL-6 cells (data not shown).

Techniques: Incubation

Journal: Leukemia

Article Title: Evidence of a role for CD44 and cell adhesion in mediating resistance to lenalidomide in multiple myeloma: therapeutic implications.

doi: 10.1038/leu.2013.174

Figure Lengend Snippet: Figure 4. CD44 expression level and lenalidomide sensitivity (A) Lenalidomide-sensitive and -resistant ANBL-6 and U266 cells were infected with Lentiviral particles containing shRNA constructs targeted to a non-specific scrambled sequence (.scr), or to CD44 (.shCD44). Stable clones with verified knockdown propagated on HA-coated plates were then exposed to lenalidomide, and viability was analyzed utilizing the WST-1 assay. Mean viability values are provided from three independent experiments ± S.D., and the student’s paired t-test was used to determine statistical significance, where “**” denotes p<0.01. (B) FACS analysis of these ANBL-6 and U266 models was then performed to examine the proportion of apoptotic (Annexin V+) cells. The fold-increase in the apoptotic cell population is shown on the right, and was obtained by normalizing the % apoptosis for scrambled controls to 1.0. (C) Lenalidomide-sensitive ANBL-6 and MM1.S cells were sorted into CD44-High and CD44-Low populations, and treated with lenalidomide at the indicated concentrations for 72 hours. Cellular viability measurements were then performed using the WST-1 assay, and all data points were normalized to the vehicle control, which was arbitrarily set at 100%. Mean viability values are provided from three independent experiments ± S.D., and the student’s paired t-test was used to determine statistical significance, where “*” denotes p<0.05, and “**” denotes p<0.01.

Article Snippet: In an attempt to show the reverse effect, we tried to overexpress the full length CD44 (Origene– NM_000610), however this overexpression appeared to be lethal by both transient transfection in HEK293 cells, and by Lentiviral transduction in lenalidomide-sensitive U266 or ANBL-6 cells (data not shown).

Techniques: Expressing, Infection, shRNA, Construct, Sequencing, Clone Assay, Knockdown, WST-1 Assay, Control

Journal: Leukemia

Article Title: Evidence of a role for CD44 and cell adhesion in mediating resistance to lenalidomide in multiple myeloma: therapeutic implications.

doi: 10.1038/leu.2013.174

Figure Lengend Snippet: Figure 6. Molecular correlates of ATRA action in lenalidomide-resistant myeloma (A) Lenalidomide-sensitive ANBL-6 cells were exposed to either vehicle, lenalidomide, ATRA, or the combination for 72 hours, and β-catenin mRNA levels were determined by qPCR using the comparative CT method. Values were normalized to GAPDH as an internal control, and the vehicle condition was arbitrarily set at 1.0 (left panel). The influence of ATRA on β-catenin protein levels was verified by Western blotting in KAS-6 lenalidomide- resistant treated with ATRA alone (middle panel), and in ANBL-6 drug-naïve cells exposed to vehicle, lenalidomide (L), ATRA (A), or both (L+A). β-actin is displayed as a loading control, and densitometry was performed to quantify β-catenin levels, which were normalized to vehicle controls arbitrarily set to 1.0. Representative blots from one of three experiments are shown. (B) Lenalidomide-sensitive and -resistant U266 cells were exposed to vehicle, lenalidomide, ATRA, or the combination for 72 hours. Western blotting was then performed to determine the total cellular content of CD44 with β-actin serving as a loading control, while quantitation was performed as described for panel A. (C) Cell surface levels of CD44 were determined by FACS utilizing an Alexa Fluor® 488 conjugated anti-CD44 antibody following exposure to ATRA at the indicated concentrations for 3, 6, 24, or 48 hours. All values were normalized to the respective vehicle control, which was arbitrarily set at 1.0, and are represented as relative CD44 cell surface levels from one of two representative and independent experiments.

Article Snippet: In an attempt to show the reverse effect, we tried to overexpress the full length CD44 (Origene– NM_000610), however this overexpression appeared to be lethal by both transient transfection in HEK293 cells, and by Lentiviral transduction in lenalidomide-sensitive U266 or ANBL-6 cells (data not shown).

Techniques: Control, Western Blot, Quantitation Assay

Journal: Leukemia

Article Title: Evidence of a role for CD44 and cell adhesion in mediating resistance to lenalidomide in multiple myeloma: therapeutic implications.

doi: 10.1038/leu.2013.174

Figure Lengend Snippet: Figure 7. Efficacy of ATRA in lenalidomide-resistant in vivo and ex vivo models (A) Immunodeficient mice were subcutaneously implanted with lenalidomide-resistant U266 cells, and after seven days were randomized to treatment with either vehicle, lenalidomide (50 mg/kg), ATRA (40 mg/kg), or the combination, with treatment given thrice weekly via intraperitoneal injection. Tumor growth was monitored by caliper measurement and calculated as tumor volume using the equation (0.4 x L x W^2). Each group had 5 mice, and the calculated p value between the control group and the combination group was 0.1. (B) Measured tumor volume from mouse xenografts from day 18 through day 28 of the experiment described in panel A. Statistically significant values are indicated by * which denotes p<0.05 as determined by the conditional χ2-test as explained in materials and methods. (C) A Kaplan-Meier plot of mouse survival data from the experiment described in panel A. (D) Purified CD138+ plasma cells obtained from patients who were immunomodulatory drug-naïve or lenalidomide-exposed and predominantly –refractory were evaluated for their surface expression of CD44 using FACS as described earlier. The left panel shows a representative histogram overlay of one lenalidomide-naive patient’s sample and another sample from a patient with lenalidomide-refractory disease. Both were probed with either the matched isotype control (dotted line) or the CD44 antibody (solid line). Mean fluorescent intensity values were obtained after normalizing to the isotype controls, and are displayed as a ratio of CD44/isotype (right panel) in 39 patients. (E) Purified CD138+ plasma cells obtained from patients who were lenalidomide refractory were treated for 72 hours with vehicle, lenalidomide, ATRA, or the combination, and viability was then analyzed using the WST-1 assay. All values are normalized to the vehicle

Article Snippet: In an attempt to show the reverse effect, we tried to overexpress the full length CD44 (Origene– NM_000610), however this overexpression appeared to be lethal by both transient transfection in HEK293 cells, and by Lentiviral transduction in lenalidomide-sensitive U266 or ANBL-6 cells (data not shown).

Techniques: In Vivo, Ex Vivo, Injection, Control, Purification, Clinical Proteomics, Expressing, WST-1 Assay