CD44 Splice Research Articles

Deciphering the role of platelet alpha granules in alveolar-capillary breakdown in severe malaria

Abstract Malaria is considered the deadliest disease in human history, killing hundreds of thousands annually. A hallmark of severe malaria is the breakdown of blood-organ barriers, such as in the lungs and brain, but the precise mechanism of how this occurs remains unknown. We determined that, unlike wildtype mice (C57BL6), mice with platelet alpha-granule deficiencies (Nbeal2−/−) did not die of malaria-associated acute respiratory distress syndrome (MA-ARDS) when infected with Plasmodium berghei NK65-Edinburgh (PbE). While wildtype mice exhibited significant alveolar-capillary breakdown by 6 days post-infection, as measured by alveolar protein concentrations and Evans blue permeation, Nbeal2−/− mice did not develop severe pathology. Flow cytometric analysis revealed that PbE-infected Nbeal2−/− mice had reduced CD8 T cell lung recruitment and activation compared to wildtypes. From this data, we concluded that a component of platelet alpha granules is crucial for activating CD8 T cells that subsequently damage the alveolar barrier. We hypothesized that the enzyme hyaluronidase 2 (HYAL2) is that crucial component and plays a 2-fold role: 1) that it directly contributes to barrier breakdown by cleaving hyaluronan (HA) from the endothelial glycocalyx (eGC), and 2) that the HA fragments released after eGC cleavage promote inflammation via CD44-binding. We found that HA is elevated in the plasma of infected mice. Preliminary data suggest that CD44 splice variants are differentially expressed in the lungs of naïve vs. PbE-infected mice, which could alter the binding kinetics with HA. Together, these data support the hypothesis that platelet HYAL2 is a critical mediator of MA-ARDS.

Abstract A013: Single-cell RNA sequencing of dedifferentiating cells from a mouse model that display oncogenic plasticity: Implications in understanding colon cancer stem cells

Abstract Characterization of colon cancer stem cells in vivo is extremely challenging. Our mouse model provides the opportunity to do so, as it displays oncogenic dedifferentiation. In this mouse model, Wnt and TGF signaling are deregulated by simultaneously mutating their transcriptional effectors, b-catenin and Smad4 respectively. This mouse model referred to as b-catenin gain-of-function and Smad4 loss-of-function (b-cateninGOF-Smad4LOF) conditional mutant displays oncogenic dedifferentiation in the intestinal epithelium. Within a week of inducing the mutation, villi cells (the mature epithelial compartment) express proliferative and stem cell markers; eventually developing adenomas (reported previously). Two striking observations, however, remain unexplained: firstly, fate reversal to stemness is seen only in a minor fraction of the epithelial cells despite the mutation being pan-epithelial; secondly, when the mutation is induced in a subset of stem cells, selective pressure is seen against the retention of mutant stem cells in the crypt s(progenitor compartment), and adenomas occur preferentially near the luminal epithelium. Our goal is to a) determine the basis for the selective pressure against mutant stem cells, and b) the differences between the normal and the b-cateninGOF-Smad4LOF mutant intestinal stem cells. To determine the gene expression profile unique to the dedifferentiating cells, we performed scRNA-seq on the dedifferentiating cells. To sort these cells–which express CD44 or proliferative markers–we integrated a reporter gene that expresses red fluorescent protein (RFP) into the b-cateninGOF-Smad4LOF mice. FACS for CD44 and RFP was performed seven days after the mutation to isolate the dedifferentiating cells from the mutant villi and the proliferating and/or CD44 positive cells in the mutant crypts. scRNA-seq confirmed enrichment of stem cell markers in the dedifferentiating cells. Gene set enrichment analyses revealed significant enrichment for the molecular signatures associated with Myc targets and active OXPHOS and Mtorc1 signaling in the dedifferentiating cells. The increase in OXPHOS is contrary to the expected metabolism in tumors. However, bulk RNA-seq for differential gene expression in the mutant villi epithelium and molecular analyses show enrichment of hypoxia gene targets and transient increase in hypoxia. These contradictory results are consistent with the spatial and temporal heterogeneity of hypoxia and metabolism observed in tumors and support the notion that intestinal stem cells rely on OXPHOS using the metabolites derived from the glycolytic metabolism of the neighboring cells. To determine the differences between normal intestinal stem cells and mutant stem cells, we cultured epithelium in 3D-matrigel to derive organoids from wild-type and mutant epithelium. The mutant organoids show higher expression of the CD44 splice variant implicated in cancer. Since stem cells are attributed to the organoid formation, our data indicate that stem cells from the mutant cells are oncogenic stem cells. Citation Format: Ansu O. Perekatt, Kylee Wrath, Zahra Hashemi, Dahlia Matouba, Christina Li. Single-cell RNA sequencing of dedifferentiating cells from a mouse model that display oncogenic plasticity: Implications in understanding colon cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A013.

CD147 mediates the CD44s-dependent differentiation of myofibroblasts driven by transforming growth factor-β1

Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-β1 and proinflammatory interleukin (IL)-1β are widely associated with fibrotic progression. TGF-β1 induces myofibroblast differentiation, while IL-1β induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-β1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-β1- and IL-1β-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-β1-induced fibroblast/myofibroblast differentiation and IL-1β-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell–cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.

Preclinical Evaluation of an Engineered Single-Chain Fragment Variable-Fragment Crystallizable Targeting Human CD44.

Glycoprotein CD44 and alternative splice variants are overexpressed in many cancers and cancer stem cells. Binding of hyaluronic acid to CD44 activates cell signaling pathways, inducing cell proliferation, cell survival, and invasion. As such, CD44 is regarded as an excellent target for cancer therapy when this interaction can be blocked. In this study, we developed a CD44-specific antibody fragment and evaluated it for imaging CD44-positive cancers using PET. Methods: A human single-chain fragment variable (scFv) was generated by phage display, using the extracellular domain of recombinant human CD44. The specificity and affinity of the scFv-CD44 were evaluated using recombinant and tumor cell-expressed CD44. Epitope mapping of the putative CD44 binding site was performed via overlapping peptide microarray. The scFv-CD44 was reformatted into a bivalent scFv-Fc-CD44, based on human IgG1-fragment crystallizable (Fc). The scFv-Fc-CD44 was radiolabeled with 64Cu and 89Zr. The purified reagents were injected into athymic nude mice bearing CD44-positive human tumors (MDA-MB-231, breast cancer, triple-negative). Biodistribution studies were performed at different times after injection of [64Cu]Cu-NOTA-scFv-Fc-CD44 or [89Zr]Zr-DFO-scFv-Fc-CD44. PET/CT imaging was conducted with [89Zr]Zr-DFO-scFv-Fc-CD44 on days 1 and 7 after injection and compared with a scFv-Fc control antibody construct targeting glycophorin A. Results: Epitope mapping of the scFv binding site revealed a linear epitope within the extracellular domain of human CD44, capable of blocking binding to native hyaluronic acid. Switching from a monovalent scFv to a bivalent scFv-Fc format improved its binding affinity toward native CD44 on human breast cancer cells by nearly 200-fold. In vivo biodistribution data showed the highest tumor uptake and tumor-to-blood ratios for [89Zr]Zr-DFO-scFv-Fc-CD44 between days 5 and 7. PET imaging confirmed excellent tumor specificity for [89Zr]Zr-DFO-scFv-Fc-CD44 when compared with the control scFv-Fc. Conclusion: We developed a CD44-specific scFv-Fc construct that binds with nanomolar affinity to human CD44. When radiolabeled with 64Cu or 89Zr, it demonstrated specific uptake in CD44-expressing MDA-MB-231 tumors. The high tumor uptake (∼56% injected dose/g) warrants clinical investigation of [89Zr]Zr-DFO-scFv-Fc-CD44 as a versatile PET imaging agent for patients with CD44-positive tumors.

Clinicopathological significance and prognostic implication of CD44 and its splice variants (v3 and v6) in colorectal cancer.

BackgroundCD44 is a marker for colorectal cancer (CRC) stem cells (CCSCs), but its prognostic value remains controversial. Furthermore, few studies have investigated the expression profile of CD44 variants in CRC. The proto-oncogene mast/stem cell growth factor receptor Kit (c-Kit) was previously found to play an important role in supporting CRC cells; however, the associations between c-Kit and CD44 (and its splice variants) remain largely unknown.MethodsA total of 148 patients with CRC were enrolled in the present study, and the levels of CD44, CD44 splice variant (v)3, CD44v6 and c-Kit were examined by immunohistochemical staining. Associations between these markers and clinicopathological parameters, as well as the extent to which these markers predict progression free-survival (PFS), were analyzed.ResultsA total of 29 (19.6%), 60 (40.5%) and 66 (44.6%) patients were CD44-, CD44v3- and CD44v6-positive, respectively. No clear c-Kit-positivity was detected in any of the patients. Analyses of clinicopathological features indicated that positive expression of CD44v3 was significantly associated with cell differentiation (P=0.03), and N (P=0.04), M (P<0.01) and tumor-node-metastasis (TNM) stages (P<0.01). Univariate analysis demonstrated that T, N, M and TNM stages (all P<0.01), and CD44 (P<0.01), CD44v3 (P=0.03) and CD44v6 (P=0.02) levels were significantly associated with PFS. Furthermore, multivariate analysis indicated that patients without CD44 expression (P<0.01) but with CD44v3 (P=0.04) and CD44v6 (P=0.02) expression exhibited significantly shortened PFS.ConclusionsCD44, CD44v3 and CD44v6 were determined to be heterogeneously expressed in CRC, and may aid in the prognostic prediction of patients with this disease.

CD44 splice variant (CD44v3) promotes progression of urothelial carcinoma of bladder through Akt/ERK/STAT3 pathways: novel therapeutic approach.

The incidence of Urothelial carcinoma of bladder (UBC) is gradually increasing by changing lifestyle and environment. The development of a tumor has been noted to be accompanied by modifications in the extracellular matrix (ECM) consisting of CD44, hyaluronic acid (HA) and its family members. The importance of CD44 splice variants and HA family members has been studied in UBC. The cohort of study included 50 UBC patients undergoing radical cystectomy and 50 healthy subjects. The molecular expression of CD44 and HA family members was determined. Effect of CD44 variant-specific silencing on downstream signaling in HT1376 cells was investigated. Combinatorial treatment of 4-MU (4-methylumbelliferone) with cisplatin or doxorubicin on chemosensitivity was also explored. Higher expression of HA, HAS2, and CD44 was observed in Indian UBC patients which also showed the trend with severity of disease. Splice variant assessment of CD44 demonstrated the distinct role of CD44v3 and CD44v6 in bladder cancer progression. shRNA-mediated downregulation of CD44v3 showed an increase effect on cell cycle, apoptosis and multiple downstream signaling cascade including pAkt, pERK and pSTAT3. Furthermore, 4-MU, an HA synthesis inhibitor, observed to complement the effect of Cisplatin or Doxorubicin by enhancing the chemosensitivity of bladder cancer cells. Our findings exhibit involvement of CD44 splice variants and HA family members in UBC and significance of 4-MU in enhancing chemosensitivity suggesting their novel therapeutic importance in disease therapeutics.

3332 Overexpression of CD44 is involved in the development of the early endometriotic lesion in a xenograft model

OBJECTIVES/SPECIFIC AIMS: Previously, we showed decreased development of endometriotic lesions in CD44 knockout mice compared to control.(1) CD44 has 10 different variants and a standard form. Menstrual endometrial cells (MECs) from women with endometriosis have increased adhesion and also express higher levels of CD44 variant 6 (v6) than v3, compared to MECs from women without endometriosis. (2) Here, we assessed the effects of CD44 standard (CD44s), CD44v3 and CD44v6 overexpression (OE) on immortalized human endometrial epithelial (iEECs) and stroma cells (hESCs) in vivo attachment in a nude mouse xenograft model. 1. Knudtson JF, Tekmal RR, Santos MT, et al. Impaired Development of Early Endometriotic Lesions in CD44 Knockout Mice. Reproductive sciences (Thousand Oaks, Calif.). 2016;23(1):87-91. 2. Griffith JS, Liu YG, Tekmal RR, Binkley PA, Holden AE, Schenken RS. Menstrual endometrial cells from women with endometriosis demonstrate increased adherence to peritoneal cells and increased expression of CD44 splice variants. Fertility and sterility. 2010;93(6):1745-1749. METHODS/STUDY POPULATION: Overexpression of CD44s, CD44v3 and CD44v6 was carried out using lipofectamine and their expression verified with qRT-PCR in iEEC and hESCs. Nude mice, 8-10 week old, were injected with estrogen 1 week prior to injection of iEECs and hESCs (n=7 per group). The cells were counted after transfection and at least 300,000 iEECs and 300,000 hESCs were injected per mouse. The transfected cells were tagged with cell tracker red (iEECs) and green (hESCs). Forty-eight hours after injection into the xenograft, the mice were sacrificed. The cells were counted using fluorescent stereo microscopy (FSM). Percent attachment was calculated based on the number of cells visualized by FSM divided by the number of transfected cells injected. Unpaired student t-test was performed to analyze differences in the percent attachment of the cells. RESULTS/ANTICIPATED RESULTS: The majority of cells were attached to the peritoneum. There was increased attachment of hESCs with OE of CD44v6 compared to control (p=0.03). CD44v6 OE did not change attachment of iEECs. There was no difference in attachment in iEECs or hESCs with OE of CD44s or CD44v3. DISCUSSION/SIGNIFICANCE OF IMPACT: Overexpression of CD44v6 increases attachment of ESCs to PMCs in an in vivo xenograft model. Menstrual endometrial cell type and CD44 variants play a complex role in the development of the early endometriotic lesion.

Mesenchymal splice isoform of CD44 (CD44s) promotes EMT/invasion and imparts stem-like properties to ovarian cancer cells.

Increased metastasis and a precipitous recurrence contribute to the lethality of ovarian cancer (OC). Several molecular mechanisms including aberrant-splicing have been closely associated with the extent of cancer progression. Numerous gene transcripts are differentially spliced in cancer cells, CD44 being one of them. CD44 splice isoforms contribute to the aggressiveness and gain of stem-like properties in different cancer types, but their role in ovarian cancer remains to be elucidated. We observed augmented CD44 levels in human ovarian cancer patient samples correlated with enhanced expression of the mesenchymal spliced variant CD44s (standard) and a concurrent decrease in the epithelial variants (CD44v). Moreover, CD44s was upregulated upon TGFβ1-induced EMT, which was mediated through the downregulation of the splicing factor, ESRP1. Furthermore, overexpression of this mesenchymal isoform in the OC cells induced EMT and invasion, followed by the gain of stem-like characteristics and chemoresistance. Since all these phenomena render lethality to this disease type, CD44s can be attributed for playing a major role in deregulated-splicing mediated ovarian cancer progression.

MET Signaling Mediates Intestinal Crypt-Villus Development, Regeneration, and Adenoma Formation and Is Promoted by Stem Cell CD44 Isoforms

Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice. We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (AhCre/Metfl/fl/LacZ) or ISC-specific disruption of MET (Lgr5Creert2/Metfl/fl/LacZ) and control mice (AhCre/Met+/+/LacZ, Lgr5Creert2/Met+/+/LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5Creert2/Metfl/fl/Apcfl/fl and Lgr5Creert2/Met+/+/Apcfl/fl mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in AhCre/Metfl/fl/Apcfl/+ mice compared with AhCre/Met+/+/Apcfl/+ control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44+/+, Cd44-/-, Cd44s/s, or Cd44v4-10/v4-10 mice). Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in AhCre/Metfl/fl/LacZ mice. Lgr5Creert2/Metfl/fl/LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5Creert2/Metfl/fl/Apcfl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (AhCre/Metfl/fl/Apcfl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44-/- mice did not expand to the same extent as crypts from Cd44+/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4-10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4-10. In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4-10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors.

Akt Signaling Is Sustained by a CD44 Splice Isoform-Mediated Positive Feedback Loop.

Tumor cells nearly invariably evolve sustained PI3K/Akt signaling as an effective means to circumvent apoptosis and maintain survival. However, for those tumor cells that do not acquire PI3K/Akt mutations to achieve this end, the underlying mechanisms have remained obscure. Here, we describe the discovery of a splice isoform-dependent positive feedback loop that is essential to sustain PI3K/Akt signaling in breast cancer. Splice isoform CD44s promoted expression of the hyaluronan synthase HAS2 by activating the Akt signaling cascade. The HAS2 product hyaluronan further stimulated CD44s-mediated Akt signaling, creating a feed-forward signaling circuit that promoted tumor cell survival. Mechanistically, we identified FOXO1 as a bona fide transcriptional repressor of HAS2. Akt-mediated phosphorylation of FOXO1 relieved its suppression of HAS2 transcription, with FOXO1 phosphorylation status maintained by operation of the positive feedback loop. In clinical specimens of breast cancer, we established that the expression of CD44s and HAS2 was positively correlated. Our results establish a positive feedback mechanism that sustains PI3K/Akt signaling in tumor cells, further illuminating the nearly universal role of this pathway in cancer cell survival. Cancer Res; 77(14); 3791-801. ©2017 AACR.

CD44 Splice Variants as Potential Players inAlzheimer's Disease Pathology.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive deficits, deposition of amyloid-β (Aβ) plaques, intracellular neurofibrillary tangles, and neuronal cell death. Neuroinflammation is commonly believed to participate in AD pathogenesis. CD44 is an inflammation-related gene encoding a widely-distributed family of alternatively spliced cell surface glycoproteins that have been implicated in inflammation, metastases, and inflammation-linked neuronal injuries. Here we investigated the expression patterns of CD44S (which does not contain any alternative exon) and CD44 splice variants in postmortem hippocampal samples from AD patients and matched non-AD controls. The expression of CD44S and CD44 splice variants CD44V3, CD44V6, and CD44V10 was significantly higher in AD patients compared to non-AD controls. Immunohistochemistry of human hippocampal sections revealed that CD44S differentially localized to neuritic plaques and astrocytes, whereas CD44V3, CD44V6, and CD44V10 expression was mostly neuronal. Consistent with these findings, we found that the expression of CD44V6 and CD44V10 was induced by Aβ peptide in neuroblastoma cells and primary neurons. Furthermore, in loss of function studies we found that both CD44V10-specific siRNA and CD44V10 antibody protected neuronal cells from Aβ-induced toxicity, suggesting a causal relationship between CD44V10 and neuronal cell death. These data indicate that certain CD44 splice variants contribute to AD pathology and that CD44V10 inhibition may serve as a new neuroprotective treatment strategy for this disease.

HnRNPM and CD44s expression affects tumor aggressiveness and predicts poor prognosis in breast cancer with axillary lymph node metastases.

HnRNPM is an essential splicing factor and its expression is closely correlated with invasion and metastasis of tumor cells. The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and CD44 splice variants have been implicated in specific oncogenic signaling pathways. To investigate the clinical significance and biological function of hnRNPM, immunohistochemistry, quantitative, and semiquantitative polymerase chain reaction, lentiviral transfection system and transwell invasion assays were performed. We found that hnRNPM expression was significantly upregulated in breast cancer tissues compared with benign breast lesions. Although there was no significant correlation between hnRNPM and total CD44 protein or mRNA level, there was a negative correlation between hnRNPM and CD44v6. HnRNPM and CD44s expression showed positive correlation and in particular, they were dually expressed in breast cancer tissues. Interestingly, cancer stem cells marker, ALDH1+ phenotype was positively associated with overexpression of CD44s or hnRNPM and negatively related to CD44v6. Patients with high hnRNPM tended to have higher levels of CD44s, shorter overall survival (OS) and higher rates of lymph node metastases (LNM). Remarkably, Kaplan-Meier and Cox regression analyses displayed that hnRNPM+ or CD44shigh was a poor prognostic factor for OS of patients with LNM. Upregulation of hnRNPM in MCF-7 cells caused a significant increase in cell invasion, and this effect may occur through the regulation of CD44s expression. In conclusion, overexpression of hnRNPM promotes breast cancer aggressiveness by regulating the level of CD44s, indicates a poor prognosis for patients with LNM, and has potential as therapeutic targets.

Abstract 5070: CD44 alternative splice variants are associated with prostate cancer cell identity and migration

Abstract Patient tumors can demonstrate tremendous cell-to-cell genetic heterogeneity while cultured cancer cell lines are often more homogeneous. This fundamental difference can undermine in vitro findings and their application to the more complex in vivo tumor environment. However, it should not be assumed that cultured cells are homogeneous even in well established and widely used cell lines. In my study of CD44 alternative splicing in the prostate cancer cell line PC3, I encountered varying CD44 alternative splicing expression profiles dependent on the origin of the PC3 cell line. CD44 is a cell surface glycoprotein that binds to components of the extracellular matrix, including hyaluronan, and is primarily involved in cell-cell and cell-matrix interactions. CD44 contains 10 variable exons that when combined in particular combinations significantly alter/guide CD44 downstream activities. In the context of cancer biology, alternative splicing of CD44 can control the epithelial to mesenchymal transition (EMT) of cancer cells and is associated with metastasis in many types cancers such as breast and prostate. To determine if cellular heterogeneity was the source of variable CD44 alternative splicing profiles, I isolated PC3 single cell clones. From these clones I identified two cell populations based on colony morphology: a compact colony subset with cells of a epithelial phenotype (PC3-EL), and a diffuse colony subset with cells of a mesenchymal phenotype (PC3-ML). I then performed reverse transcription-PCR (rt-PCR) on cDNA generated from these two populations with primers that specifically amplify the CD44 variable region to generate a fingerprint of CD44 alternative splicing. I found that PC3 cells with an epithelial phenotype express multiple CD44 variant exons (CD44v), including the epithelium-associated CD44v1,v8-10 variant (CD44E). However, phenotypically mesenchymal PC3 cells show a complete loss of CD44 splice variants and primarily express standard CD44v1 (CD44S). These differences in CD44 mRNA alternative spicing are manifested at the protein level as phenotypically epithelial PC3 cells express predominantly high molecular weight CD44 variants. I then monitored the association of CD44v expression on cell migration and determined that PC3 cells that express CD44E are non-motile, while PC3 cells that predominantly express CD44S are highly motile. Acquisition of CD44S over CD44E can signal EMT, an early and critical step in cancer metastasis. These data indicate that the CD44 alternative splice fingerprint may provide a predictive biomarker for EMT and the acquisition of other early pro-metastatic features in prostate cancer cells. Citation Format: David J. Bond, John D. Lewis. CD44 alternative splice variants are associated with prostate cancer cell identity and migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5070.

CD44 Splice Variant v8-10 as a Marker of Serous Ovarian Cancer Prognosis.

CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue-specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10) has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemical (IHC) analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20%) compared to those with the lowest expression (bottom 10 and 20%) (p = 0.0181, p = 0.0262 respectively). Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p<0.05). Therefore, presence of transmembrane CD44v8-10 on the surface of primary tumor cells may be a marker of a highly epithelial tumor with better prognosis while enzymatic cleavage of CD44v8-10, as detected by presence of the soluble extracellular domain in ascites fluid, may be indicative of a more metastatic disease and worse prognosis.

The CD44s splice isoform is a central mediator for invadopodia activity.

The ability for tumor cells to spread and metastasize to distant organs requires proteolytic degradation of extracellular matrix (ECM). This activity is mediated by invadopodia, actin-rich membrane protrusions that are enriched for proteases. However, the mechanisms underlying invadopodia activity are not fully understood. Here, we report that a specific CD44 splice isoform, CD44s, is an integral component in invadopodia. We show that CD44s, but not another splice isoform CD44v, is localized in invadopodia. Small hairpin (sh)RNA-mediated depletion of CD44s abolishes invadopodia activity, prevents matrix degradation and decreases tumor cell invasiveness. Our results suggest that CD44s promotes cortactin phosphorylation and recruits MT1-MMP (also known as MMP14) to sites of matrix degradation, which are important activities for invadopodia function. Importantly, we show that depletion of CD44s inhibits breast cancer cell metastasis to the lung in animals. These findings suggest a crucial mechanism underlying the role of the CD44s splice isoform in breast cancer metastasis.

Aberrant Splicing of Estrogen Receptor, HER2, and CD44 Genes in Breast Cancer.

Breast cancer (BC) is the most common cause of cancer-related death among women under the age of 50 years. Established biomarkers, such as hormone receptors (estrogen receptor [ER]/progesterone receptor) and human epidermal growth factor receptor 2 (HER2), play significant roles in the selection of patients for endocrine and trastuzumab therapies. However, the initial treatment response is often followed by tumor relapse with intrinsic resistance to the first-line therapy, so it has been expected to identify novel molecular markers to improve the survival and quality of life of patients. Alternative splicing of pre-messenger RNAs is a ubiquitous and flexible mechanism for the control of gene expression in mammalian cells. It provides cells with the opportunity to create protein isoforms with different, even opposing, functions from a single genomic locus. Aberrant alternative splicing is very common in cancer where emerging tumor cells take advantage of this flexibility to produce proteins that promote cell growth and survival. While a number of splicing alterations have been reported in human cancers, we focus on aberrant splicing of ER, HER2, and CD44 genes from the viewpoint of BC development. ERα36, a splice variant from the ER1 locus, governs nongenomic membrane signaling pathways triggered by estrogen and confers 4-hydroxytamoxifen resistance in BC therapy. The alternative spliced isoform of HER2 lacking exon 20 (Δ16HER2) has been reported in human BC; this isoform is associated with transforming ability than the wild-type HER2 and recapitulates the phenotypes of endocrine therapy-resistant BC. Although both CD44 splice isoforms (CD44s, CD44v) play essential roles in BC development, CD44v is more associated with those with favorable prognosis, such as luminal A subtype, while CD44s is linked to those with poor prognosis, such as HER2 or basal cell subtypes that are often metastatic. Hence, the detection of splice variants from these loci will provide keys to understand the pathogenesis, predict the prognosis, and choose specific therapies for BC.

Malignant Reprogramming of Progenitors into Leukemia Stem Cells Is Enhanced By Upregulation of CD44 Transcript Variant 3 in Malignant Microenvironments

IntroductionMalignant reprogramming, first described in chronic myeloid leukemia (CML), occurs upon activation of the Wnt/b-catenin pathway in granulocyte-macrophage progenitors (GMPs) that gain the capacity to self-renew and contribute to the emergence of BCR-ABL1 tyrosine kinase inhibitor (TKI) resistant blast crisis CML. Deregulation of the Wnt/b-catenin target gene, CD44, plays a vital role in leukemia stem cell (LSC) maintenance in the malignant microenvironment in mouse models of CML. However, extensive alternative mRNA splicing in humans results in expression of multiple CD44 isoforms, some of which have been implicated in cancer invasion and metastasis. In this study we investigated the role of CD44 splice variant expression on human blast crisis LSC maintenance in the malignant niche.Methods and ResultsCD44 Isoform Expression AnalysisTo investigate the splice isoform expression pattern of CD44, whole transcriptome RNA sequencing (RNA Seq; Illumina HiSeq 2000) was performed on FACS sorted chronic phase (CP; n=8) and blast crisis (BC; n=8) CML progenitors (CD34+CD38+Lin-) as well as their normal counterparts from cord blood (CB) (n=7) and adult peripheral blood (NPB; n=4). While whole gene expression analysis revealed upregulation of CD44 in blast crisis compared with chronic phase and normal progenitors, a plethora of CD44 transcript variants were also detected including variants 3, 4 (CD44s), 5, 6, 7, 8. Notably, RNA Seq isoform analysis detected a higher expression of CD44 transcript variant 3 in BC compared to CP and CB and NPB. Moreover, CD44 transcript variant 3 gene expression was highly expressed in undifferentied human embryonic stem cells (hESCs) while differentiated hESCs (embryoid bodies) had low expression, suggesting CD44 transcript variant 3 to be important for pluripotency.Lentiviral CD44 Variant 3 OverexpressionTo directly determine the impact of CD44 variant 3 expression on malignant reprogramming of CP progenitors into self-renewing LSC, we developed a lentiviral human CD44 variant 3 overexpression vector and transduced CP CML progenitors. Transduced CP progenitors harbored increased expression of migration specific markers, such as osteopontin and ICAM1, as well as an upregulation of the pro-survival long isoforms of BCL2 family members BCLX and MCL1, thereby enhancing survival and replating in hematopoietic progenitor assays. Moreover, hESCs transduced with CD44 transcript variant 3 showed upregulation of pro-survival BCL2 isoforms, enhanced proliferation and as well as maintenance of an undifferentiated state, suggesting that CD44 transcript variant 3 promotes pluripotency.Targeted Inhibition of CD44 variant 3 Expressing LSCHumanized RAG2-/-gc-/-mice engrafted with CD34+ BC CML patient samples showed a significant reduction of human progenitor cells post treatment with a clinical grade CD44 mAb, both alone and in combination with Dasatinib in all hematopoietic niches. Bone marrow and spleen samples from primary transplanted mice show a reduced gene expression level of CD44 and CD44 transcript variant 3 upon combination treatment of CD44 and Dasatinib. Most importantly, serial transplantation of progenitors treated with the CD44 mAb as well as in combination with Dasatinib revealed a significant reduction in LSC self-renewal capacity commensurate with a reduction in CD44 variant 3 expression.ConclusionsUpregulation of an embryonic splice variant of CD44, variant 3, expands pluripotent stem cell populations and promotes malignant reprogramming of CML progenitors into self-renewing LSC. Treatment with a humanized CD44 specific mAb sensitizes CML LSC residing in malignant niches to Dasatinib. From these results CD44 mAb appears to be an excellent antibody for future combination clinical studies aimed at eradicating therapy resistant blast crisis LSC in CML. In addition, these observations strongly suggest that CD44 transcript variant 3 upregulation serves as a biomarker of progression from CP to BC as well as the generation of TKI resistant LSCs, with the potential of being a more specific target for future combination therapies. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1969: Overexpression of specific CD44 variants mediate endocrine insensitivity and invasion in breast cancer cells

Abstract The majority of breast cancers express the oestrogen receptor and are potentially amenable to antihormone therapy; however the clinical effectiveness of these agents is limited by the phenomenon of acquired resistance which is associated with disease relapse and poor prognosis. In vitro evidence suggests that acquired resistance to tamoxifen and fulvestrant results in a significant gain to the migratory and invasive nature of breast cancer cells which may augment their metastatic potential in vivo. We have previously demonstrated that the CD44 receptor is overexpressed in acquired endocrine resistance and contributes to a gain in aggressive phenotype and poor outcome clinically. However, little is known regarding the effects of CD44 splice variants in this context. Here we have explored the role of CD44 variant 3 (v3) and CD44 variant 6 (v6) in endocrine-resistant and endocrine-sensitive breast cancer models. CD44v3 and CD44v6 isoform expression was assessed in cell models of acquired tamoxifen (Tam-R) and fulvestrant (Fas-R) resistance along with their endocrine-sensitive MCF-7 counterparts by microarray analysis, Western blotting, immunofluorescence and immunohistochemistry. The ability of CD44v3 and CD44v6 to modulate tumor cell growth (cell counting assay), migration (fibronectin motility assay), invasion (Matrigel Boyden chamber assay), adhesion (matrix adhesion assay) and response to endocrine agents were determined using cells in which CD44v3 or CD44v6 expression was modulated by overexpression (MCF-7) or by siRNA (Tam-R, Fas-R). Changes in cellular signalling pathways as a result of CD44 modulation and activation were assessed using Western blotting. CD44v3 and CD44v6 were overexpressed at gene and protein level in Tam-R and Fas-R cells versus endocrine-sensitive MCF-7 cells. Inhibition of CD44 expression (all isoforms) in resistant cells resulted in a loss of migratory, proliferative and invasive phenotype. Overexpression of CD44v6 in MCF-7 cells resulted in a gain in EGFR signalling and enhanced their intrinsic invasive capacity and promoted a loss of endocrine response. In contrast, overexpression of CD44v3 decreased EGFR pathway activity in MCF-7 cells and did not augment their invasive capacity or alter their endocrine sensitivity. Our data suggests that upregulation of CD44v6 in acquired resistant breast cancer may contribute to their gain in aggressive phenotype and loss of endocrine response through transactivation of the EGFR pathway. Future therapeutic targetting of CD44v6 may prove to be an effective strategy alongside EGFR targeted agents in delaying/preventing acquired resistance in breast cancer. Citation Format: Rebecca L. Bellerby, Christopher Smith, Julia Gee, Tracey Martin, Peter Barrett-Lee, Stephen Hiscox. Overexpression of specific CD44 variants mediate endocrine insensitivity and invasion in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1969. doi:10.1158/1538-7445.AM2014-1969

Abstract 211: Sulfasalazine (SSZ) works cancer stem-like cells (CSCs) via inhibiting xCT signal pathway: Phase 1 study in patients with gastric cancer (EPOC 1205)

Abstract Back ground: CD44 is an adhesion molecule expressed in cancer stem-like cells (CSCs). Our group recently reported that CD44 splice variant (CD44v) is expressed in CSCs and interacts with xCT, a glutamate-cystine transporter, keeping high levels of the intracellular reduced glutathione (GSH). Thus, CSCs with a high expression of CD44v have an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS), resulting in resistance to various therapeutic stresses (Cancer Cell 2011; Cancer Res 2013). Sulfasalazine (SSZ) as an xCT inhibitor suppressed CD44v-dependent tumor growth and increased sensitivity to cytotoxic drugs in vivo study. Methods: A phase 1 dose escalation study in patients with advanced gastric cancer who had received one or more standard chemotherapies was conducted to determine the optimal dose, change in CD44v expression and intra-tumor level of GSH pre- and post SSZ exposure and pharmacokinetics. SSZ was given fourth-daily oral administration with 2 weeks as one cycle, which were continued until progression of disease, unacceptable toxicity, or other discontinuation criteria were met. A 3+3 escalation was used to evaluate a MTD. Tumor tissues were obtained pre- and post SSZ administration to evaluate expression of CD44v and intra-tumor level of GSH by immunohistochemistry and boron doped diamond microelectrode (Sci Rep 2012), respectively. Results: Eleven patients were dosed from 8 g to 12 g/day; median age: 71 years (61-78); median number of prior chemotherapies: 3 (1-4). There was two DLT of grade 3 anorexia and nausea among patients who were treated with 12 g/day. One additional patients required frequent dose interruption with grade 2 anorexia and nausea. Therefore 12g/day was judged as MTD. No DLT was observed among patients with 8g/day. Among the 5 patients with high CD44v expression, 4 patients achieved reduced expression of CD44v after the administration of SSZ for 2 weeks. GSH was also apparently decreased in 2 of 5 patients. Further exploratory biomarker analyses are still ongoing. The individual variability of SSZ exposure was explainable in terms of the genotypes of ABCG2 and NAT2 which influence SSZ pharmacokinetics. Conclusions: Optimal dose of SSZ was considered as 8g/day. Down regulation of CD44v expression and decreased level of GSH is confirmed as a pharmacodynamic marker of drug-on-target effect and mode of action of SSZ for CSCs, which warrants further investigation for combination with chemotherapy or other targeting agents. Clinical trial information: UMIN000010254 Citation Format: Kohei Shitara, Shunji Takahashi, Takako Nakajima, Hironaka Shuichi, Osamu Nagano, Chiyo Imamura, Taisei Mushiroda, Yasuaki Einaga, Miki Fukunani, Akihiro Sato, Atsushi Ohtsu, Hideyuki Saya, Toshihiko Doi. Sulfasalazine (SSZ) works cancer stem-like cells (CSCs) via inhibiting xCT signal pathway: Phase 1 study in patients with gastric cancer (EPOC 1205). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 211. doi:10.1158/1538-7445.AM2014-211

Abstract 4798: The niche specific role of CD44 splice isoform expression in blast crisis leukemia stem cell generation

Abstract Introduction Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm initiated in hematopoietic stem cells by expression of the BCR-ABL1 fusion oncogene and its protein product, which enhances ABL1 kinase activity. Targeted BCR-ABL tyrosine kinase inhibitors (TKIs), such as Imatinib, and the second generation TKIs like Nilotinib as well as the dual specific SCR and ABL inhibitor Dasatinib have significantly slowed disease progression by eradicating the bulk of BCR-ABL1 expressing cells in the circulation. However, progression to a therapeutically resistant blast crisis (BC) phase is driven by CD34+CD38+Lin- progenitors that co-opt stem cell properties, such as enhanced self-renewal and survival, albeit in a deregulated manner. These BC leukemia stem cells (BC LSC) harbor enhanced BCL2 and beta-catenin expression. Seminal research shows that a key Wnt/beta-catenin target gene, CD44, plays a vital role in cancer stem cell survival and retention in the malignant microenvironment. Because alternative mRNA splicing in humans generates many CD44 isoforms, we investigated CD44 variant expression and retention of human BC LSC in specific hematopoietic niches. Methods and Results We performed whole transcriptome RNA sequencing (RNA Seq) of FACS sorted progenitors (Lin-CD34+CD38+) from chronic phase (CP)(n=8) and blast crisis (BC)(n=8) CML patients as well as from normal cord blood (CB) (n=7) and adult peripheral blood (NPB)(n=4). A number of CD44 transcript variants were detected: 3, 4 (CD44s), 5, 6, 7, 8 plus additional variants. RNA seq analysis uncovered a higher overall CD44 expression in BC compared to CP progenitors. Notably, one isoform of CD44, variant 3, was highly upregulated in BC compared to CP progenitors and minimally expressed by their normal counterparts. Moreover, BC progenitors that engrafted in RAG2-/-γc-/- harbored CD44v3 expression in the bone marrow and the splenic niche and this differential BC LSC isoform expression was reduced after dasatinib treatment. In addition, BC LSC in the spleen showed a reduction of migration specific markers, such as RHAMM, ICAM-1 and Osteopontin, compared with the bone marrow resident BC LSC. Furthermore, a comparative splice isoform specific qRT-PCR analysis of CD44 variants expression in young versus old bone marrow showed no correlation with aging. In fact, human embryonic stem cells harbored variant 3 expression. Conclusions These data suggest that CD44 transcript variant 3 upregulation occurs following malignant reprogramming of hematopoietic progenitors that enables them to adopt features of an embryonic transcriptional program in select microenvironments. Detection of CD44 variant 3 has the potential to more precisely identify patients at risk for relapse and progression, suggest that combined therapeutic strategies involving a BCR-ABL specific TKI and a CD44 monoclonal antibody may decrease relapse and BC transformation rates. Citation Format: Frida L. Holm, Eva Hellqvist, Cayla Mason, Christian Barrett, Kelly A. Frazer, Anil Sadarangani, Catriona HM Jamieson. The niche specific role of CD44 splice isoform expression in blast crisis leukemia stem cell generation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4798. doi:10.1158/1538-7445.AM2014-4798