CD44 Variant Exon 6 Isoform Expression as a Potential Predictor of Lymph Node Metastasis in Invasive Breast Carcinoma of No Special Type

Background. Invasive breast carcinoma of no special type (IBC-NST) is the most widespread invasive carcinoma subtype causing primarily regional metastases of the lymphatic node (LNM). The capacity of CD44 variant exon 6 (CD44v6) expression as an LNM predictor biomarker in IBC-NST was explored. Methods. We conducted a cross-sectional research with 48 paraffin blocks containing IBC-NST primary tumors that were divided into two groups by LNM. The assessment has been carried out in terms of age, tumor size, tumor grade, lymphovascular invasion (LVI), and CD44v6 expression. The expression of CD44v6 was analyzed on the grounds of immunohistochemical (IHC) staining, while other data were taken from archives. Statistical analysis is carried out by univariate, multivariate, and AUROC. Results. CD44v6 exhibits a dominant expression in IBC-NST tumor cells. Univariate analysis revealed a significant association between CD44v6 and LNM status ( p = 0.001 ). Multiple logistic regression results showed that CD44v6 expression and LVI were significantly associated with LNM with OR 10.7 (95% CI: 2.43 to 47.08) and 6.22 (95% CI: 1.4 to 27.88), respectively. CD44v6 expression was able to discriminate against LNM with AUROC 0.863 ± 0.053 (95% CI: 0.759 to 0.967) at the H-score cut-off 133.889 (75% sensitivity and 83.3% specificity). Conclusion. CD44v6 expression and LVI are potential predictors of LNM in IBC-NST. The H-score cut-off of the CD44v6 expression can also be used as a threshold for classification in further investigation.

Hyaluronic Acid - 2-Deoxy-D-Glucose Conjugate Act as a Promising Targeted Drug Delivery Option for the Treatment of COVID-19

In the present study, we propose Hyaluronic acid - 2-Deoxy-D-Glucose (HA-2DG) conjugate as a novel drug for the treatment of COVID-19. In silico molecular docking studies HA-2DG and 2-Deoxy-D-Glucose (2DG) against four different SARS-CoV-2 viral protein (Mpro, RdRp, PLpro and S protein) revealed that HA-2DG conjugate showed better binding affinity (-6.2, -7.2, -7.0 and 6.4 Kcal/mol) with all four screened SASR-CoV-2 viral targets than the antimetabolite drug 2DG alone (-4.8, -4.9, -4.6 and 4.7 Kcal/mol) respectively. ADMET analysis of 2DG and HA-2DG revealed that HA-2DG possessed reduced toxicity than 2DG alone. The study also revealed that the HA-2DG conjugate has multiple advantages of efficient drug delivery to its CD44 variant isoform receptors of the lower respiratory tract, highest interactive binding affinity with SARS-CoV-2 protein targets. Moreover, the HA-2DG drug conjugate possesses added advantages of good biodegradability, biocompatibility, no toxicity and non-immunogenicity. In conclusion, our study suggested that further in-vitro and in-vivo examination of HA-2DG drug conjugate will be useful to establish a promising early-stage antiviral drug for the novel treatment of COVID-19.

Exosome-delivered CD44v6/C1QBP complex drives pancreatic cancer liver metastasis by promoting fibrotic liver microenvironment

ObjectivePancreatic ductal adenocarcinoma (PDAC) shows a remarkable predilection for liver metastasis. Pro-oncogenic secretome delivery and trafficking via exosomes are crucial for pre-metastatic microenvironment formation and metastasis. This study aimed to explore the underlying mechanisms of how PDAC-derived exosomes (Pex) modulate the liver microenvironment and promote metastasis.DesignC57BL/6 mice were ‘educated’ by tail vein Pex injection. The intrasplenic injection liver metastasis and PDAC orthotopic transplantation models were used to evaluate liver metastasis. Stable cell lines CD44v6 (CD44 variant isoform 6) or C1QBP (complement C1q binding protein) knockdown or overexpression was established using lentivirus transfection or gateway systems. A total of 142 patients with PDAC in Huashan Hospital were retrospectively enrolled. Prognosis and liver metastasis were predicted using Kaplan-Meier survival curves and logistic regression models.ResultsPex tail vein injection induced the deposition of liver fibrotic extracellular matrix, which promoted PDAC liver metastasis. Specifically, the exosomal CD44v6/C1QBP complex was delivered to the plasma membrane of hepatic satellite cells (HSCs), leading to phosphorylation of insulin-like growth factor 1 signalling molecules, which resulted in HSC activation and liver fibrosis. Expression of Pex CD44v6 and C1QBP in PDAC patients with liver metastasis was significantly higher than in PDAC patients without liver metastasis, and simultaneous high expression of exosomal CD44v6 and C1QBP correlated with a worse prognosis and a higher risk of postoperative PDAC liver metastasis.ConclusionThe Pex-derived CD44v6/C1QBP complex is essential for the formation of a fibrotic liver microenvironment and PDAC liver metastasis. Highly expressed exosomal CD44v6 and C1QBP are promising biomarkers for predicting prognosis and liver metastasis in patients with PDAC.

TRAF4/6 Is Needed for CD44 Cleavage and Migration via RAC1 Activation

The hyaluronan receptor CD44 can undergo proteolytic cleavage in two steps, leading to the release of its intracellular domain; this domain is translocated to the nucleus, where it affects the transcription of target genes. We report that CD44 cleavage in A549 lung cancer cells and other cells is promoted by transforming growth factor-beta (TGFβ) in a manner that is dependent on ubiquitin ligase tumor necrosis factor receptor-associated factor 4 or 6 (TRAF4 or TRAF6, respectively). Stem-like A549 cells grown in spheres displayed increased TRAF4-dependent expression of CD44 variant isoforms, CD44 cleavage, and hyaluronan synthesis. Mechanistically, TRAF4 activated the small GTPase RAC1. CD44-dependent migration of A549 cells was inhibited by siRNA-mediated knockdown of TRAF4, which was rescued by the transfection of a constitutively active RAC1 mutant. Our findings support the notion that TRAF4/6 mediates pro-tumorigenic effects of CD44, and suggests that inhibitors of CD44 signaling via TRAF4/6 and RAC1 may be beneficial in the treatment of tumor patients.

The Harmonious Interplay of Amino Acid and Monocarboxylate Transporters Induces the Robustness of Cancer Cells

There is a growing body of evidence that metabolic reprogramming contributes to the acquisition and maintenance of robustness associated with malignancy. The fine regulation of expression levels of amino acid and monocarboxylate transporters enables cancer cells to exhibit the metabolic reprogramming that is responsible for therapeutic resistance. Amino acid transporters characterized by xCT (SLC7A11), ASCT2 (SLC1A5), and LAT1 (SLC7A5) function in the uptake and export of amino acids such as cystine and glutamine, thereby regulating glutathione synthesis, autophagy, and glutaminolysis. CD44 variant, a cancer stem-like cell marker, stabilizes the xCT antiporter at the cellular membrane, and tumor cells positive for xCT and/or ASCT2 are susceptible to sulfasalazine, a system Xc(-) inhibitor. Inhibiting the interaction between LAT1 and CD98 heavy chain prevents activation of the mammalian target of rapamycin (mTOR) complex 1 by glutamine and leucine. mTOR signaling regulated by LAT1 is a sensor of dynamic alterations in the nutrient tumor microenvironment. LAT1 is overexpressed in various malignancies and positively correlated with poor clinical outcome. Metabolic reprogramming of glutamine occurs often in cancer cells and manifests as ASCT2-mediated glutamine addiction. Monocarboxylate transporters (MCTs) mediate metabolic symbiosis, by which lactate in cancer cells under hypoxia is exported through MCT4 and imported by MCT1 in less hypoxic regions, where it is used as an oxidative metabolite. Differential expression patterns of transporters cause functional intratumoral heterogeneity leading to the therapeutic resistance. Therefore, metabolic reprogramming based on these transporters may be a promising therapeutic target. This review highlights the pathological function and therapeutic targets of transporters including xCT, ASCT2, LAT1, and MCT.

Role of metaplasia during gastric regeneration

Spasmolytic polypeptide/trefoil factor 2 (TFF2)-expressing metaplasia (SPEM) is a mucous-secreting reparative lineage that emerges at the ulcer margin in response to gastric injury. Under conditions of chronic inflammation with parietal cell loss, SPEM has been found to emerge and evolve into neoplasia. Cluster-of-differentiation gene 44 (CD44) is known to coordinate normal and metaplastic epithelial cell proliferation. In particular, CD44 variant isoform 9 (CD44v9) associates with the cystine-glutamate transporter xCT, stabilizes the protein, and provides defense against reactive oxygen species (ROS). xCT stabilization by CD44v9 leads to defense against ROS by cystine uptake, glutathione (GSH) synthesis, and maintenance of the redox balance within the intracellular environment. Furthermore, p38 signaling is a known downstream ROS target, leading to diminished cell proliferation and migration, two vital processes of gastric epithelial repair. CD44v9 emerges during repair of the gastric epithelium after injury, where it is coexpressed with other markers of SPEM. The regulatory mechanisms for the emergence of CD44v9 and the role of CD44v9 during the process of gastric epithelial regeneration are largely unknown. Inflammation and M2 macrophage infiltration have recently been demonstrated to play key roles in the induction of SPEM after injury. The following review proposes new insights into the functional role of metaplasia in the process of gastric regeneration in response to ulceration. Our insights are extrapolated from documented studies reporting oxyntic atrophy and SPEM development and our current unpublished findings using the acetic acid-induced gastric injury model.

Interplay between Podoplanin, CD44s and CD44v in Squamous Carcinoma Cells

Podoplanin and CD44 are transmembrane glycoproteins involved in inflammation and cancer. In this paper, we report that podoplanin is coordinately expressed with the CD44 standard (CD44s) and variant (CD44v) isoforms in vivo—in hyperplastic skin after a pro-inflammatory stimulus with 12-O-tetradecanoylphorbol-13-acetate (TPA)—and in vitro—in cell lines representative of different stages of mouse-skin chemical carcinogenesis, as well as in human squamous carcinoma cell (SCC) lines. Moreover, we identify CD44v10 in the mouse-skin carcinogenesis model as the only CD44 variant isoform expressed in highly aggressive spindle carcinoma cell lines together with CD44s and podoplanin. We also characterized CD44v3-10, CD44v6-10 and CD44v8-10 as the major variant isoforms co-expressed with CD44s and podoplanin in human SCC cell lines. Immunofluorescence confocal microscopy experiments show that these CD44v isoforms colocalize with podoplanin at plasma membrane protrusions and cell–cell contacts of SCC cells, as previously reported for CD44s. Furthermore, CD44v isoforms colocalize with podoplanin in chemically induced mouse-skin SCCs in vivo. Co-immunoprecipitation experiments indicate that podoplanin physically binds to CD44v3-10, CD44v6-10 and CD44v8-10 isoforms, as well as to CD44s. Podoplanin–CD44 interaction is mediated by the transmembrane and cytosolic regions and is negatively modulated by glycosylation of the extracellular domain. These results point to a functional interplay of podoplanin with both CD44v and CD44s isoforms in SCCs and give insight into the regulation of the podoplanin–CD44 association.