CD44ICD promotes breast cancer stemness via PFKFB4-mediated glucose metabolism.

CD44 is a single-pass cell surface glycoprotein that is distinguished as the first molecule used to identify cancer stem cells in solid tumors based on its expression. In this regard, the CD44high cell population demonstrates not only the ability to regenerate a heterogeneous tumor, but also the ability to self-regenerate when transplanted into immune-deficient mice. However, the exact role of CD44 in cancer stem cells remains unclear in part because CD44 exists in various isoforms due to alternative splicing. Methods: Gain- and loss-of-function methods in different models were used to investigate the effects of CD44 on breast cancer stemness. Cancer stemness was analyzed by detecting SOX2, OCT4 and NANOG expression, ALDH activity, side population (SP) and sphere formation. Glucose consumption, lactate secretion and reactive oxygen species (ROS) levels were detected to assess glycolysis. Western blot, immunohistochemical staining, ELISA and TCGA dataset analysis were performed to determine the association of CD44ICD and PFKFB4 with clinical cases. A PFKFB4 inhibitor, 5MPN, was used in a xenograft model to inhibit breast cancer development. Results: In this report, we found that the shortest CD44 isoform (CD44s) inhibits breast cancer stemness, whereas the cleaved product of CD44 (CD44ICD) promotes breast cancer stemness. Furthermore, CD44ICD interacts with CREB and binds to the promoter region of PFKFB4, thereby regulating PFKFB4 transcription and expression. The resultant PFKFB4 expression facilitates the glycolysis pathway (vis-à-vis oxidative phosphorylation) and promotes stemness of breast cancer. In addition, we found that CD44ICD and PFKFB4 expressions are generally up-regulated in the tumor portion of breast cancer patient samples. Most importantly, we found that 5MPN (a selective inhibitor of PFKFB4) suppresses CD44ICD-induced tumor development. Conclusion: CD44ICD promotes breast cancer stemness via PFKFB4-mediated glycolysis, and therapies that target PFKFB4 (e.g., 5MPN therapy) may lead to improved outcomes for cancer patients

CD44v/CD44s expression patterns are associated with the survival of pancreatic carcinoma patients

BACKGROUND AND PURPOSE: CD44 variants have been associated with tumor invasion and metastasis, but CD44 expression patterns have not been systematically investigated in pancreatic carcinoma. This study systematically investigated whether CD44 expression patterns are involved in pancreatic carcinoma metastasis and prognosis. METHODS: We applied primers specific for all CD44 variants and CD44s to analyze the expression patterns of CD44 (CD44v2-CD44v10 and CD44s) using quantitative real-time PCR (qRT-PCR). We then further evaluated their roles in pancreatic carcinoma metastasis and prognosis using clinical survival analysis. RESULTS: Increased CD44v expression and decreased CD44s expression were found in metastatic pancreatic carcinoma in three different cell lines and in human tumor tissue. Clinical analysis showed that CD44v6(+) and CD44v9(+) were correlated with lymph node metastasis, liver metastasis and TNM stage. However, CD44s(−) was associated with liver metastasis, tumor differentiation and TNM stage. Survival analysis showed that patients with CD44v6(+)/CD44s(−) or CD44v6(+)/CD44s(−) had lower overall survival (OS) rates, although the individual expression of CD44v6, CD44v9 and CD44s was also related to decreased OS rates. Univariate analysis showed that lymph node metastasis; vessel invasion; hepatic metastases; TNM stage; and individual or co-expression of CD44v6, CD44v9 and CD44s were risk factors affecting survival. Multivariate analysis showed that CD44v6(+)/CD44s(−) was an independent predictor of survival. CONCLUSIONS: We found that CD44v6(+), CD44v9(+) and CD44s(−) were associated with pancreatic carcinoma metastasis and progression and that CD44v6(+)/CD44s(−) was an independent risk factor affecting survival in pancreatic carcinoma. Therefore, the different expression patterns of CD44v/CD44s may determine pancreatic carcinoma prognosis. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1579257224116287

Stem-like and highly invasive prostate cancer cells expressing CD44v8-10 marker originate from CD44-negative cells

In human prostate cancer (PCa), the neuroendocrine cells, expressing the prostate cancer stem cell (CSC) marker CD44, may be resistant to androgen ablation and promote tumor recurrence. During the study of heterogeneity of the highly aggressive neuroendocrine PCa cell lines PC3 and DU-145, we isolated and expanded in vitro a minor subpopulation of very small cells lacking CD44 (CD44neg). Unexpectedly, these sorted CD44neg cells rapidly and spontaneously converted to a stable CD44high phenotype specifically expressing the CD44v8-10 isoform which the sorted CD44high subpopulation failed to express. Surprisingly and potentially interesting, in these cells expression of CD44v8-10 was found to be induced in stem cell medium. CD44 variant isoforms are known to be more expressed in CSC and metastatic cells than CD44 standard isoform. In agreement, functional analysis of the two sorted and cultured subpopulations has shown that the CD44v8-10pos PC3 cells, resulting from the conversion of the CD44neg subpopulation, were more invasive in vitro and had a higher clonogenic potential than the sorted CD44high cells, in that they produced mainly holoclones, known to be enriched in stem-like cells. Of interest, the CD44v8-10 is more expressed in human PCa biopsies than in normal gland. The discovery of CD44v8-10pos cells with stem-like and invasive features, derived from a minoritarian CD44neg cell population in PCa, alerts on the high plasticity of stem-like markers and urges for prudency on the approaches to targeting the putative CSC

Nuclear hyaluronidase 2 drives alternative splicing of CD44 pre-mRNA to determine profibrotic or antifibrotic cell phenotype

The cell surface protein CD44 is involved in diverse physiological processes, and its aberrant function is linked to various pathologies such as cancer, immune dysregulation, and fibrosis. The diversity of CD44 biological activity is partly conferred by the generation of distinct CD44 isoforms through alternative splicing. We identified an unexpected function for the ubiquitous hyaluronan-degrading enzyme, hyaluronidase 2 (HYAL2), as a regulator of CD44 splicing. Standard CD44 is associated with fibrotic disease, and its production is promoted through serine-arginine–rich (SR) protein–mediated exon exclusion. HYAL2 nuclear translocation was stimulated by bone morphogenetic protein 7, which inhibits the myofibroblast phenotype. Nuclear HYAL2 displaced SR proteins from the spliceosome, thus enabling HYAL2, spliceosome components (U1 and U2 small nuclear ribonucleoproteins), and CD44 pre-mRNA to form a complex. This prevented double-exon splicing and facilitated the inclusion of CD44 exons 11 and 12, which promoted the accumulation of the antifibrotic CD44 isoform CD44v7/8 at the cell surface. These data demonstrate previously undescribed mechanisms regulating CD44 alternative splicing events that are relevant to the regulation of cellular phenotypes in progressive fibrosis

Distinguishing mechanisms underlying EMT tristability

Abstract Background The Epithelial-Mesenchymal Transition (EMT) endows epithelial-looking cells with enhanced migratory ability during embryonic development and tissue repair. EMT can also be co-opted by cancer cells to acquire metastatic potential and drug-resistance. Recent research has argued that epithelial (E) cells can undergo either a partial EMT to attain a hybrid epithelial/mesenchymal (E/M) phenotype that typically displays collective migration, or a complete EMT to adopt a mesenchymal (M) phenotype that shows individual migration. The core EMT regulatory network - miR-34/SNAIL/miR-200/ZEB1 - has been identified by various studies, but how this network regulates the transitions among the E, E/M, and M phenotypes remains controversial. Two major mathematical models – ternary chimera switch (TCS) and cascading bistable switches (CBS) - that both focus on the miR-34/SNAIL/miR-200/ZEB1 network, have been proposed to elucidate the EMT dynamics, but a detailed analysis of how well either or both of these two models can capture recent experimental observations about EMT dynamics remains to be done. Results Here, via an integrated experimental and theoretical approach, we first show that both these two models can be used to understand the two-step transition of EMT - E→E/M→M, the different responses of SNAIL and ZEB1 to exogenous TGF-β and the irreversibility of complete EMT. Next, we present new experimental results that tend to discriminate between these two models. We show that ZEB1 is present at intermediate levels in the hybrid E/M H1975 cells, and that in HMLE cells, overexpression of SNAIL is not sufficient to initiate EMT in the absence of ZEB1 and FOXC2. Conclusions These experimental results argue in favor of the TCS model proposing that miR-200/ZEB1 behaves as a three-way decision-making switch enabling transitions among the E, hybrid E/M and M phenotypes

Value of tissue markers p27kip1, MIB-1, and CD44s for the pre-operative prediction of tumour features in screen-detected prostate cancer

The pre-operative prediction of prognostic tumour features in the radical prostatectomy specimen using routine clinicopathological variables remains limited. The present study evaluated the predictive value of the cell-cycle protein p27kip1, the proliferation marker MIB-1, and the cell-adhesion protein CD44s, determined on the diagnostic needle biopsy of asymptomatic men screened for prostate cancer. Of 81 screen-detected prostate cancers, representative biopsy cores and matched radical prostatectomy specimens were immunohistochemically stained for these tissue markers. Conventional pre-operative and post-operative clinicopathological variables were assessed and cancers were divided according to a validated tumour classification model (potentially harmless, clinically significant). Low (<50%) p27kip1 expression, high (≥10%) MIB-1 expression, and low (<25%) CD44s expression were considered adverse prognostic signs. Binary logistic regression analysis was performed to assess the most valuable predictors of clinically significant disease. An adverse prognostic immunostaining assessment on the biopsy was found in 10 (12.3%), 17 (21.0%), and 25 (30.9%) cases for p27kip1, MIB-1, and CD44s, respectively. The concordance in tissue marker assessment between the biopsy specimen and matched radical prostatectomy specimens was low for all three. The positive predictive value (PPV) of p27kip1 was 90.0%, remarkably higher than that of MIB-1 and CD44s (41.2% and 52.0%, respectively), indicating that a low radical prostatectomy p27kip1 score is expected if the biopsy p27kip1 score is low. Logistic regression analysis revealed that biopsy Gleason score (p<0.01) and p27kip1 assessment (p<0.01) remained the only significant predictors of clinically significant disease. All cases with low p27kip1 expression were found to have clinically significant disease after radical prostatectomy. The assessment of p27kip1 in the biopsy specimen might thus assist in distinguishing between potentially aggressive and potentially non-aggressive disease in prostate cancer screening. Copyrigh

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression

Splicing factor ESRP1 controls ER-positive breast cancer by altering metabolic pathways

The epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) control the epithelial-to-mesenchymal transition (EMT) splicing program in cancer. However, their role in breast cancer recurrence is unclear. In this study, we report that high levels of ESRP1, but not ESRP2, are associated with poor prognosis in estrogen receptor positive (ER+) breast tumors. Knockdown of ESRP1 in endocrine-resistant breast cancer models decreases growth significantly and alters the EMT splicing signature, which we confirm using TCGA SpliceSeq data of ER+ BRCA tumors. However, these changes are not accompanied by the development of a mesenchymal phenotype or a change in key EMT-transcription factors. In tamoxifen-resistant cells, knockdown of ESRP1 affects lipid metabolism and oxidoreductase processes, resulting in the decreased expression of fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), and phosphoglycerate dehydrogenase (PHGDH) at both the mRNA and protein levels. Furthermore, ESRP1 knockdown increases the basal respiration and spare respiration capacity. This study reports a novel role for ESRP1 that could form the basis for the prevention of tamoxifen resistance in ER+ breast cancer