Dissecting the influence of Neolithic demic diffusion on Indian Y-chromosome pool through J2-M172 haplogroup

The global distribution of J2-M172 sub-haplogroups has been associated with Neolithic demic diffusion. Two branches of J2-M172, J2a-M410 and J2b-M102 make a considerable part of Y chromosome gene pool of the Indian subcontinent. We investigated the Neolithic contribution of demic dispersal from West to Indian paternal lineages, which majorly consists of haplogroups of Late Pleistocene ancestry. To accomplish this, we have analysed 3023 Y-chromosomes from different ethnic populations, of which 355 belonged to J2-M172. Comparison of our data with worldwide data, including Y-STRs of 1157 individuals and haplogroup frequencies of 6966 individuals, suggested a complex scenario that cannot be explained by a single wave of agricultural expansion from Near East to South Asia. Contrary to the widely accepted elite dominance model, we found a substantial presence of J2a-M410 and J2b-M102 haplogroups in both caste and tribal populations of India. Unlike demic spread in Eurasia, our results advocate a unique, complex and ancient arrival of J2a-M410 and J2b-M102 haplogroups into Indian subcontinent

The heparan sulfate proteoglycan syndecan-1 regulates colon cancer stem cell function via a focal adhesion kinase—Wnt signaling axis

In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan‐1 (Sdc‐1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc‐1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor‐initiating cell phenotype. Sdc‐1 small‐interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT‐29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex‐determining region Y)‐box 2, KLF2, and TCF4/TCF7L2. Sdc‐1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial‐to‐mesenchymal transition‐related gene expression. Sdc‐1‐depleted HT‐29 xenograft growth was increased compared to controls. Decreased Sdc‐1 expression was associated with an increased activation of β1‐integrins, focal adhesion kinase (FAK), and wingless‐type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc‐1‐depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc‐1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness‐associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence.DatabasesThe GEO accession number of the Affymetrix transcriptomic screening is GSE58751

Proteoglycans and glycosaminoglycans as regulators of cancer stem cell function and therapeutic resistance

In contrast to the bulk of the tumor, a subset of cancer cells called cancer stem cells (or tumor-initiating cells) is characterized by self-renewal, unlimited proliferative potential, expression of multidrug-resistance proteins, active DNA repair capacity, apoptosis resistance, and a considerable developmental plasticity. Due to these properties, cancer stem cells display increased resistance to chemo- and radiotherapy. Recent findings indicate that aberrant functions of proteoglycans and glycosaminoglycans contribute substantially to the cancer stem cell phenotype and therapeutic resistance. In this review, we summarize how the diverse functions of the glycoproteins and carbohydrates facilitate acquisition and maintenance of the cancer stem cell phenotype, and how this knowledge can be exploited to develop novel anti-cancer therapies. For example, the large transmembrane chondroitin sulfate proteoglycan NG2 / CSPG4 marks stem cell populations in brain tumors. Cell surface heparan sulfate proteoglycans of the syndecan and glypican families modulate the stemness-associated Wnt, hedgehog and notch signaling pathways, whereas the interplay of hyaluronan in the stem cell niche with cancer stem cell CD44 determines maintenance of stemness and promotes therapeutic resistance. A better understanding of the molecular mechanisms by which proteoglycans and glycosaminoglycans regulate cancer stem cell function will aid the development of targeted therapeutic approaches which could avoid relapse after an otherwise successful conventional therapy. Chimeric antigen receptor T cells, proteoglycan-primed dendritic cells, proteoglycan-targeted antibody-drug conjugates and inhibitory peptides and glycans have already shown highly promising results in preclinical models.Fil: Vitale, Daiana Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; ArgentinaFil: Kumar Katakam, Sampath. Münster University Hospital; AlemaniaFil: Greve, Burkhard. Münster University Hospital; AlemaniaFil: Jang, Bohee. Ewha Womans University; Corea del SurFil: Oh, Eok Soo. Ewha Womans University; Corea del SurFil: Alaniz, Laura Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; ArgentinaFil: Götte, Martin. Münster University Hospital; Alemani

Syndecan-1 (CD138) Modulates Triple-Negative Breast Cancer Stem Cell Properties via Regulation of LRP-6 and IL-6-Mediated STAT3 Signaling

Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a coreceptor for growth factors and chemokines and is a molecular marker associated with epithelial-mesenchymal transition during development and carcinogenesis. Resistance of Syndecan-1-deficient mice to experimentally-induced tumorigenesis has been linked to altered Wnt-responsive precursor cell pools, suggesting a potential role of Syndecan-1 in breast cancer cell stem function. However, the precise molecular mechanism is still elusive. Here, we decipher the functional impact of Syndecan-1 knockdown using RNA interference on the breast cancer stem cell phenotype of human triple-negative MDA-MB-231 and hormone receptor-positive MCF-7 cells in vitro employing an analytical flow cytometric approach. Successful Syndecan-1 siRNA knockdown was confirmed by flow cytometry. Side population measurement by Hoechst dye exclusion and Aldehyde dehydrogenase-1 activity revealed that Syndecan-1 knockdown in MDA-MB-231 cells significantly reduced putative cancer stem cell pools by 60 % and 27%, respectively, compared to controls. In MCF-7 cells, Syndecan-1 depletion reduced the side population by 40 % and Aldehyde dehydrogenase-1 by 50%, repectively. In MDA-MB-231 cells, the CD44(+)CD24(-/low) phenotype decreased significantly by 6 % upon siRNA-mediated Syndecan-1 depletion. Intriguingly, IL-6, its receptor sIL-6R, and the chemokine CCL20, implicated in regulating stemness-associated pathways, were downregulated by>40 % in Syndecan-1-silenced MDA-MB-23

The Heparan Sulfate Sulfotransferases HS2ST1 and HS3ST2 Are Novel Regulators of Breast Cancer Stem-Cell Properties

Heparan sulfate (HS) is a glycosaminoglycan found mainly in its protein-conjugated form at the cell surface and the extracellular matrix. Its high sulfation degree mediates functional interactions with positively charged amino acids in proteins. 2-O sulfation of iduronic acid and 3-O sulfation of glucosamine in HS are mediated by the sulfotransferases HS2ST and HS3ST, respectively, which are dysregulated in several cancers. Both sulfotransferases regulate breast cancer cell viability and invasion, but their role in cancer stem cells (CSCs) is unknown. Breast CSCs express characteristic markers such as CD44+/CD24−/low, CD133 and ALDH1 and are involved in tumor initiation, formation, and recurrence. We studied the influence of HS2ST1 and HS3ST2 overexpression on the CSC phenotype in breast cancer cell lines representative of the triple-negative (MDA-MB-231) and hormone-receptor positive subtype (MCF-7). The CD44+/CD24−/low phenotype was significantly reduced in MDA-MB-231 cells after overexpression of both enzymes, remaining unaltered in MCF-7 cells. ALDH1 activity was increased after HS2ST1 and HS3ST2 overexpression in MDA-MB-231 cells and reduced after HS2ST1 overexpression in MCF-7 cells. Colony and spheroid formation were increased after HS2ST1 and HS3ST2 overexpression in MCF-7 cells. Moreover, MDA-MB-231 cells overexpressing HS2ST1 formed more colonies and could not generate spheres. The phenotypic changes were associated with complex changes in the expression of the stemness-associated notch and Wnt-signaling pathways constituents, syndecans, heparanase and Sulf1. The results improve our understanding of breast CSC function and mark a subtype-specific impact of HS modifications on the CSC phenotype of triple-negative and hormone receptor positive breast cancer model cell lines

Syndecan-1 (CD138) Modulates Triple-Negative Breast Cancer Stem Cell Properties via Regulation of LRP-6 and IL-6-Mediated STAT3 Signaling

<div><p>Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a coreceptor for growth factors and chemokines and is a molecular marker associated with epithelial-mesenchymal transition during development and carcinogenesis. Resistance of Syndecan-1-deficient mice to experimentally-induced tumorigenesis has been linked to altered Wnt-responsive precursor cell pools, suggesting a potential role of Syndecan-1 in breast cancer cell stem function. However, the precise molecular mechanism is still elusive. Here, we decipher the functional impact of Syndecan-1 knockdown using RNA interference on the breast cancer stem cell phenotype of human triple-negative MDA-MB-231 and hormone receptor-positive MCF-7 cells in vitro employing an analytical flow cytometric approach. Successful Syndecan-1 siRNA knockdown was confirmed by flow cytometry. Side population measurement by Hoechst dye exclusion and Aldehyde dehydrogenase-1 activity revealed that Syndecan-1 knockdown in MDA-MB-231 cells significantly reduced putative cancer stem cell pools by 60% and 27%, respectively, compared to controls. In MCF-7 cells, Syndecan-1 depletion reduced the side population by 40% and Aldehyde dehydrogenase-1 by 50%, repectively. In MDA-MB-231 cells, the CD44(+)CD24(-/low) phenotype decreased significantly by 6% upon siRNA-mediated Syndecan-1 depletion. Intriguingly, IL-6, its receptor sIL-6R, and the chemokine CCL20, implicated in regulating stemness-associated pathways, were downregulated by >40% in Syndecan-1-silenced MDA-MB-231 cells, which showed a dysregulated response to IL-6-induced shifts in E-cadherin and vimentin expression. Furthermore, activation of STAT-3 and NFkB transcription factors and expression of a coreceptor for Wnt signaling, LRP-6, were reduced by >45% in Syndecan-1-depleted cells compared to controls. At the functional level, Syndecan-1 siRNA reduced the formation of spheres and cysts in MCF-7 cells grown in suspension culture. Our study demonstrates the viability of flow cytometric approaches in analyzing cancer stem cell function. As Syndecan-1 modulates the cancer stem cell phenotype via regulation of the Wnt and IL-6/STAT3 signaling pathways, it emerges as a promising novel target for therapeutic approaches.</p> </div

Syndecan−1 modulates activation of the STAT3 and NFκB signaling pathways and expression of LRP-6 in MDA−MB−231 breast cancer cells.

<p>Lysates of control and Syndecan−1 silenced cells were collected and 30-50µg protein/lane was immunoblotted and probed with the indicated antibodies. A) Western blot analysis reveals reduction of the phosphorylated form of STAT3 in Syndecan−1−silenced cells compared to controls. B) Syndecan−1 depletion leads to a significant reduced activation of NFκB. Immunoblot band intensities were normalized for tubulin expression and the data were analyzed using the paired Student's <i>t</i>-test. Data shown are triplicates from a single experiment representative of three independent experiments. **=p≤0.01, n≥3, error bars=SEM. C) Lysates of control and Syndecan−1 silenced cells were collected and 30-50µg protein/lane was immunoblotted and probed with an antibody recognizing LRP-6 (left panel). Immunoblot band intensities were normalized for tubulin expression and the data were analyzed using the paired Student's <i>t</i>-test (right panel). Data shown are triplicates from a single experiment representative of three independent experiments. **=p≤0.01, n≥3, error bars=SEM.</p

Syndecan-1 silencing impairs the formation of spheres and differentiation into cysts in MCF-7 cells.

<p>A) MCF-7 with sphere formation capacity were enriched for and transfected with a control siRNA or Syndecan-1 siRNA, followed by placing the cells in non-adherent culture conditions that promote sphere formation from single cells. After 4 days in suspension without siRNA it’s evident that Syndecan-1 knockdown is affecting proliferation. At this step it is already visible that the spheres are not only smaller but also more irregular compared to controls (upper panel). After 1 week, the spheres formed by Syndecan-1 transfected MCF-7 are less abundant, smaller and there are much more aggregates compared to controls (central panel). After 1 week in suspension culture, MCF-7 cells start to form cysts (bottom panel). The Syndecan-1-transfected cells show a drastically reduced cyst formation capability. The insert shows a Syndecan-1-depleted sphere that is generating a small “sphere/cyst hybrid”. Scale bar = 500µm (upper, central panels); 200µm (lower panel). B) Quantitative analysis of the sphere formation efficiency in control and Syndecan-1 siRNA-treated MCF-7 cells. Sphere number was determined 1 week after plating 3.000 transfected cells. Syndecan-1 siRNA-treatment results in a significant reduction of sphere formation efficiency (P<0.001, n=6).</p

Characterization of SP cells concerning their CD44/CD24 expression.

<p>The combination of SP- and CD44/CD24 measurement revealed a proportion of 99.2% of SP cells with the CD44+/CD24(-/low) phenotype after control siRNA treatment while the Syndecan-1 knockdown approach showed that this proportion decreased only slightly to 98.7% (A). Influence of Syndecan-1 knockdown on CD24 expression. Compared to the control-siRNA treated cells, the Syndecan-1-siRNA transfected cells showed a 20% (±8.0%) increase in CD24 expression (*=P<0.05, n=3) (B).</p

siRNA-mediated knockdown of Syndecan-1 downregulates expression of IL-6R, IL-6 and CCL20 and dysregulates epithelial and mesenchymal marker protein expression in MDA-MB-231 breast cancer cells.

<p>A) left panel: RT-PCR analysis of IL-6R expression in MDA-MB-231 cells subjected to Syndecan-1 siRNA knockdown. Following total RNA isolation, mRNA was reverse transcribed and used as a template for PCR amplification of IL-6R. Right panel: PCR band intensities were normalized for actin expression and the data were analyzed using the paired Student's <i>t</i>-test. B) Left panel: Western blot analysis reveals reduction of IL-6R following Syndecan-1 silencing. Lysates of control and Syndecan-1 silenced cells were collected and 30-50µg protein/lane was immunoblotted and probed with sIL-6R antibody. Right panel: Immunoblot band intensities were normalized for tubulin expression and the data were analyzed using the paired Student's <i>t</i>-test. Data shown are triplicates from a single experiment representative of three independent experiments. C) left panel: RT-PCR analysis of IL-6 expression. Right panel: semiquantitative densitometric analysis (see panel A). D) left panel: RT-PCR analysis of CCL20 expression. Right panel: semiquantitative densitometric analysis (see panel A). *=p<0.05, ***=p<0.001, n≥3, error bars=SEM. E,F) The influence of IL-6 treatment on the expression of the epithelial marker E-cadherin (E) and the mesenchymal marker vimentin (F) was studied by Western blotting. Cells were stimulated by 50ng/ml IL-6 24h after transfection with siRNA for 4h and 19h. In control cells, IL-6 treatment for 19h promoted EMT. Syndecan-1 depletion resulted in significant downregulation of E-cadherin expression. IL-6 treatment of Syndecan-1 depleted cells for 4h resulted in marker expression changes suggestive of enhanced mesenchymal-to-epithelial transition. (E,F) Upper panels = representative Western blots, lower panels = quantitative analysis. n≥3,*=P<0.05. G) Confocal immunofluorescence microscopy of phalloidin-labeled actin filaments reveals increased formation of actin stress fibers, filopodia (*) and lamellopodia (#) in Syndecan-1 siRNA-treated compared to control siRNA treated MCF-7 cells. </p