Abstract
The heparan sulfate proteoglycan Syndecan-1 binds cytokines, morphogens and extracellular matrix components, regulating cancer stem cell properties and invasiveness. Syndecan-1 is modulated by the heparan sulfate-degrading enzyme heparanase, but the underlying regulatory mechanisms are only poorly understood. In colon cancer pathogenesis, complex changes occur in the expression pattern of Syndecan-1 and heparanase during progression from well-differentiated to undifferentiated tumors. Loss of Syndecan-1 and increased expression of heparanase are associated with a change in phenotypic plasticity and an increase in invasiveness, metastasis and dedifferentiation. Here we investigated the regulatory and functional interplay of Syndecan-1 and heparanase employing siRNA-mediated silencing and plasmid-based overexpression approaches in the human colon cancer cell line Caco2. Heparanase expression and activity were upregulated in Syndecan-1 depleted cells. This increase was linked to an upregulation of the transcription factor Egr1, which regulates heparanase at the promoter level. Inhibitor experiments demonstrated an impact of focal adhesion kinase, Wnt and ROCK-dependent signaling on this process. siRNA-depletion of Syndecan-1, and upregulation of heparanase increased the colon cancer stem cell phenotype based on sphere formation assays and phenotypic marker analysis (Side-population, NANOG, KLF4, NOTCH, Wnt, and TCF4 expression). Syndecan-1 depletion increased invasiveness of Caco2 cells in vitro in a heparanase-dependent manner. Finally, upregulated expression of heparanase resulted in increased resistance to radiotherapy, whereas high expression of enzymatically inactive heparanase promoted chemoresistance to paclitaxel and cisplatin. Our findings provide a new avenue to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence.
Highlights
In the tumor microenvironment (TME), repopulation of cells after radiotherapy and chemotherapy represents a mechanism of resistance and tumor recurrence [1]
To analyze the interdependence of Sdc-1 and HPSE expression, we tested the impact of Sdc-1 depletion on expression of the transcription factor Egr1, a known regulator of HPSE expression [21, 41]. qPCR and Western blot analysis revealed an upregulation of Egr1 in Sdc-1-depleted cells (Figures 2A,B)
The increased activity of focal adhesion kinase (FAK) in Sdc-1-depleted cells was abolished by early growth response 1 (EGR1) siRNA knockdown (Figure 2E), whereas application of a FAK inhibitor resulted in an inhibition of Sdc-1-dependent EGR1 and HPSE expression (Figures 2F,G), indicating a mechanistic involvement of this pathway
Summary
In the tumor microenvironment (TME), repopulation of cells after radiotherapy and chemotherapy represents a mechanism of resistance and tumor recurrence [1]. Heparanase (HPSE), matrix metalloproteinases, and sulfatases, are highly expressed in many cancers, whereas some heparan sulfate sulfotransferases are silenced [5,6,7] Proteoglycandegrading enzymes such as HPSE, the only mammalian endoglycosidase capable of cleaving heparan sulfate, regulate ECM dynamics that are under the tight homeostatic control of several signaling pathways [7, 8]. Invading colon carcinoma cells showed decreased expression of Sdc-1 [13] and increased expression of HPSE [14, 15]. Transcriptional studies show that loss of Sdc-1 [13, 16] and enhanced expression of HPSE [17,18,19] correlate with tumor growth, invasion, metastatic potential, and reduced postoperative survival of cancer patients [20]. In multiple myeloma, high HPSE expression is linked to poor prognosis, and contributes to disease pathogenesis by inducing
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