HSulf-1 modulates FGF2- and hypoxia-mediated migration and invasion of breast cancer cells

Abstract

HSulf-1 modulates the sulfation states of heparan sulfate proteoglycans critical for heparin binding growth factor signaling. In the present study, we show that HSulf-1 is transcriptionally deregulated under hypoxia in breast cancer cell lines. Knockdown of HIF-1α rescued HSulf-1 downregulation imposed by hypoxia, both at the RNA and protein levels. Chromatin immunoprecipitation with HIF-1α and HIF-2α antibodies confirmed recruitment of HIF-α proteins to the two functional hypoxia-responsive elements on the native HSulf-1 promoter. HSulf-1 depletion in breast cancer cells resulted in an increased and sustained bFGF2 (basic fibroblast growth factor) signaling and promoted cell migration and invasion under hypoxic conditions. In addition, FGFR2 (fibroblast growth factor receptor 2) depletion in HSulf-1-silenced breast cancer cells attenuated hypoxia-mediated cell invasion. Immunohistochemical analysis of 53 invasive ductal carcinomas and their autologous metastatic lesions revealed an inverse correlation for the expression of HSulf-1 to CAIX in both the primary tumors (P ≥ 0.0198) and metastatic lesions (P ≥ 0.0067), respectively, by χ(2) test. Finally, HSulf-1 expression levels in breast tumors by RNA in situ hybridization showed that high HSulf-1 expression is associated with increased disease-free and overall survival (P ≥ 0.03 and P ≥ 0.0001, respectively). Collectively, these results reveal an important link between loss of HSulf-1 under hypoxic microenvironment and increased growth factor signaling, cell migration, and invasion.

Figure 1. Hypoxic conditions, HIF-1α stabilizing agents diminish HSulf-1 levels in breast cancer cells

(A) Top Panel, Immunoblot analysis of HSulf-1 expression in breast cancer cell lines with β-actin as a loading control (bottom Panel). (B) Immunoblot analysis with indicated antibodies and (C) Quantitative Real-time PCR expression of HSulf-1 in MCF10DCIS cells unexposed or exposed to hypoxia (3% oxygen) for 16 hours. *p value =<0.03. Immunoblot analysis of MCF10DCIS cells treated with DFO with indicated antibodies (D) and (E) of MCF10DCIS cells exposed to hypoxia (3%) following transient transfection of HIF-1α siRNA and control scrambled siRNA oligos with indicated antibodies. HSulf-1 levels as determined by densitometry are indicated below. (F) Immunoblot of MCF10DCIS cells following transient transfection of full length HIF-1α with indicated antibodies.

Figure 2. Regulation of HSulf-1 by HIF-2α in 786-O cells

(A) Immunoblot analysis of MCF10DCIS cells transfected with increasing amounts of full length myc-tag HIF-2α plasmid with indicated antibodies. (B) Real time PCR analysis of 786-O RCC cells and 786-O cells stably expressing VHL were either left untreated or exposed to hypoxia for 16 hours or treated with DFO (100μM) for 16 hours. (C) Immunoblot analysis of Non-targeted control shRNA (NTC) or HIF-2α shRNA targeted stable clones HB17 and HB19 in 786-O cells with indicated antibodies and (D) Real time PCR analysis using HSulf-1 and 18S specific primers. *P value <0.05 **p value <0.04.

Figure 3. Recruitment of HIF-1α to functional hypoxia responsive elements (HRE) in HSulf-1 promoter

Immunoblot analysis following transient transfection of control and/ dominant negative HIF-1α. in MCF10DCIS cells (A) and/or (B), cells were exposed to hypoxia for 16 hours and were subjected to QRT-PCR analysis using HSulf-1 and 18S primers. * p value =<0.05. (C) Schematic representation of potential HIF-1α binding sites in HSulf-1 promoter (A, inset). Firefly/luciferase activity of 293T cells either left untreated or treated with DFO (100μM) for 16 hours using dual luciferase reporter assay following co-transfection with either HSulf-1 wild type luciferase reporter constructs 1 and 2 respectively with Renilla luciferase vector and (D) with plasmids containing mutations in the putative HIF-1α binding sites. Results are shown as mean ± SEM of triplicate samples. *p values= <0.002. (E) HIF-1α was subjected to chromatin immunoprecipitation (ChIP) analysis using Anti- HIF-1α antibody or rabbit IgG in MCF10DCIS cells untreated or treated with 100μM DFO for 16 hours. ChIP assays using P1, P2 and P3, P4 primers, encompassing the HSulf-1 HRE 1 and HRE 2 regions respectively was performed to demonstrate in vivo recruitment of HIF-1α to HSulf-1 promoter. (F) 786-O and 786-O VHL cells were harvested and HIF-2α was subjected to ChIP analysis using Anti- HIF-2α antibody or rabbit IgG. Input control DNA was diluted 5 fold prior to PCR amplification using P1 and P2 primers, encompassing the HSulf-1 HRE 1 region was performed to demonstrate in vivo recruitment of HIF-2α to HSulf-1 promoter.

Figure 4. Downregulation of HSulf-1 leads to increased cell migration and invasion

(A) Immunoblot analysis of Non-targeted control shRNA (NTC) or HSulf-1 shRNA targeted stable batch clones HL-55 and HL-58 were exposed to normoxia and/or hypoxia treatment for 16 hrs with indicated antibodies. Values below top panel indicate densitometry analysis of the blot. Middle panel shows stabilization of HIF-1α under hypoxia. (B) NTC or HSulf-1 shRNA targeted stable batch clones, HL-55 and HL-58 were subjected to transwell cell migration assay (B) or transwell invasion assay (C) under both normoxic and hypoxic conditions for 24 hours. *p value =<0.03 (compare NTC to HL-55 and HL58, normoxia) **p value =<0.02 (compare NTC to HL-55 and HL58, hypoxia). (D) HIF-1α expression was downregulated in MCF10DCIS cells with shRNA (HL718) or NTC shRNA (batch clones), followed by hypoxia treatment. Immunoblot analysis with anti-HSulf-1 antibodies shows downregulation of HIF-1α rescues HSulf-1 expression under hypoxia (Compare lanes 2 and 4, Top Panel). (E and F) Transwell migration assay of HL-718 and NTC MCF10DCIS cells against HIF-1α. * p value =<0.04 (D) or transwell invasion assay, * p value =<0.06 (E) under both normoxic and hypoxic conditions. The cells in eight different fields were counted.

Figure 5. Increased bFGF2 signaling following HSulf-1 depletion

(A) NTC or HSulf-1 shRNA targeted stable batch clones HL-55 and HL-58 were exposed to normoxia and/or hypoxia treatment for 16 hrs and were immediately exposed to bFGF2 (10ng/ml) for indicated intervals of time. Immunoblot analysis with indicated antibodies shows sustained activation of p-ERK in HSulf-1 depleted clones exposed to hypoxia (compare lanes 3 and 4 to 7 and 8). (B) MCF10DCIS cells were transfected with control vector or pcDNA3.1 HSulf-1-myc/his plasmids. After 36 hours, cells were exposed to hypoxia. Cell surface proteins on MCF10DCIS cells were labeled with membrane impermeable sulfo-NHS-LC-biotin at 4°C after exposure to hypoxia (16h, 3% oxygen) or left untreated and/or, (C) were serum starved for 16 hours and then subjected to bFGF2 (10ng/ml) for 30 minutes. Biotinylated proteins were immunoprecipitated with streptavidin-beads and subjected to immunoblot analysis with anti-phospho FGFR2, FGFR2 and anti-strepavidin-HRP antibodies. Whole cell lysates were probed with anti-myc (9E10) antibody to detect transfected HSulf-1 and anti-α-tubulin antibody as a loading control.

Figure 6

(A) NTC or FGFR2 shRNA targeted batch clones, shRNA1 and shRNA2 were harvested and subjected to immunoblot analysis with indicated antibodies (A, inset) and to cell invasion assay (A) in the presence of bFGF2 (10ng/ml) [* p value =<0.005 (compare NTC to sh1 and sh2, control), ** p value =<0.05(compare NTC to sh1 and sh2, bFGF2)] or under hypoxic conditions for 24 hrs (B), [* p value =<0.05 (compare NTC to sh1 and sh2, normoxia), ** p value =<0.05(compare NTC to sh1 and sh2, hypoxia)]. NTC or HSulf-1 shRNA targeted stable batch clones, HL-55 were transduced with FGFR2 shRNA1 and subjected to (C) immunoblot analysis with anti- FGFR2 and anti-α-tubulin antibodies, and (D) to cell invasion assay. * p value =<0.02 (compare NTC to NTC-FGFR2 shRNA1, normoxia), ** p value =<0.05(compare NTC to NTC-FGFR2 shRNA1, hypoxia), # p value =<0.01 (compare HL55 to HL-55-FGFR2 shRNA1, normoxia), ## p value =<0.02 (compare HL55 to HL-55-FGFR2 shRNA1, hypoxia).

Figure 7

(A), Immunohistochemical analysis showing different levels of HSulf-1 in normal breast and primary tumors (0=absent, 1=low, 2=moderate and 3= high). (B and C) Representative photomicrographs (40×) of HSulf-1 and CAIX immunostaining in autologous primary tumors and metastatic lesions. A lower level of HSulf-1 expression was associated with higher levels of CAIX staining and vice versa observed in matched primary and metastatic breast tumors.