title: Silencing of HSulf-2 expression in MCF10DCIS.com cells attenuate ductal carcinoma in situ progression to invasive ductal carcinoma in vivo.
abstract: BACKGROUND
  Ductal carcinoma in situ (DCIS) of the breast is a heterogeneous group of proliferative cellular lesions that have the potential to become invasive. Very little is known about the molecular alterations involved in the progression from DCIS to invasive ductal carcinoma (IDC). Heparan endosulfatase (HSulf-2) edits sulfate moieties on heparan sulfate proteoglycans (HSPGs) and has been implicated in modulating heparin binding growth factor signaling, angiogenesis and tumorigenesis. However, the role of HSulf-2 in breast cancer progression is poorly understood. MCF10DCIS.com cells (referred as MCF10DCIS) express HSulf-2 and form comedo type DCIS and progress to IDC when transplanted in immune-deficient mice and, therefore, is an ideal model to study breast cancer progression. We evaluated the role of HSulf-2 in progression from DCIS to IDC using mouse fat pad mammary xenografts.
  
  
  METHODS
  Non-target control (NTC) and HSulf-2 knockdown in MCF10DCIS breast cancer cells were achieved by NTC shRNA and two different lentiviral shRNA against HSulf-2 respectively. Xenografts were established by injecting NTC and HSulf-2 deficient MCF10DCIS cells in mouse mammary fat pads. Xenografts were subjected to H&E staining for morphological analysis, TUNEL and Propidium iodide staining (to determine the extent of apoptosis), Western blot analysis and zymography.
  
  
  RESULTS
  Using a mouse mammary fat pad derived xenograft model, we observed that compared to control treated xenografts, down-regulation of HSulf-2 was associated with significant delays in growth at Week 7 (P-value < 0.05). Histological examination of the tumors demonstrated substantial differences in comedo necrosis, with marked luminal apoptosis and up-regulation of apoptotic markers Bim, cleaved PARP and cleaved caspase 3 in HSulf-2 depleted xenografts. Furthermore, HSulf-2 depleted xenografts retained the basement membrane integrity with decreased activity and expression of matrix metalloproteinase 9 (MMP-9), an enzyme critical for degradation of extracellular matrix compared to nontargeted control.
  
  
  CONCLUSIONS
  Our data suggest that HSulf-2 expression may be critical for human breast cancer progression. Down-regulation of HSulf-2 leads to retention of comedo type DCIS and delays the progression of DCIS to IDC. Further studies are necessary to determine if therapeutic targeting of HSulf-2 expression might delay the progression of DCIS to IDC.
authors.affiliation: Department of Experimental Pathology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA.
authors.forename: Ashwani
authors.initials: A
authors.lastname: Khurana
authors.forename: Hiedi
authors.initials: H
authors.lastname: McKean
authors.forename: Hyunseok
authors.initials: H
authors.lastname: Kim
authors.forename: Sung-Hoon
authors.initials: SH
authors.lastname: Kim
authors.forename: Jacie
authors.initials: J
authors.lastname: mcguire
authors.forename: Lewis R
authors.initials: LR
authors.lastname: Roberts
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Goetz
authors.forename: Viji
authors.initials: V
authors.lastname: Shridhar
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D044584:Carcinoma, Ductal / Q000235:genetics* / Q000473:pathology; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000235:genetics* / Q000473:pathology*; D045744:Cell Line, Tumor; D018450:Disease Progression; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000235:genetics / Q000473:pathology; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D013429:Sulfatases; D015238:Sulfotransferases / Q000235:genetics* / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D015238:Sulfotransferases; C470792:SULF2 protein, human; D013429:Sulfatases; D020780:Matrix Metalloproteinase 9