Role of human HGFIN/nmb in breast cancer

Abstract

Introduction: HGFIN, previously identified as nmb, and its homolog osteoactivin are single transmembrane proteins that are expressed in differentiated immune cells. These proteins exhibit properties that could potentiate tumorigenesis or decrease invasiveness. These seemingly opposing roles of HGFIN suggest that this protein might be central to malignancies and might also behave as a tumor suppressor. Consistent with the reported roles for HGFIN is the fact that this gene is regulated by p53 through multiple binding sites in the 5' flanking region, and is expressed in osteoblasts.

Methods: This study used siRNA to knock-out HGFIN in non-tumorigenic breast cells and ectopically expressed HGFIN in breast cancer cells. In addition, in situ hybridization studies analyzed primary breast tissues from archived breast surgeries. Reporter gene assays studied the untranslated exon 1 of HGFIN.

Results: HGFIN expression led to reduced cell growth of breast cancer cells and reduced migration. At the molecular level, reporter gene analyses determined the untranslated exon 1 to be a negative regulator of the upstream enhancing effect. Ectopic expression of wild-type p53 in breast cancer cells that expressed endogenous mutant p53 resulted in increased HGFIN reporter gene activities.

Conclusion: As the majority of cancer cells have mutations in p53, further studies on the relationship between p53 and HGFIN expression, and its role in tumor genesis and bone invasion, might uncover novel therapy targets for breast and other cancers. The results show a central role for p53 in HGFIN expression, which appears to determine the behavior of the cancer cells.

Figure 1

HGFIN expression in breast cells and tissues. (a) RT-PCR for HGFIN and GAPDH in non-tumorigenic (NT) (MCF10 and MCF12A) and tumorigenic (T) (DU4475) breast cells. P1, P10 and P38 are the non-identifier codes for patients. (b) Total RNA was analyzed by northern analyses for HGFIN from MCF12A non-tumorigenic cells and tumorigenic cell lines (HCC70 and T47D). Membranes were stripped and re-probed for 18S rRNA. (c) and (d) Representative in situ hybridizations for HGFIN from benign breast tissue, n = 25 (c) and malignant cells, n = 50 (d). Arrow shows dense staining for alkaline phosphatase (blue) in the benign section. The images were acquired with 100×/0.3 NA objectives.

Figure 2

Transformation of HGFIN knock-out MCF12A. (a) Representative colonies in 5-day clonogenic assays in methylcellulose with MCF-12A as untransfected, stably transfected with pPMSKH1, or pPMSKH1-HGFIN siRNA. (b) The total number of colonies in methylcellulose cultures, plated with 100 cells/dish are presented as mean ± SD, n = 5. (c) Growth curves were established with MCF-12A as untransfected or stably transfected pPMSKH1 or pPMSKH1-HGFIN siRNA. The total numbers of viable cells were counted at weekly intervals and the results presented as mean ± SD, n = 5. *p < 0.05 vs culture with pPMSKH1-HGFIN siRNA.

Figure 3

HGFIN imparts contact-dependent growth by T47D. (a) Representative (n = 5) colonies from clonogenic assays with T47D, as untransfected T47D (top panel); ectopically expressed for HGFIN (middle panel) or transfected with vector alone (lower panel). (b) T47D, untransfected or stably transfected with pFLAG-HGFIN were studied in clonogenic assays with 100 cells/35 mm² dishes. At day 5, the total number of colonies were counted and are presented as mean ± SD, n = 5. *p < 0.05 vs untransfected and vector transfectants.

Figure 4

Role of HGFIN in cell migration. MCF12A and MCF10 were knock-out for HGFIN, and T47D were ectopically expressed for HGFIN. All cell types were studied in cell migration assays. Controls were performed with MCF12A and MCF10 as untransfected, vector transfectants or transfectants with HGFIN mutant siRNA. Controls for T47D used untransfected cells or vector transfectants. All values for the control groups were pooled and presented together in single bars. The data are presented as mean ± SD, n = 5.

Figure 5

Role of exon 1 in the activity of the 5' flanking region of HGFIN. (a) Cartoon of the upstream region of HGFIN. TS, transcription start site. (b) Non-tumorigenic (MCF12A) and tumorigenic (T47D, HCC70, MDA-MB-231) breast cells were co-transfected with pGL3-HGFIN-RM/2.0 or -RM/2.0E and pGal. Luciferase activities were normalized with β-galactosidase activities and the data are presented as the mean ± SD, n = 6. *p < 0.05 vs HGFIN-RM/2.0; **p > 0.05 vs HGFIN-RM/2.0E, MDA-MB-231. (c) T47D were co-transfected with HGFIN-RM/2.0 or HGFIN-RM/2.0E, and wild-type or mutant p53 expression vectors. Controls were transfected with vector alone. Luciferase activities were determined 16 h after transfection. The data are presented as mean ± SD, n = 6.

Figure 6

HGFIN reporter gene activity in MDA-MB-231, ectopically expressed for p53. (a) MDA-MB-231 was co-transfected with pLuc and/or pME18S-SCX3 expression p53, mutant or vector alone. K562 transfected with pLuc served as control. The results are presented as mean ± SD, n = 5. (b) MDA-MB-231 was co-expressed with HGFIN-RM/2.0 or HGFIN-RM/2.0E and/or p53 expression vector, pME18S-SCX3. Controls were co-transfected with p53 mutants or vector alone. The results were normalized with β-galactosidase and are presented as mean ± SD, n = 6. *p < 0.05 vs vector/untransfected and mutant p53.