The Expression of Fascin, an Actin-Bundling Motility Protein, Correlates with Hormone Receptor–Negative Breast Cancer and a More Aggressive Clinical Course

186 Vol. 11, 186–192, January 1, 2005 Clinical Cancer Research

Brian J. Yoder,1 Elisa Tso,2 Marek Skacel,1 site, but rather from local invasion and/or distant metastasis Jim Pettay,1 Shannon Tarr,1 Thomas Budd,2 (2, 3). Metastasis is a complex process involving neovascula- Raymond R. Tubbs,1 Josephine C. Adams,3 and rization, stromal invasion by cancer cells, and infiltration into 1 vascular and lymphatic spaces, survival in the circulation, David G. Hicks extravasation, and growth at a secondary site (2–7). The cellular 1 The Cleveland Clinic Foundation, Department of Anatomic Pathology, mechanisms, which mediate these processes, are poorly 2Taussig Cancer Center, and 3Department of Cell Biology, Lerner Research Institute, Cleveland, Ohio understood, but one requirement is enhancement of cell motility. Augmented movement of cancer cells has been reported to correlate with greater metastatic potential in animal models and a ABSTRACT poor prognosis in human cancer (2). Multiple cellular and extra- The invasion and metastasis of tumor cells is a major cellular factors regulate cell motility, through actions on the cause of mortality in cancer patients. In the current study, we assembly of the actin cytoskeleton. Fascin, is a cytoplasmic investigated the expression of fascin, an actin-bundling protein that functions to bundle cytoplasmic actin filaments, a motility-associated protein, in 210 invasive breast carcino- process which is critical in determining the distribution and ac- mas with corresponding 5-year clinical follow-up. Fascin tivity of the actin cytoskeleton (8). Recently, fascin has emerged expression was compared with hormone receptor (ER/PR) as a key bundling protein in diverse forms of actin-based status, HER2 status, cancer grade, cancer stage, metastasis motility-structures (8). Fascin, is normally expressed in neuronal pattern, disease-free survival, and overall survival. Fascin and mesenchymal cells and is low or absent in epithelia (8, 9). expression was seen in 16% (33/210) of the cases and However, striking up-regulation of fascin has been reported in correlated with ER negativity (22/33, P < 0.001), PR several human epithelial tumors including breast, colon, lung, negativity (21/33, P < 0.001), Bloom-Richardson grade 3 and ovarian carcinomas (10–15). Initial findings implicate fascin (19/29, P < 0.001), and advanced stage (stage 3 or 4, as a potentially significant mediator of tumor cell invasion, and P = 0.04). There was no correlation between fascin ex- an aggressive clinical course. pression and HER2 status or pattern of metastases. Pa- A preliminary study of 58 patients with breast cancer tients whose tumors were positive for fascin showed both a showed that fascin expression correlated significantly with tumor decreased mean disease-free survival (74.44 versus 100.52 grade, DNA ploidy, and correlated inversely with estrogen months, P = 0.002) and mean overall survival (77.58 versus receptor (ER) and progesterone receptor (PR) expression (10). 98.98 months, P = 0.002), independent of tumor stage and These observations suggest that fascin may contribute to the HER2 status, but not independent of ER/PR status or cancer disease progression of some breast cancers. It has been well grade. Given fascin’s role in altering cell motility, over- established in the literature that hormone receptor (ER/PR)– expression may contribute to a more aggressive clinical negative breast cancers have a more aggressive clinical course course in ER/PR-negative breast cancers. If so, then fascin with decreased disease-free and overall survival, compared with may represent a new molecular target for therapeutic hormone receptor–positive tumors (16–18). Furthermore, intervention in patients with ER-negative breast cancer. in vitro experiments have shown that hormone receptor– negative breast cancer cells have increased cell motility and INTRODUCTION increased invasiveness (19, 20). These published observations Breast cancer is one of the leading causes of cancer suggest a possible relationship between hormone receptor neg- mortality for women in the United States today. The American ativity, enhanced cell motility, and fascin expression in invasive Cancer Society estimates that over 200,000 new cases and human breast carcinomas. 40,000 cancer deaths will occur annually (1). In general, most Studies of breast cancer cells in culture have suggested that individuals with cancer die not from the tumor in the primary fascin expression may be regulated, in part, through the overexpression of the membrane receptor tyrosine kinase HER2. MDA-MB435 tumor cells stably transected with HER2 Received 7/8/04; revised 8/11/04; accepted 8/16/04. show a marked increase in mRNA and protein levels of fascin. In Grant support: Internal Research Program Council grant RPC #07384. correlation, cell motility was increased (21). Other studies have The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked shown that the motility responses of fascin-positive breast cancer advertisement in accordance with 18 U.S.C. Section 1734 solely to cells to insulin-like growth factor I depend on fascin protrusions indicate this fact. (22). The current study sought to further investigate the Requests for reprints: D.G. Hicks, Department of Anatomic Pathology, relationship between fascin expression and ER, PR, and HER2 The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: 216-444-5466; Fax: 216-445-6967; E-mail: [email protected]. status as well as explore possible relationships between fascin D2005 American Association for Cancer Research. expression and patient prognosis and patterns of metastasis.

MATERIALS AND METHODS if at least one of the two tissue cores contained at least 5% of Tissue Microarrays. A series of tissue microarrays invasive tumor cells with 2+ or 3+ membranous staining. 2 (TMA) were constructed containing over 210 consecutive pri- Statistics. Bivariate analysis was done via v analysis mary invasive breast carcinomas diagnosed at The Cleveland (fascin positivity versus ER positivity, PR positivity, HER2 Clinic Foundation between 1995 and 1996. The constructed status, tumor stage, and tumor grade) or via one-way ANOVA TMA series consisted of various 8 Â 12 arrays of 1.5 mm tissue with post hoc analysis (percentage of ER-positive or PR-positive cores from archived formalin-fixed, paraffin-embedded surgical cells versus intensity of fascin staining). Survivability data was blocks. Two separate tissue cores of invasive carcinoma, totaling calculated via the generation of Kaplan-Meier curves. Deaths a surface area of 3.5 mm2 , represented each surgical case in the due to causes other than breast cancer were treated as censored TMA series. Each separate tissue core was assigned a unique observations. Subsequent multivariate analysis (death due to TMA location number, which was subsequently linked to an breast cancer versus fascin positivity and either ER, PR, HER2, Institutional Review Board–approved database containing tumor stage, or tumor grade) was done via Cox’s proportional corresponding to a 5-year clinical follow-up. hazards model. All statistics were carried out using Statistical Immunohistochemistry. Fascin, ER, PR, and HER2 Package for the Social Sciences software. Statistical significance expression was assessed via immunohistochemistry. Immuno- was assumed if P < 0.05. Mean follow-up of the study histochemistry was carried out using the fully automated population was 67 months (1-106 months). Ventana Benchmark system. Briefly, a 4-Am-thick unstained section of each TMA were placed onto electrostatically charged RESULTS glass slides and baked to allow for tissue adherence. The glass Characteristics of the Study Population and TMA slides were pretreated with the recommended pretreatment Validation. A summary of the study population is listed in solution provided by Ventana for tissue deparaffinization and Table 1. There were 210 consecutive, newly diagnosed patients antigen retrieval. After primary antibody incubation, antigen with adequate formalin-fixed, paraffin-embedded tissue at our detection was done via peroxidase/3,3V-diaminobenzidine after a institution from 1995 through 1996. The majority of these (168) secondary biotinylated antibody/streptavidin amplification step. For double-labeled sections, alkaline phosphatase red was used for secondary antigen detection. Lastly, a hematoxylin counter- Table 1 Clinical and pathologic characteristics for the study population (210 patients) stain was applied. Immunohistochemistry scoring was done by two independent observers (B.Y., D.H.) completely blinded to Criteria Number % the clinical outcome data. Age (y) The expression of fascin (DAKO, clone 55K-2) was first < 50 66/210 31 tested on positive control tissue (cytoplasmic staining of Reed- > 50 144/210 69 Tumor size Sternberg cells in Hodgkin’s disease) using serial dilutions to T1 128/210 61 determine the optimal antibody concentration (1:50). Each T2 56/210 27 separate tissue core was scored on a 0 to 3+ intensity scale T3 11/210 5 (1+ = weak cytoplasmic and membrane staining, 2+ = T4 11/210 5 Unknown 4/210 2 moderately intense staining, and 3+ = strong staining), and the Estrogen receptor results entered into the research database. An individual case was Negative 51/210 24 considered to be positive if at least one of the two tissue cores Positive 159/210 76 contained at least 10% of invasive tumor cells with 1 to 3+ Progesterone receptor cytoplasmic and membranous staining. Negative 85/210 40 Positive 125/210 60 ER and PR status was re-tested on the TMA series and the HER2 status (immunohistochemistry) results compared with the reported values on the surgical report Negative (0 to 1+) 191/210 91 of each case. Immunohistochemistry of primary antibodies for Overexpressed (2 to 3+) 19/210 9 ER (Ventana, clone 6F11) and PR (Ventana, clone 16) were Bloom-Richardson grade* I 28/168 16 (13) prediluted by the manufacturer for optimal antigen detection. II 87/168 52 (41) The percentage of ER-positive and PR-positive invasive cancer III 53/168 32 (26) cells (nuclear staining) within each separate tissue core was Infiltrating lobular/other 42/210 20 entered into the research database. A case was considered to be Axillary lymph node status ER-positive or PR-positive if the average percentage of positive Nx 19/210 9 invasive cancer cells was > 5%. N0 113/210 54 N1 56/210 27 The HER2 status of each case was assessed via immuno- N2 18/210 8 histochemistry using the TMA series, as testing was not N3 4/210 2 routinely done on breast cancer cases at the original time of Recurrence diagnosis. The immunohistochemistry primary antibody for None 159/210 76 Local recurrence only 0/210 0 HER2 (Ventana, clone CB11) was prediluted by the manufac- Visceral only 13/210 6 turer for optimal antigen detection. HER2 scoring (0 to 3+) was Bone only 7/210 3 based upon previously published scoring schemes (22, 23), and Mixed visceral and bone 31/210 15 the score of each separate tissue core was entered into the *Infiltrating lobular and rare infiltrating ductal variants not assigned research database. A case was considered to be HER2-amplified a Bloom-Richardson grade.

were infiltrating ductal carcinoma. The remaining 42 cases consisted of 32 infiltrating lobular carcinomas, 4 cancers with mixed ductal and lobular features, 3 mucinous carcinomas, and 3 medullary carcinomas. The majority were T1 (128) or T2 (56) in size and ER/PR-positive (76% and 60%, respectively). HER2 amplification was detected via immunohistochemistry in 19 (9%) cases. The traditional clinicopathologic prognostic variables, including tumor stage, grade, ER/PR status, and HER2 status of the study population correlated well with survival, as displayed in Fig. 1. The mean survival for ER-positive patients was 110.42 F 1.56 months versus 76.56 F 4.01 months for ER- negative patients (P = 0.002). PR-positive patients likewise had an increased mean survival of 95.45 F 1.50 months versus 91.13 F 3.36 months for PR-negative patients (P = 0.007, data not shown). HER2 nonamplified patients showed an increased mean survival of 98.28 F 1.65 months versus 79.69 F 7.80 months for HER2-amplified patients (P = 0.023). Finally, the mean survival time inversely correlated with both Bloom-Richardson grade and cancer stage (grade 1 = 94.78 F 3.16 months, grade 2 = 92.70 F 2.24 months, grade 3 = 85.12 F 3.26 months, stage 1 = 102.54 F 1.53 months, stage 2 = 96.30 F 1.21 months, stage 3 = 72.38 F 4.85 months, and stage 4 = 39.63 F 11.27 months). There was an excellent concordance between the reported ER/PR results done on the full histologic sections from 1995 and 1996 and the re-tested ER/PR status done on the TMA series (j = 0.91, P < 0.001). Additional steps to reduce sampling error in the TMA series included the use of larger 1.5 mm cores, as well as utilizing two separate tissue cores per case, each tissue core from a different area of the primary tumor. Using two separate 1.5 mm cores from each case allows for a total surface area of 3.5 mm2, which in our experience, is more than twice the area necessary for adequate tumor sampling (24) and more than 10 times the surface area necessary reported by others (25). Fascin Expression Correlates With Hormone Receptor– Negative Breast Cancers. Normal breast ductal epithelium was negative for fascin, although supporting myoepithelium frequently shows weak to moderate staining (Fig. 2A). Fascin expression was observed in a cytoplasmic and membranous pattern in 33 (16%) of the 210 primary invasive carcinomas. Fig. 2B displays typical fascin-positive cells in one of the tissue cores from the TMA series. Fascin expression correlated with both ER-negative (22/33, P < 0.001) and PR-negative (21/33, P < 0.001) tumors. Fig. 3A graphically shows the disproportionate distribution of fascin-positive tumors between hormone receptor–positive and hormone receptor–negative tumors. There was no correlation between fascin expression and HER2 status (P = 0.189). Fascin-positive tumors tended to be Bloom-Richardson grade 3 (19/29, P < 0.001) and of advanced stage (stage 3 or 4, P = 0.046). The correlative findings with fascin expression are summarized in Table 2. To further characterize the relationship between fascin and the hormone receptor status, the percentage of ER-positive cells per case was plotted against the intensity of fascin expression. In addition, selected slides from the TMA series were concurrently double-labeled for both ER (brown) expression and fascin (red) Fig. 1 Kaplan-Meier curves of the study population showing that the traditional clinicopathologic prognostic variables correlated well with expression. Fig. 3B graphically exhibits the existence of an survival, including ER status (A), HER2 status (B), tumor grade (C), and inverse relationship between the intensity of fascin expression tumor stage (D). All P values were statistically significant (< 0.01), and the percentage of ER-positive cells (P < 0.001, ANOVA). including PR status (data not shown).

and tumor size (T1, T2, T3, or T4), lymph node status (N1, N2, or N3), the occurrence of metastases, the site of metastases (lung, liver, bone, central nervous system, and soft tissue), or the pattern of metastases (visceral dominate, bone dominate, and local recurrence).

DISCUSSION In the current study, fascin expression was seen in 16% of invasive human breast cancers and correlated with hormone receptor–negative cases and a more aggressive clinical course for these patients. Fascin plays an important role in the assembly of cell-motility structures, which have been shown to be critical in cancer invasion and metastasis (2). Therefore, it is possible that fascin expression in cancer cells may lead to a more clinically aggressive course through augmented cell motility and enhanced metastatic potential, a finding supported by in vitro observations (11, 26). Evidence for the Up-regulation of Fascin in Human Carcinomas. Fascin is typically expressed at very low levels in normal epithelia (10–15). Likewise, in the current study, normal breast ductal epithelium was negative for fascin, whereas supporting myoepithelium frequently showed weak to moderate staining. Reports in the literature have documented high levels of

Fig. 2 Normal breast ductal epithelium is negative for fascin, whereas myoepithelial cells show moderate staining (A, Â400). Typical 3+ immunohistochemistry fascin-positive breast cancer case observed in the TMA series with neighboring endothelial cells serving as an internal positive control (B, Â400).

Double labeling for ER and fascin revealed that although 11 cases were dual-positive for both fascin and ER, all of the individual ER-positive cells were fascin-negative, and all of the individual fascin-positive cells were ER-negative (Fig. 4). Tumors which failed to express (< 5%) either fascin or ER were rare (data not shown). Fascin-Positive Breast Cancers Have Worse Prognosis. Five-year clinical follow-up was available for all 210 patients in the study, including time of recurrence, sites of metastases, and cause of death. Fascin-positive tumors show both a decreased disease-free and overall survival compared with fascin-negative tumors (Fig. 5). The mean disease-free survival for fascin- positive tumors was 73.85 months versus 100.42 months for fascin-negative tumors (P < 0.001). The mean overall survival for fascin-positive tumors was 77.98 months versus 98.82 months for fascin-negative tumors (P = 0.002). Subsequent Fig. 3 Fascin expression correlates with hormone receptor negativity. multivariate analysis revealed that fascin expression is an The numbers of fascin-positive cases were disproportionally higher in ER-negative (22) and PR-negative (21) cases compared with ER-positive independent prognostic predictor from PR (P = 0.028), HER2 (11) and PR-positive cases (12), P < 0.001 (A). Likewise, there was an (P = 0.0010), and stage (P = 0.0235), but not independent of ER inverse linear relationship between the percentage of ER-positive cells or grade. There was no correlation between fascin expression per case and the intensity of fascin staining, P < 0.001 (B).

Table 2 Fascin expression correlation with clinical and pathologic parameters ER negativity (22/33 cases, P < 0.001) PR negativity (21/33 cases, P < 0.001) Bloom-Richardson grade 3 (19/29 ductal cancers, P < 0.001) Advanced stage (stage 3 or 4, P = 0.046) Shortened mean overall survival (77.58 months versus 98.98 months, P = 0.002) Shortened mean disease-free survival (74.44 months versus 100.52 months, P < 0.001) No correlation with HER2 status (P = 0.189) No correlation with tumor size (T1, T2, T3, or T4) No correlation with nodal status (N1, N2, or N3) No correlation with metastasis or patterns of metastasis NOTE: Immunohistochemistry revealed 33 out of 210 cases positive for fascin expression.

fascin protein expression in carcinomas of the breast (10), ovary (15), colon (11), skin (12), and lung (13, 14). The expression of fascin in lung cancer correlated with a shorter survival and was an independent prognostic predictor of unfavorable clinical course of the disease (13, 14). Overall, in ovarian tumors, the expression of fascin in cultured tumor cells was significantly associated with the ability of these cells to grow intraperitoneally in a xenograft animal model (15). Similarly, in the current study, we have shown that a subset of invasive breast cancers reveal a marked overexpression of fascin, which was associated with an aggressive clinical course and poor disease-free and overall survival. Fascin Expression and Hormone Receptor Status in Breast Cancer. Hormone receptor–negative breast cancers traditionally have a worse prognosis and fewer available Fig. 5 Kaplan-Meier curves showing decreased overall (A)and disease-free (B) survival of fascin-positive tumors, P < 0.01. Subsequent multivariate analysis revealed that fascin expression is an independent prognostic predictor from PR (P = 0.028), HER2 (P = 0.0010), and stage (P = 0.0235), but is not independent of ER or grade.

treatment options (ineffectiveness of hormonal therapy) compared with hormone receptor–positive tumors (16–18). It is interesting that hormone receptor–negative breast cancers also display increased cell motility in vitro (19, 20, 27). In a study examining the ability of breast cancer cell lines to penetrate into a collagen-fibroblast matrix, cells expressing mRNA for estrogen receptor showed a noninvasive phenotype, whereas cells lacking estrogen receptor mRNA were shown to be highly invasive (20). Similarly, in a modified Boyden chamber assay for invasion, estradiol induced inhibition of breast cancer cell invasion and motility, and a similar inhibitory effect for estradiol was found when the wild-type estrogen receptor-a was stably transfected into the ER-negative MDA-MB231 and 3Y1-Ad12 cells (19). These authors state that the mechanism of the inhibitory effect of estrogen receptor on cell motility is unknown. However, the results presented here suggest that regulatory interactions between the estrogen receptor and fascin gene expression could explain, in part, the alterations of in vitro invasion and cell Fig. 4 Double labeling of fascin in red and ER in brown from three motility. These findings, along with the data presented here, separate TMA tissue cores, fascin-positive/ER-negative (A, Â400), suggest a connection between the expression of fascin and fascin-positive/ER-positive (B, Â400), and fascin-negative/ER-positive the absence of hormone receptors, increased cell motility, and (C, Â400). The dual-positive case (B) has no individual cell that coexpress fascin and ER. The ER-positive case shows no fascin-positive decreased survival in human breast cancers. This notion is tumor cells although it displays fascin-positive internal control supported by fascin expression being a poorer prognostic factor, endothelial cells (C). which was not independent of ER negativity or high tumor grade

in the current study. Furthermore, whereas 11 of the 33 fascin- cell motility, the data presented here suggests that fascin positive tumors were also ER-positive, these tumors tended to expression may contribute to a more aggressive clinical course show a lower level of ER expression and double-labeling studies and thus an enhanced metastatic potential in ER/PR-negative (simultaneous staining for ER and fascin in the same section) breast cancer. If fascin is a downstream mediator contributing to showed that on an individual tumor cell basis, fascin and ER a more aggressive clinical course through enhanced cell motility were never coexpressed in the same tumor cell. Clearly, more and metastatic potential, then this protein may represent a new studies need to be done to further elaborate the regulatory molecular target for therapeutic intervention in this subset of relationship between fascin expression and hormone receptor patients with hormone receptor–negative breast cancer. expression in human breast cancer. If the expression of fascin in human breast cancers proves similar in prospective studies, it may represent a potential therapeutic target for patients with REFERENCES hormone receptor–negative breast cancer. 1. Greenlee RT, Murray T, Bolden S, Wingo PA. Cancer statistics, 2000. Current Molecular Understanding of the Role of Fascin CA Cancer J Clin 2000;50:7–33. in Cell Motility. Fascin is a 55-kDa globular actin-bundling 2. Woodhouse EC, Chuaqui RF, Liotta LA. General mechanisms of protein that is principally expressed in normal mesenchymal, metastasis. Cancer 1997;80:1529–37. endothelial, dendritic, and neuronal cells (8). 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Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. The Expression of Fascin, an Actin-Bundling Motility Protein, Correlates with Hormone Receptor −Negative Breast Cancer and a More Aggressive Clinical Course

Brian J. Yoder, Elisa Tso, Marek Skacel, et al.

Clin Cancer Res 2005;11:186-192.

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