Abstract
Background
Fascin, an actin bundling protein, plays a critical role in cell motility due to formation of actin rich protrusions called filopodia, important in cell migration, invasion and metastatic spread. Fascin overexpression has been associated with epithelial to mesenchymal transition and correlates with progression and unfavourable prognosis in breast carcinoma.
Objective
To evaluate fascin expression by immunohistochemistry and correlate the expression pattern with clinicopathological parameters in breast cancer in African-American (AA) women, in whom triple negative breast cancer (TNBC), an aggressive subtype, is more prevalent.
Methods
Tissue microarrays were constructed from formalin-fixed, paraffin-embedded blocks of tumour tissue from primary breast carcinomas in 202 AA women. Immunohistochemical detection of fascin was correlated with four major subtypes of breast carcinoma (luminal A, luminal B, human epidermal growth factor receptor 2 and triple negative (TN)) and other clinicopathological factors, including age, grade, tumour size, stage, regional lymph node status and survival.
Results
We observed a significant association between fascin expression and TN subtype, oestrogen receptor (ER) negativity, progesterone receptor (PR) negativity, Elston–Nottingham (EN) grade 3 and decreased overall survival. There was also a significant association between expression of CK 5/6, a marker of basal-like phenotype, and fascin expression.
Conclusion
These results suggest that fascin is a marker for TN subtype having a basal-like phenotype and decreased overall survival. Fascin may represent a target for therapy in TNBC in AA women.
INTRODUCTION
Although the incidence of breast cancer in African-American (AA) women is lower than that in the Caucasian population, mortality due to breast cancer is higher.1 This may be attributed to a higher incidence of triple-negative breast cancer (TNBC) in AA women compared to Caucasian American (CA) women. In a recent study of 375 761 invasive breast cancers, evaluating incidence rates in a population-based tumour registry, the total cumulative incidence of TNBC in AA was 208/100 000 and in CA women was 113/100 000.2 Recent epidemiological studies show that TNBC was more than twice as likely to occur in AA women at a younger age than in CA women; the proportion with TNBC in AA women was 21–47% and in CA women it was 8–22%.3–8 In previous studies at our institution, triple negative (TN) subtype was the most common subtype in AA women less than 35 years of age.9,10 TNBCs have a more aggressive clinical course with higher recurrence rate and decreased disease-free survival. Irrespective of stage at diagnosis, women with TNBC showed poorer survival than women with other breast cancer subtypes.11 Other factors which could contribute to increased mortality in AA women with breast carcinoma, include younger age, higher grade and later stage at diagnosis.2,5,6
The aggressive nature of the disease may be a consequence of epithelial to mesenchymal transition (EMT), a complex process in which epithelial cells lose their intercellular adhesive properties and acquire spindle cell morphology. This transition leads to a propensity for invasion and metastasis, mediated by enhanced cell motility.12–14 Fascin, a major actin filament-crosslinking protein encoded by the FSCN1 gene (gene ID: 6624), increases cell motility by reorganising the distribution and activity of the actin cytoskeleton. In tumour cells, fascin tightly bundles actin filaments to form finger-like plasma membrane protrusions, invadopodia, associated with invasion.15,16
Fascin is normally expressed during embryonic development; however, in adult life expression is limited predominantly to neuronal and dendritic cells with very weak expression in normal epithelia.17 Fascin expression is upregulated in various epithelial malignancies, including high grade breast carcinoma.17–20 Fascin expression has been correlated with hormone receptor negative and basal-like breast carcinomas, which are associated with increased aggression and poor prognosis.17,19,20 The role of fascin in breast cancer in a large cohort of AA women has not been evaluated in any published study. In the present study, we determined the association between immunohistochemical expression of fascin and the four major subtypes of breast carcinoma (luminal A, luminal B, human epidermal growth factor receptor 2 and TN) and other clinicopathological factors including age, grade, tumour size, stage, regional node status and survival in AA women. We also analysed fascin expression in basal-like phenotype, characterised by expression of CK 5/6.21
MATERIALS AND METHODS
Tissue samples
This study was reviewed and formally exempted by the Howard University Institutional Review Board (IRB-10-MED-24). We analysed invasive ductal carcinomas (IDCs) of breast from 202 AA women diagnosed and treated at the Howard University Hospital between 2000 and 2010, from which adequate tumour tissue could be obtained. Demographic and clinical information was obtained through the Howard University Cancer Center Tumor Registry.
Tissue microarrays
A series of tissue microarrays (TMAs) was constructed (Pantomics, Richmond, California, USA). The TMAs consisted of 10×16 arrays of 1.0-mm tissue cores from well preserved, morphologically representative tumour cells in archived, formalin-fixed, paraffin-embedded (FFPE) surgical blocks from primary IDCs in 202 AA women. A precision tissue arrayer (Beecher Instruments, Silver Spring, Maryland, USA) with two separate core needles for punching the donor and recipient blocks was used. The device also had a micrometer-precise coordinate system for tissue assembly on a multi-tissue block. Two separate tissue cores of IDC represented each surgical case in the TMA. Each tissue core was assigned a unique TMA location number, which was subsequently linked to an Institutional Review Board approved database containing demographic and clinical data. Using a microtome, 5-μm sections were cut from the TMA blocks and mounted onto superfrost plus microscope slides.
Immunohistochemistry
Immunohistochemistry (IHC) was performed on TMA sections of FFPE tumour tissue. Following deparaffinisation and rehydration of the tissue sections, heat-induced epitope retrieval at pH 9.0 was performed. A prediluted anti-fascin mouse monoclonal antibody (clone 55k-2, isotype IgG1; Cell Marque, Rocklin, California, USA) was used at a concentration of 0.01 μg/mL. TMAs were stained with a ready-to-use anti-CK 5/6 mouse monoclonal primary antibody (clone D5/16B4, isotype IgG1; Ventana Medical Systems, OroValley, Arizona, USA). Primary antibody detection was carried out using a polymer-based detection system with staining development achieved by incubation with 3,3′-diaminobenzadine (DAB) and DAB Enhancer (Envision Plus, DAKO, Carpinteria, California, USA). IHC staining was performed at Quest Diagnostics (Chantilly, Virginia, USA).
Immunohistochemically stained sections were scored by two independent observers (TN and AE) blinded to the clinical outcome. Individual tissue cores were scored for intensity of reactivity (0, no staining; 1+, weak cytoplasmic and membrane staining; 2+, moderately intense staining; and 3+, strong staining) and the percentage of reactive cells. The results were entered into a secure research database. An H-score was derived from the results of these measurements.22 An individual case was considered positive for fascin (figure 1) if at least one of the two cores had ≥10% of tumour cells with any intensity of cytoplasmic staining.19
Figure 1.
Fascin expression in breast ductal carcinomas. (A) Triple-negative tumour core showing diffuse and strong cytoplasmic fascin expression. (B) Luminal B tumour core showing moderate and focal cytoplasmic fascin expression. (C) Luminal A tumour core negative for fascin expression. (Left, ×40; right, ×400.)
Breast subtypes were defined using immunohistochemical expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) based on criteria established in the literature. Luminal A was characterised by strong expression of ER and/or PR (H-score ≥200) and HER2 negativity with low proliferation rate (Ki67 expression ≤14%). Luminal B was characterised by expression of ER and/or PR (H-score <200) and HER2 negativity with increased proliferation identified by Ki-67 expression >14% or by triple positive expression of ER, PR and HER2.23,24 The HER2 subtype was hormone receptor negative with only HER2 positivity. The triple-negative subtype lacked expression of ER, PR and HER2.25 Cytoplasmic expression of CK 5/6 in ≥10% tumour cells (figure 2) was used to determine basal-like phenotype.21
Figure 2.
CK 5/6 expression in breast ductal carcinomas. (A) Triple-negative tumour core showing diffuse and strong cytoplasmic CK 5/6 expression (×200). (B) Luminal A tumour core showing no expression for CK 5/6. CK 5/6 expression seen in normal residual breast myoepithelial cells (×200).
Statistical analysis
The χ2 test or t test, as appropriate, was used to examine bivariate association between fascin and CK 5/6 expression with clinicopathological variables. Age was categorised into two groups with a cut-off of 50 years. The logistic regression analysis is used to examine independent association between fascin expression and TNBC in models that adjust for significant clinicopathological variables. Variables that met a <0.1 significance in the bivariate analysis, were included in the logistic regression models to identify independent association of TNBC with fascin expression. Results are reported as ORs along with calculated 95% CIs.
Kaplan–Meier estimates of overall survival and disease-free survival were plotted and the log-rank test was performed to compare estimates among groups. The calculation of time-to-relapse was determined by review of pathological and radiological findings obtained at follow-up. Cox proportional hazard regression analysis was utilised to examine the independent effect of fascin expression on overall survival and disease-free survival in models that adjust for stage, subtype, grade and lymph node status.
The measured utility of fascin expression in predicting TN subtype was examined by calculating sensitivity, specificity, and area under the receiver operating characteristic curve (AUC). Data are reported as mean±SD or n (%). Estimates were considered statistically significant for two-tailed values of p<0.05. All analyses were carried out using the SPSS V.20.
RESULTS
Characteristics of the study population
Clinical and pathological characteristics of the study population are summarised in table 1. Data consisted of 202 AA women diagnosed with IDC with a mean age of 57.48±13.20 years (range 29–96); 71.8% were older than 50 years. The majority of patients (74.8%) had no cancer recurrence. The most prevalent breast cancer subtype was luminal A (53.0%), followed by TN (32.7%). Using CK5/6 expression, 75% of the TNBCs were basal-like. Grade 3 tumours comprised 67.3% of the tumours in the study population.
Table 1.
Clinical and pathological characteristics of study population
| Number | % | |
|---|---|---|
| Age (years) | ||
| <50 | 57 | 28.2 |
| >50 | 145 | 71.8 |
| ER status | ||
| Positive | 116 | 57.4 |
| Negative | 86 | 42.6 |
| PR status | ||
| Positive | 96 | 47.5 |
| Negative | 106 | 52.5 |
| HER2 status | ||
| Positive | 29 | 14.4 |
| Negative | 173 | 85.6 |
| Subtype* | ||
| Luminal A | 89 | 44.1 |
| Luminal B | 28 | 13.9 |
| Her2 | 18 | 8.9 |
| Triple-negative | 67 | 33.1 |
| Stage at diagnosis | ||
| I | 61 | 30.2 |
| II | 82 | 40.6 |
| III | 42 | 20.8 |
| IV | 12 | 5.9 |
| Unknown | 5 | 2.5 |
| Grade | ||
| 1 | 9 | 4.5 |
| 2 | 57 | 28.2 |
| 3 | 136 | 67.3 |
| Recurrence | ||
| None | 151 | 74.8 |
| Loco-regional | 9 | 4.5 |
| Distant | 22 | 10.9 |
| Never disease-free | 10 | 5.0 |
| Never disease-free and distant metastasis | 8 | 4.0 |
| Unknown | 2 | 1.0 |
| Vital status | ||
| Alive | 131 | 64.9 |
| Dead | 71 | 35.1 |
Luminal A: ER+ and/or PR+, HER2− with Ki67 ≤14%; luminal B: ER+ and/or PR+, HER2− with Ki67 >14% and ER+ or PR+, HER2+; triple-negative: ER−, PR−, HER2−; HER2+: ER−, PR−, HER2+.
ER, oestrogen receptor; PR, progesterone receptor.
Results show a significant difference in median survival times for stage (stages I, II, III and IV were, respectively, 119.87 ±6.60, 106.36±7.92, 88.36±9.67 and 26.75±10.58 months, p<0.0001), tumour size (<20 mm, 20–50 mm and >50 mm were, respectively, 112.52±6.16, 96.02±8.31 and 68.57 ±11.45 months, p<0.0001) and lymph node involvement (without involvement and with involvement were, respectively, 113.87±5.87 and 81.23±6.33 months, p=0.001). No statistically significant correlation was seen in median survival times for grade and subtype.
Fascin expression in the IDCs
In the present study 40.6% (82/202) of cases showed expression of fascin. Fascin expression was significantly associated with TN subtype (OR=18.08, 95% CI=8.04 to 40.67, p<0.0001), ER negativity (OR=12.40, 95% CI=6.32 to 24.33, p<0.0001), PR negativity (OR=7.37, 95% CI=3.83 to 14.17, p<0.0001) and EN grade 3 (OR=4.77, 95% CI=2.35 to 9.71, p<0.0001). No association was seen between fascin expression and age, tumour size, stage at presentation and lymph node status. In the logistic regression model with adjustment for grade, distant metastasis and recurrence, TN subtype was independently associated with fascin expression (OR=16.45, 95% CI=6.19 to 43.69, p<0.0001; table 2).
Table 2.
Association of fascin expression with clinicopathological parameters
| Fascin status |
Unadjusted* |
Adjusted† |
||||
|---|---|---|---|---|---|---|
| Negative | Positive | OR (95% CI) | p Value | OR (95% CI) | p Value | |
| Age | ||||||
| <50 years | 35 | 29 | 1.0 | |||
| >50 years | 83 | 53 | 0.77 (0.42 to 1.40) | 0.309 | – | – |
| ER status | ||||||
| Positive | 96 | 20 | 1.0 | |||
| Negative | 24 | 62 | 12.4 (6.3 to 24.32) | <0.0001 | – | – |
| PR status | ||||||
| Positive | 79 | 17 | 1.0 | |||
| Negative | 41 | 65 | 7.36 (3.83 to 14.17) | <0.0001 | – | – |
| HER2 | ||||||
| Negative | 100 | 74 | 1.0 | |||
| Positive | 20 | 8 | 0.54 (0.23 to 1.29) | 0.163 | – | – |
| Subtype | ||||||
| Luminal A | 71 | 18 | 1.0 | 1.0 | ||
| Luminal B | 26 | 2 | 0.30 (0.07 to 1.40) | 0.13 | 0.29 (0.5 to 1.46) | 0.13 |
| Her2+ | 11 | 7 | 2.51 (0.85 to 7.39) | 0.095 | 2.0 (0.55 to 7.21) | 0.28 |
| Triple-negative | 12 | 55 | 18.07 (8.04 to 40.67) | <0.0001 | 16.45 (6.19 to 43.69) | <0.0001 |
| Stage at diagnosis | ||||||
| I | 37 | 24 | 1.0 | |||
| II | 48 | 34 | 1.09 (0.56 to 2.15) | 0.799 | – | – |
| III | 26 | 16 | 0.94 (0.42 to 2.13) | 0.898 | – | – |
| IV | 4 | 8 | 3.08 (0.84 to 11.38) | 0.091 | – | – |
| Size | ||||||
| <20 mm | 49 | 31 | 1.0 | |||
| 20–50 mm | 40 | 31 | 1.22 (0.64 to 2.35) | 0.54 | – | – |
| >50 mm | 15 | 15 | 1.58 (0.68 to to 3.68) | 0.288 | – | – |
| Unknown | 6 | 5 | – | – | – | |
| Grade | ||||||
| 1+2 (ref) | 54 | 12 | 1.0 | |||
| 3 | 66 | 70 | 4.77 (2.35 to 9.71) | <0.0001 | 0.6 (0.22 to 1.62) | 0.31 |
| Lymph nodes | ||||||
| Negative | 56 | 42 | 1.0 | |||
| Positive | 44 | 30 | 0.90 (0.49 to 1.68) | 0.76 | – | – |
| Distant metastases | ||||||
| No | 104 | 65 | 1.0 | |||
| Yes | 14 | 17 | 1.94 (0.90 to 4.21) | 0.092 | 0.65 (0.08 to 5.25) | 0.69 |
| Unknown | 2 | 0 | – | – | – | – |
| Recurrence | ||||||
| No | 92 | 54 | 1.0 | |||
| Yes | 15 | 17 | 1.93 (0.89 to 4.18) | 0.095 | 0.39 (0.71 to 2.2) | 0.29 |
Bivariate analysis based on χ2.1
Adjusted based on logistic regression model adjusted for subtype, grade, distant metastasis, recurrence. ER and PR were not adjusted since they determine the subtype.
ER, oestrogen receptor; PR, progesterone receptor.
Fascin expression was associated with decreased overall survival (p=0.005): mean overall survival was 116.47±7.42 months for fascin-negative tumours and 86.15±6.72 months for fascin-positive cancers. There was a trend noted for reduced disease-free survival (p=0.062; figure 3). In the Cox proportional hazard regression model that adjusts for stage, subtype, grade and lymph node metastasis, results showed a strong independent association of fascin expression with a decrease in overall survival (HR=0.34, 95% CI=0.16 to 0.69, p=0.003), and a marginal association with disease-free survival (HR=0.31, 95% CI=0.09 to 1.06, p=0.064; table 3).
Figure 3.
Correlation of fascin expression with survival. Kaplan–Meier curves shows significant correlation of fascin expression with overall survival (left) and a trend towards disease-free survival (right).
Table 3.
Cox regression model of the effect of fascin express on overall survival and disease-free survival
| Overall survival |
Disease-free survival |
|||
|---|---|---|---|---|
| HR (95% CI) | p Value | HR (95% CI) | p Value | |
| Fascin expression | 0.34 (0.16 to 0.69) | 0.003 | 0.31 (0.09 to 1.06) | 0.064 |
| Subtype | ||||
| Luminal A | 1.0 | 1.0 | ||
| Luminal B | 0.80 (0.31 to 2.05) | 0.65 | 5.74 (1.55 to 21.22) | 0.009 |
| Her2+ | 1.28 (0.49 to 3.37) | 0.61 | 1.44 (0.23 to 8.94) | 0.69 |
| Triple-negative | 0.88 (0.41 to 1.9) | 0.76 | 1.91 (0.47 to 7.7) | 0.35 |
| Stage at diagnosis | ||||
| I | 1.0 | 1.0 | ||
| II | 1.45 (0.63 to 3.34) | 0.377 | 1.29 (0.37 to 4.42) | 0.68 |
| III | 2.41 (0.91 to 6.36) | 0.074 | 2.36 (1.01 to 10.19) | 0.24 |
| IV | 17.57 (4.96 to 62.17) | <0.001 | 12.99 (1.01 to 166.39) | 0.04 |
| High grade | 1.40 (0.69 to 2.86) | 0.34 | 1.06 (0.35 to 3.22) | 0.91 |
| Lymph node involvement | 0.68 (0.33 to 1.43) | 0.31 | 0.63 (0.20 to 1.95) | 0.43 |
Fascin expression intensity and extent (H-score) in the subtypes
Markedly increased intensity and extent of fascin expression (high H-score) were observed in the TN subtype (mean score 159±111.03) when compared with all other subtypes combined (mean score 19.59±58.08) and with each other subtype individually (luminal A mean score 10.79±34.94, luminal B mean score 12.68±50.19 and HER2 mean score 73.89±112.52; figure 4).
Figure 4.
Fascin expression (H-score) correlation with subtypes. Graph shows mean H-scores for each subtype with 95% CIs.
Measured utility of fascin in predicting TNBC
The sensitivity of fascin expression for predicting TNBC is 82.1% (95% CI=70.4 to 90.0) while its specificity is 80% (95% CI=72.1 to 86.2). Thus, fascin expression misses 33% of the truly TNBC patients and incorrectly identifies 10% of the non-TNBC as TNBC. The AUC is 81% (95% CI=74.6 to 86.2). The prevalence of TNBC among AA women is very high, almost 99.99%.
CK 5/6 expression in IDCs
In the present study, 71.6% (48/67) of TNBCs had a basal-like phenotype, determined by CK 5/6 expression. CK 5/6 expression was significantly associated with fascin expression (OR=7.95, 95% CI=3.93 to 16.10, p<0.0001), TN subtype (OR=32.64, 95% CI=13.88 to 76.76, p<0.0001), ER negativity (OR=14.33, 95% CI=6.43 to 31.95, p<0.0001), PR negativity (OR=9.10, 95% CI=4.02 to 20.6, p<0.0001) and HER2 negativity (OR=12.48, 95% CI=1.66 to 94.20, p=0.002) (table 4).
Table 4.
CK 5/6 expression with clinicopathological variables
| CK 5/6 status |
Unadjusted* |
|||
|---|---|---|---|---|
| Negative | Positive | OR (95% CI) | p Value | |
| ER status | ||||
| Positive | 107 | 9 | 1.0 | |
| Negative | 39 | 47 | 14.33 (6.43 to 31.95) | <0.0001 |
| PR status | ||||
| Positive | 88 | 8 | 1.0 | |
| Negative | 58 | 48 | 9.10 (4.02 to 20.6) | <0.0001 |
| HER2 status | ||||
| Positive | 27 | 1 | 1.0 | |
| Negative | 119 | 55 | 12.47 (1.66 to 94.20) | 0.002 |
| Subtype | ||||
| Other subtypes | 125 | 9 | 1.0 | |
| Triple-negative | 19 | 48 | 32.64 (13.88 to 76.76) | <0.0001 |
Bivariate analysis based on χ2.1
ER, PR and HER2 were not adjusted since they determine the subtype.
ER, oestrogen receptor; PR, progesterone receptor.
Co-expression of fascin and CK 5/6
Fascin expression was present in 85% (41/48) of the TNBCs having basal-like phenotype, determined by CK 5/6 expression. Co-expression of fascin and CK 5/6 was significantly associated with TN subtype (OR=400.00, 95% CI=74.53 to 2146.91, p<0.0001), ER negativity (OR=104.71, 95% CI=23.09 to 474.89, p<0.0001), PR negativity (OR=86.82, 95% CI=11.46 to 657.93, p<0.0001) and EN grade 3 (OR=6.66, 95% CI=2.33 to 17.54, p<0.0001). There was no association with stage, lymph node status or tumour size.
DISCUSSION
The significance of fascin expression as a marker of TN subtype in breast cancers from AA women has not been previously evaluated. Fascin was chosen for analysis due to its critical role in cell motility, mediated by formation of cell membrane protrusions, invadopodia, at the advancing edge of cancers.26 The number of studies evaluating the efficacy of fascin as a marker of TNBC is limited. Our study is the first to involve a large cohort of AA women with breast cancer.
The results of our study found a significant association of fascin expression with TN subtype, ER negativity, PR negativity, EN grade 3 and decreased overall survival. Our study also showed that fascin expression was an independent prognostic variable. In our AA population, fascin expression had high specificity and sensitivity in TN subtype. Expression of CK 5/6, a marker of EMT, showed statistical significance with TN subtype (p<0.0001), ER negativity (p<0.0001), PR negativity (p<0.0001) and absence of HER2 amplification (p=0.002). In the present study, 71.6% of TNBCs had a basal-like phenotype, determined by CK 5/6 expression; 85% of the TNBCs having basal-like phenotype, confirmed by CK 5/6 expression, also demonstrated increased fascin expression. Breast cancers co-expressing fascin and CK5/6 were likely to be TN subtype (p<0.0001), show absence of hormone expression (p<0.0001) and HER2 amplification (p<0.0001), and display EN grade 3 differentiation (p<0.0001).
It has been estimated that approximately 70% of TNBCs are basal-like with a stem-like phenotype, a feature linked to EMT and aggressive cancers.12 In vitro studies have also demonstrated fascin’s role in promoting breast cancer progression by activating EMT and augmenting a stem cell population; these studies have also convincingly linked fascin overexpression in invasive breast cancer cell lines with cell migration, proliferation and invasion.27
A few studies have evaluated fascin expression in non-AA women, in all of which the TN subtype was less prevalent and fascin expression was much lower. In two studies evaluating fascin in CA with breast cancer, 15.7% and 20.7%, respectively, showed increased fascin expression.17,19 Two studies conducted in Madrid, Spain revealed fascin expression in 25.4% and 20.7% of cases, with 11.9% and 44.6% having a basal-like phenotype, respectively.12,20 One study in Saudi Arabia showed fascin expression in 31.0%, with 63.6% having a stem cell phenotype.28 Interestingly, a study in China looking at fascin expression in invasive breast cancers revealed weak fascin expression in only 6.7%.29 The present study, comprised exclusively of IDCs in AA women, found a high association of fascin expression with TN subtype (55/67, 82%), of which 85% (41/48) had a basal-like phenotype, characterised by CK 5/6 expression. In addition, TN subtype demonstrated markedly increased intensity and extent of fascin expression (high H-score) when compared to all other subtypes. However, technical variability between different laboratories and differential expression among racial groups could be partially responsible for differing results.
Fascin has been shown to have an integral function in many processes linked to cancer progression. Modification of the extracellular matrix is an important step in EMT, which involves reorganisation of the actin cytoskeleton.30 Fascin mediates this critical action by bundling actin to form protrusive filopodia.31 Upregulation of EMT mesenchymal markers, including vimentin, and reduction in epithelial markers, including E-cadherin membranous expression, occurs in concert with enhanced fascin expression.12,32 Fascin spikes in breast cancer cells have been linked to insulin growth factor-1 receptor (IGF-1R) activation.33 Other studies have shown some evidence that the tumour necrosis factor-α (TNF-α) signalling pathway may lead to fascin overexpression in cancer cells.16 A brisk lymphocytic stromal reaction and increased cytokine production have been found in TNBCs exhibiting a basal-like phenotype. Cytokines IL-6 and oncostatin M have been shown to induce expression of fascin by STAT3 mediated transcriptional activation of the fascin gene.34 IGF-1R and cytokine-mediated induction of fascin as well as TNF-α and human T-cell factor/human lymphoid enhancer factor (TCF/LEF) signalling pathways could be pathogenetically related to increased fascin expression in TNBCs. Additional studies need to be conducted to prove these hypotheses.
Metastasis has also been shown to be related to fascin, which further confirms its importance in breast cancer having an adverse prognosis. Fascin has been found to upregulate several factors essential for metastasis including NF-κB, urokinase-type plasminogen activator (uPA) and matrix metalloproteases (MMP)-2 and MMP-9. These findings give credence to its role in metastatic spread.28
Recent studies have shown that migrastatin and its synthetic analogue, makroketone, powerful inhibitors of tumour metastasis in animal models, act by binding to critical actin binding sites on fascin; this step blocks fascin cross-linkage of actin and invadopodia formation, which are steps instrumental in cell invasion and dissemination. Thus, fascin represents a potential molecular target for therapy in TNBCs, which are difficult to treat due to a lack of targeted agents.35,36
Lack of statistically significant correlation between fascin expression with other unfavourable indicators such as tumour size, lymph node status and disease-free survival may be related to a limited follow-up in some cases. A comparison with CA women in our patient base was not possible due to the rarity of this population at our hospital. However, in our exclusively AA cohort, the TN subtype was significantly associated with basal-like phenotype, with 75% of TNBC demonstrating CK 5/6 expression; 85% of the TNBC with basal-like phenotype showed fascin expression, which was statistically significant.
In summary, we have confirmed that fascin expression is strongly associated with TNBC, having a basal-like phenotype. The TN subtype with basal-like phenotype correlates with progression and decreased survival in AA women. Invasion and metastasis are important factors in poor outcome in breast cancer and are directly linked to EMT, in which fascin plays a key role by remodelling the actin cytoskeleton, leading to enhanced cell motility, essential to cell migration and invasion. Fascin also plays a pivotal role in TGF-β and canonical Wnt signalling pathways, both of which are instrumental in cell proliferation. IGF-1R and cytokines such as IL-6 may also be involved by inducing fascin expression. Fascin is of paramount importance in cancer invasion and dissemination. TNBC, often demonstrating a basal-like phenotype linked to EMT, is more common in AA women and is associated with adverse prognosis. TNBC has limited treatment options. The present study revealed a highly significant correlation between fascin expression and TNBC, having a basal-like phenotype, and thus provides supportive evidence that fascin represents a potentially significant marker for targeted therapy, especially in AA women.
Take-home messages.
-
‣
Fascin plays a critical role in cell motility and is a mediator of epithelial to mesenchymal transition in aggressive carcinomas.
-
‣
Fascin expression is upregulated in triple negative breast cancers (TNBCs) with basal-like phenotype in African-American women.
-
‣
Fascin is potential marker for TNBC, with basal phenotype and a target for therapy.
Acknowledgements
We would like to thank Tejaswita Karve, PhD, of Georgetown University for her assistance in preparation of the manuscript.
Funding This project has been funded in whole or in part with Federal funds from the National Center for Research Resources (NCRR- UL1RR031975), National Institutes of Health, through the Clinical and Translational Science Awards Program (CTSA), a trademark of DHHS, part of the Roadmap Initiative, ‘Re-Engineering the Clinical Research Enterprise’; from the RCMI Program at Howard University, Division of Research Infrastructure, National Center for Research Resources, NIH (NCRR-G12 RR003048) and the Howard University Cancer Center/Johns Hopkins Cancer Center Partnership, National Cancer Institute, (NCI-U54 CA091431).
Footnotes
Competing interests None.
Ethics approval The Office of Regulatory Research Compliance, Howard University, Washington DC, USA.
Provenance and peer review Not commissioned; externally peer reviewed.
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