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Folate receptor-α (FOLR1) expression and function in triple negative tumors.

Necela BM, Crozier JA, Andorfer CA, Lewis-Tuffin L, Kachergus JM, Geiger XJ, Kalari KR, Serie DJ, Sun Z, Moreno-Aspitia A, Aspita AM, O'Shannessy DJ, Maltzman JD, McCullough AE, Pockaj BA, Cunliffe HE, Ballman KV, Thompson EA, Perez EA - PLoS ONE (2015)

Bottom Line: FOLR1 expression varied within breast tumor subtypes; triple negative/basal tumors were significantly associated with increased expression of FOLR1 mRNA, compared to ER+ and HER2+ tumors.FOLR1 expression did not correlate to common clinicopathological parameters such as tumor stage and nodal status.Loss of FOLR1 resulted in growth inhibition, whereas FOLR1 overexpression promoted folate uptake and growth advantage in low folate conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, United Sates of America.

ABSTRACT
Folate receptor alpha (FOLR1) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense overexpression of FOLR1 in breast tumors and poor prognosis, yet there is limited examination of the distribution of FOLR1 across clinically relevant breast cancer subtypes. To explore this further, we used RNA-seq data from multiple patient cohorts to analyze the distribution of FOLR1 mRNA across breast cancer subtypes comprised of estrogen receptor positive (ER+), human epidermal growth factor receptor positive (HER2+), and triple negative (TNBC) tumors. FOLR1 expression varied within breast tumor subtypes; triple negative/basal tumors were significantly associated with increased expression of FOLR1 mRNA, compared to ER+ and HER2+ tumors. However, subsets of high level FOLR1 expressing tumors were observed in all clinical subtypes. These observations were supported by immunohistochemical analysis of tissue microarrays, with the largest number of 3+ positive tumors and highest H-scores of any subtype represented by triple negatives, and lowest by ER+ tumors. FOLR1 expression did not correlate to common clinicopathological parameters such as tumor stage and nodal status. To delineate the importance of FOLR1 overexpression in triple negative cancers, RNA-interference was used to deplete FOLR1 in overexpressing triple negative cell breast lines. Loss of FOLR1 resulted in growth inhibition, whereas FOLR1 overexpression promoted folate uptake and growth advantage in low folate conditions. Taken together, our data suggests patients with triple negative cancers expressing high FOLR1 expression represent an important population of patients that may benefit from targeted anti-FOLR1 therapy. This may prove particularly helpful for a large number of patients who would typically be classified as triple negative and who to this point have been left without any targeted treatment options.

No MeSH data available.


Related in: MedlinePlus

Folate receptor overexpression increases cell growth and folate uptake.(A). Growth of HS578T breast cancer cells engineered to stably overexpress FOLR1 or empty vector (NT). Cells were cultured in low (0–40 nM) and super-physiological (160 nM) concentrations of folic acid and growth determined after 120 hr using the MTT assay. Error bars represent ± SD. **Statistical significance calculated by two sided unpaired t-test, assuming unequal variances. (B). BrdU incorporation in HS578T/NT and HS578T/FOLR1 cells grown in 10 nM folic acid. **Statistical significance calculated by two sided unpaired t-test, assuming unequal variances. (C). Folate uptake in HS578T/NT versus HS578T/FOLR1 cells. Briefly, flow cytometry was used to measure internalized fluorescent folate (fluorescent tagged folate agent, FolateRSense 690) after 1 hr of exposure. Dot plots show forward scatter area (FSC-A) vs. FolateRSense fluorescence (APC). The percent of cells with internalized fluorescent folate and the mean fluorescence per cell type are shown.
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pone.0122209.g007: Folate receptor overexpression increases cell growth and folate uptake.(A). Growth of HS578T breast cancer cells engineered to stably overexpress FOLR1 or empty vector (NT). Cells were cultured in low (0–40 nM) and super-physiological (160 nM) concentrations of folic acid and growth determined after 120 hr using the MTT assay. Error bars represent ± SD. **Statistical significance calculated by two sided unpaired t-test, assuming unequal variances. (B). BrdU incorporation in HS578T/NT and HS578T/FOLR1 cells grown in 10 nM folic acid. **Statistical significance calculated by two sided unpaired t-test, assuming unequal variances. (C). Folate uptake in HS578T/NT versus HS578T/FOLR1 cells. Briefly, flow cytometry was used to measure internalized fluorescent folate (fluorescent tagged folate agent, FolateRSense 690) after 1 hr of exposure. Dot plots show forward scatter area (FSC-A) vs. FolateRSense fluorescence (APC). The percent of cells with internalized fluorescent folate and the mean fluorescence per cell type are shown.

Mentions: To confirm the requirement of FOLR1 for cellular growth by a different means, we overexpressed the folate receptor in HS578T cells, a TNBC cell line with lower levels of FOLR1 (Fig. 6A). FOLR1 overexpression promoted growth in sub- physiological concentrations (10–40 nM) of extracellular folate (Fig. 7A) compared to control. The increased growth rate of FOLR1 overexpressing cells at sub- physiological concentrations of folate was reflected in an increase in DNA synthesis (Fig. 6B). No significant difference in growth was observed among cells grown in supra-physiological concentrations of folate (160 nM). To determine whether the growth advantage induced by FOLR1 overexpression was due to an increase in folate uptake, we measured folate internalization by flow cytometry using a fluorescent tagged folate agent, FolateRSense 690 (Perkin Elmer). After 1 hr of exposure to FolateRSense in folate- free media, cells were washed and acid stripped to remove surface bound folate, and internalized fluorescent folate was measured via flow cytometry. HS578T cells overexpressing FOLR1 displayed significantly greater folate uptake as shown by an increase in the number of cells with internalized FolateRSense (0.9% vs, 95%) and an increase in mean fluorescence (Fig. 7C).


Folate receptor-α (FOLR1) expression and function in triple negative tumors.

Necela BM, Crozier JA, Andorfer CA, Lewis-Tuffin L, Kachergus JM, Geiger XJ, Kalari KR, Serie DJ, Sun Z, Moreno-Aspitia A, Aspita AM, O'Shannessy DJ, Maltzman JD, McCullough AE, Pockaj BA, Cunliffe HE, Ballman KV, Thompson EA, Perez EA - PLoS ONE (2015)

Folate receptor overexpression increases cell growth and folate uptake.(A). Growth of HS578T breast cancer cells engineered to stably overexpress FOLR1 or empty vector (NT). Cells were cultured in low (0–40 nM) and super-physiological (160 nM) concentrations of folic acid and growth determined after 120 hr using the MTT assay. Error bars represent ± SD. **Statistical significance calculated by two sided unpaired t-test, assuming unequal variances. (B). BrdU incorporation in HS578T/NT and HS578T/FOLR1 cells grown in 10 nM folic acid. **Statistical significance calculated by two sided unpaired t-test, assuming unequal variances. (C). Folate uptake in HS578T/NT versus HS578T/FOLR1 cells. Briefly, flow cytometry was used to measure internalized fluorescent folate (fluorescent tagged folate agent, FolateRSense 690) after 1 hr of exposure. Dot plots show forward scatter area (FSC-A) vs. FolateRSense fluorescence (APC). The percent of cells with internalized fluorescent folate and the mean fluorescence per cell type are shown.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4376802&req=5

pone.0122209.g007: Folate receptor overexpression increases cell growth and folate uptake.(A). Growth of HS578T breast cancer cells engineered to stably overexpress FOLR1 or empty vector (NT). Cells were cultured in low (0–40 nM) and super-physiological (160 nM) concentrations of folic acid and growth determined after 120 hr using the MTT assay. Error bars represent ± SD. **Statistical significance calculated by two sided unpaired t-test, assuming unequal variances. (B). BrdU incorporation in HS578T/NT and HS578T/FOLR1 cells grown in 10 nM folic acid. **Statistical significance calculated by two sided unpaired t-test, assuming unequal variances. (C). Folate uptake in HS578T/NT versus HS578T/FOLR1 cells. Briefly, flow cytometry was used to measure internalized fluorescent folate (fluorescent tagged folate agent, FolateRSense 690) after 1 hr of exposure. Dot plots show forward scatter area (FSC-A) vs. FolateRSense fluorescence (APC). The percent of cells with internalized fluorescent folate and the mean fluorescence per cell type are shown.
Mentions: To confirm the requirement of FOLR1 for cellular growth by a different means, we overexpressed the folate receptor in HS578T cells, a TNBC cell line with lower levels of FOLR1 (Fig. 6A). FOLR1 overexpression promoted growth in sub- physiological concentrations (10–40 nM) of extracellular folate (Fig. 7A) compared to control. The increased growth rate of FOLR1 overexpressing cells at sub- physiological concentrations of folate was reflected in an increase in DNA synthesis (Fig. 6B). No significant difference in growth was observed among cells grown in supra-physiological concentrations of folate (160 nM). To determine whether the growth advantage induced by FOLR1 overexpression was due to an increase in folate uptake, we measured folate internalization by flow cytometry using a fluorescent tagged folate agent, FolateRSense 690 (Perkin Elmer). After 1 hr of exposure to FolateRSense in folate- free media, cells were washed and acid stripped to remove surface bound folate, and internalized fluorescent folate was measured via flow cytometry. HS578T cells overexpressing FOLR1 displayed significantly greater folate uptake as shown by an increase in the number of cells with internalized FolateRSense (0.9% vs, 95%) and an increase in mean fluorescence (Fig. 7C).

Bottom Line: FOLR1 expression varied within breast tumor subtypes; triple negative/basal tumors were significantly associated with increased expression of FOLR1 mRNA, compared to ER+ and HER2+ tumors.FOLR1 expression did not correlate to common clinicopathological parameters such as tumor stage and nodal status.Loss of FOLR1 resulted in growth inhibition, whereas FOLR1 overexpression promoted folate uptake and growth advantage in low folate conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, United Sates of America.

ABSTRACT
Folate receptor alpha (FOLR1) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense overexpression of FOLR1 in breast tumors and poor prognosis, yet there is limited examination of the distribution of FOLR1 across clinically relevant breast cancer subtypes. To explore this further, we used RNA-seq data from multiple patient cohorts to analyze the distribution of FOLR1 mRNA across breast cancer subtypes comprised of estrogen receptor positive (ER+), human epidermal growth factor receptor positive (HER2+), and triple negative (TNBC) tumors. FOLR1 expression varied within breast tumor subtypes; triple negative/basal tumors were significantly associated with increased expression of FOLR1 mRNA, compared to ER+ and HER2+ tumors. However, subsets of high level FOLR1 expressing tumors were observed in all clinical subtypes. These observations were supported by immunohistochemical analysis of tissue microarrays, with the largest number of 3+ positive tumors and highest H-scores of any subtype represented by triple negatives, and lowest by ER+ tumors. FOLR1 expression did not correlate to common clinicopathological parameters such as tumor stage and nodal status. To delineate the importance of FOLR1 overexpression in triple negative cancers, RNA-interference was used to deplete FOLR1 in overexpressing triple negative cell breast lines. Loss of FOLR1 resulted in growth inhibition, whereas FOLR1 overexpression promoted folate uptake and growth advantage in low folate conditions. Taken together, our data suggests patients with triple negative cancers expressing high FOLR1 expression represent an important population of patients that may benefit from targeted anti-FOLR1 therapy. This may prove particularly helpful for a large number of patients who would typically be classified as triple negative and who to this point have been left without any targeted treatment options.

No MeSH data available.


Related in: MedlinePlus