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Reversible inhibition of lysine specific demethylase 1 is a novel anti-tumor strategy for poorly differentiated endometrial carcinoma.

Theisen ER, Gajiwala S, Bearss J, Sorna V, Sharma S, Janat-Amsbury M - BMC Cancer (2014)

Bottom Line: These effects were largely dose-dependent.Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen.Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT, USA. margit.janat-amsbury@hsc.utah.edu.

ABSTRACT

Background: Endometrial cancer is the most common gynecologic malignancy. Type II endometrial carcinoma is often poorly differentiated and patients diagnosed with Type II disease (~11%) are disproportionately represented in annual endometrial cancer deaths (48%). Recent genomic studies highlight mutations in chromatin regulators as drivers in Type II endometrial carcinoma tumorigenesis, suggesting the use of epigenetic targeted therapies could provide clinical benefit to these patients. We investigated the anti-tumor efficacy of the LSD1 inhibitor HCI2509 in two poorly differentiated Type II endometrial cancer cell lines AN3CA and KLE.

Methods: The effects of HCI2509 on viability, proliferation, anchorage-independent growth, global histone methylation, LSD1 target gene induction, cell cycle, caspase activation and TUNEL were assayed. KLE cells were used in an orthotopic xenograft model to assess the anti-tumor activity of HCI2509.

Results: Both AN3CA and KLE cells were sensitive to HCI2509 treatment with IC50s near 500 nM for cell viability. Inhibition of LSD1 with HCI2509 caused decreased proliferation and anchorage independent growth in soft agar, elevated global histone methylation, and perturbed the cell cycle in both cell lines. These effects were largely dose-dependent. HCI2509 treatment also caused apoptotic cell death. Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen. Tumor burden was distributed log-normally. Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

Conclusions: Our findings demonstrate the anti-cancer properties of the LSD1 inhibitor HCI2509 on poorly differentiated endometrial carcinoma cell lines, AN3CA and KLE. HCI2509 showed single-agent efficacy in orthotopic xenograft studies. Continued studies are needed to preclinically validate LSD1 inhibition as a therapeutic strategy for endometrial carcinoma.

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HCI2509 impairs cell viability, proliferation and transformation in Type II EC cell lines. (A, B) Dose–response curves showing the effects of 96-hour HCI2509 or medroxyprogesterone 17-acetate (MPA) treatment on cell viability of (A) AN3CA and (B) KLE cells normalized to vehicle controls. EC50s and 95% CI’s were calculated using GraphPad Prism 6.0 and are reported where the R2 > 0.9. Data points are reported as mean and standard deviation (n = 3). (C, D) Proliferation (3 T5) assays showing cell doubling times for (C) AN3CA and (D) KLE cells with vehicle and increasing doses of HCI2509. Data points are reported as mean and standard deviation (n = 3). (E, F) Quantification of colonies formed by (E) AN3CA or (F) KLE cells in soft agar with either vehicle or HCI2509 treatment at varying concentrations. Error bars indicate SD of duplicate assays.
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Fig1: HCI2509 impairs cell viability, proliferation and transformation in Type II EC cell lines. (A, B) Dose–response curves showing the effects of 96-hour HCI2509 or medroxyprogesterone 17-acetate (MPA) treatment on cell viability of (A) AN3CA and (B) KLE cells normalized to vehicle controls. EC50s and 95% CI’s were calculated using GraphPad Prism 6.0 and are reported where the R2 > 0.9. Data points are reported as mean and standard deviation (n = 3). (C, D) Proliferation (3 T5) assays showing cell doubling times for (C) AN3CA and (D) KLE cells with vehicle and increasing doses of HCI2509. Data points are reported as mean and standard deviation (n = 3). (E, F) Quantification of colonies formed by (E) AN3CA or (F) KLE cells in soft agar with either vehicle or HCI2509 treatment at varying concentrations. Error bars indicate SD of duplicate assays.

Mentions: We first validated previous data suggesting that Type II endometrial carcinoma cells were sensitive to LSD1 inhibition with HCI2509 [35]. Both AN3CA and KLE cell lines exhibited a dose-dependent decrease in cell viability after 96 hours of treatment with HCI2509 (Figure 1A, B) with EC50 values determined at 499 nM and 435 nM, respectively (Figure 1A, B). In separate experiments, treatment with medroxyprogesterone 17-acetate (MPA) showed no effect on cell viability, confirming that both cell lines exhibit resistance to hormone treatment (Figure 1A, B). Having determined the EC50 we next tested the effect of HCI2509 on population doubling times using a 3T5 proliferation assay in treatment conditions below and above the EC50 (Figure 1C, D). HCI2509 decreased proliferation rates in a dose dependent manner in both AN3CA and KLE cell lines. Interestingly, even the lowest tested treatment concentration (0.3 X IC50) resulted in cytostasis in KLE cells. At and above the IC50, both cell lines exhibited negative growth, suggesting cell death.Figure 1


Reversible inhibition of lysine specific demethylase 1 is a novel anti-tumor strategy for poorly differentiated endometrial carcinoma.

Theisen ER, Gajiwala S, Bearss J, Sorna V, Sharma S, Janat-Amsbury M - BMC Cancer (2014)

HCI2509 impairs cell viability, proliferation and transformation in Type II EC cell lines. (A, B) Dose–response curves showing the effects of 96-hour HCI2509 or medroxyprogesterone 17-acetate (MPA) treatment on cell viability of (A) AN3CA and (B) KLE cells normalized to vehicle controls. EC50s and 95% CI’s were calculated using GraphPad Prism 6.0 and are reported where the R2 > 0.9. Data points are reported as mean and standard deviation (n = 3). (C, D) Proliferation (3 T5) assays showing cell doubling times for (C) AN3CA and (D) KLE cells with vehicle and increasing doses of HCI2509. Data points are reported as mean and standard deviation (n = 3). (E, F) Quantification of colonies formed by (E) AN3CA or (F) KLE cells in soft agar with either vehicle or HCI2509 treatment at varying concentrations. Error bars indicate SD of duplicate assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197342&req=5

Fig1: HCI2509 impairs cell viability, proliferation and transformation in Type II EC cell lines. (A, B) Dose–response curves showing the effects of 96-hour HCI2509 or medroxyprogesterone 17-acetate (MPA) treatment on cell viability of (A) AN3CA and (B) KLE cells normalized to vehicle controls. EC50s and 95% CI’s were calculated using GraphPad Prism 6.0 and are reported where the R2 > 0.9. Data points are reported as mean and standard deviation (n = 3). (C, D) Proliferation (3 T5) assays showing cell doubling times for (C) AN3CA and (D) KLE cells with vehicle and increasing doses of HCI2509. Data points are reported as mean and standard deviation (n = 3). (E, F) Quantification of colonies formed by (E) AN3CA or (F) KLE cells in soft agar with either vehicle or HCI2509 treatment at varying concentrations. Error bars indicate SD of duplicate assays.
Mentions: We first validated previous data suggesting that Type II endometrial carcinoma cells were sensitive to LSD1 inhibition with HCI2509 [35]. Both AN3CA and KLE cell lines exhibited a dose-dependent decrease in cell viability after 96 hours of treatment with HCI2509 (Figure 1A, B) with EC50 values determined at 499 nM and 435 nM, respectively (Figure 1A, B). In separate experiments, treatment with medroxyprogesterone 17-acetate (MPA) showed no effect on cell viability, confirming that both cell lines exhibit resistance to hormone treatment (Figure 1A, B). Having determined the EC50 we next tested the effect of HCI2509 on population doubling times using a 3T5 proliferation assay in treatment conditions below and above the EC50 (Figure 1C, D). HCI2509 decreased proliferation rates in a dose dependent manner in both AN3CA and KLE cell lines. Interestingly, even the lowest tested treatment concentration (0.3 X IC50) resulted in cytostasis in KLE cells. At and above the IC50, both cell lines exhibited negative growth, suggesting cell death.Figure 1

Bottom Line: These effects were largely dose-dependent.Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen.Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT, USA. margit.janat-amsbury@hsc.utah.edu.

ABSTRACT

Background: Endometrial cancer is the most common gynecologic malignancy. Type II endometrial carcinoma is often poorly differentiated and patients diagnosed with Type II disease (~11%) are disproportionately represented in annual endometrial cancer deaths (48%). Recent genomic studies highlight mutations in chromatin regulators as drivers in Type II endometrial carcinoma tumorigenesis, suggesting the use of epigenetic targeted therapies could provide clinical benefit to these patients. We investigated the anti-tumor efficacy of the LSD1 inhibitor HCI2509 in two poorly differentiated Type II endometrial cancer cell lines AN3CA and KLE.

Methods: The effects of HCI2509 on viability, proliferation, anchorage-independent growth, global histone methylation, LSD1 target gene induction, cell cycle, caspase activation and TUNEL were assayed. KLE cells were used in an orthotopic xenograft model to assess the anti-tumor activity of HCI2509.

Results: Both AN3CA and KLE cells were sensitive to HCI2509 treatment with IC50s near 500 nM for cell viability. Inhibition of LSD1 with HCI2509 caused decreased proliferation and anchorage independent growth in soft agar, elevated global histone methylation, and perturbed the cell cycle in both cell lines. These effects were largely dose-dependent. HCI2509 treatment also caused apoptotic cell death. Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen. Tumor burden was distributed log-normally. Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

Conclusions: Our findings demonstrate the anti-cancer properties of the LSD1 inhibitor HCI2509 on poorly differentiated endometrial carcinoma cell lines, AN3CA and KLE. HCI2509 showed single-agent efficacy in orthotopic xenograft studies. Continued studies are needed to preclinically validate LSD1 inhibition as a therapeutic strategy for endometrial carcinoma.

Show MeSH
Related in: MedlinePlus