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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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Dose-dependent cell cycle perturbation in Type II EC cell lines with HCI2509 treatment. (A, B) Cell cycle populations of (A) AN3CA and (B) KLE cell lines after exposure to vehicle, 300 nM, 1 μM, and 3 μM HCI2509 for 48 hours. 2 × 104 counts and 1 × 104 counts were used for AN3CA and KLE cells, respectively. Data is representative of four biological replicates. Mean and standard deviation are plotted (* p < 0.05, ** p < 0.01).
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Fig3: Dose-dependent cell cycle perturbation in Type II EC cell lines with HCI2509 treatment. (A, B) Cell cycle populations of (A) AN3CA and (B) KLE cell lines after exposure to vehicle, 300 nM, 1 μM, and 3 μM HCI2509 for 48 hours. 2 × 104 counts and 1 × 104 counts were used for AN3CA and KLE cells, respectively. Data is representative of four biological replicates. Mean and standard deviation are plotted (* p < 0.05, ** p < 0.01).

Mentions: The observation of decreased proliferative rates prompted us to test the effect of HCI2509 treatment on cell cycle progression in both AN3CA and KLE cells. Cell cycle analysis was performed with either vehicle or HCI2509 exposure at 300 nM, 1 μM, or 3 μM for 48 hours. AN3CA cells showed a dose-dependent increase in the percentage of cells in S-phase (Figure 3A). This was accompanied by a decrease in the G0/G1 population. In a time-course experiment 3 μM HCI2509 shows an accumulation of cells in the G0/G1 population from 6–12 hours before developing the increased S-phase fraction at 24 and 48 hours (Additional file 2: Figure S3A). KLE cells show a similar accumulation in early S-phase with increasing concentrations of HCI2509 (Figure 3B). Unlike the AN3CA data, the increase in the S-phase fraction occurs at the expense of the G2/M population of cells. The time-course experiment with KLE cells in 3 μM HCI2509 interestingly never passed through the same distribution as observed for 1 μM HCI2509 at 48 hours, and failed to show any obvious change until 48 hours (Additional file 2: Figure S3B). These data suggest that LSD1 inhibition with HCI2509 perturbs cell cycle progression in both Type II endometrial carcinoma cell lines, most likely through an accumulation in early S-phase.Figure 3


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)

Dose-dependent cell cycle perturbation in Type II EC cell lines with HCI2509 treatment. (A, B) Cell cycle populations of (A) AN3CA and (B) KLE cell lines after exposure to vehicle, 300 nM, 1 μM, and 3 μM HCI2509 for 48 hours. 2 × 104 counts and 1 × 104 counts were used for AN3CA and KLE cells, respectively. Data is representative of four biological replicates. Mean and standard deviation are plotted (* p < 0.05, ** p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Dose-dependent cell cycle perturbation in Type II EC cell lines with HCI2509 treatment. (A, B) Cell cycle populations of (A) AN3CA and (B) KLE cell lines after exposure to vehicle, 300 nM, 1 μM, and 3 μM HCI2509 for 48 hours. 2 × 104 counts and 1 × 104 counts were used for AN3CA and KLE cells, respectively. Data is representative of four biological replicates. Mean and standard deviation are plotted (* p < 0.05, ** p < 0.01).
Mentions: The observation of decreased proliferative rates prompted us to test the effect of HCI2509 treatment on cell cycle progression in both AN3CA and KLE cells. Cell cycle analysis was performed with either vehicle or HCI2509 exposure at 300 nM, 1 μM, or 3 μM for 48 hours. AN3CA cells showed a dose-dependent increase in the percentage of cells in S-phase (Figure 3A). This was accompanied by a decrease in the G0/G1 population. In a time-course experiment 3 μM HCI2509 shows an accumulation of cells in the G0/G1 population from 6–12 hours before developing the increased S-phase fraction at 24 and 48 hours (Additional file 2: Figure S3A). KLE cells show a similar accumulation in early S-phase with increasing concentrations of HCI2509 (Figure 3B). Unlike the AN3CA data, the increase in the S-phase fraction occurs at the expense of the G2/M population of cells. The time-course experiment with KLE cells in 3 μM HCI2509 interestingly never passed through the same distribution as observed for 1 μM HCI2509 at 48 hours, and failed to show any obvious change until 48 hours (Additional file 2: Figure S3B). These data suggest that LSD1 inhibition with HCI2509 perturbs cell cycle progression in both Type II endometrial carcinoma cell lines, most likely through an accumulation in early S-phase.Figure 3

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