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Mirk kinase inhibition blocks the in vivo growth of pancreatic cancer cells.

Deng X, Friedman E - Genes Cancer (2014)

Bottom Line: EHT5372 treatment in vivo led to an increased fraction of Ki67 positive, cycling cells in Panc1 xenografts whose size was reduced.Mirk inhibition led to a roughly four-fold increase in tumor αSMA-positive fibroblasts and large stromal collagen-rich infiltrates in the pancreas that can restrain tumor growth.The mTOR inhibitor RAD001 alone, or together with EHT5372, reduced pancreatic cancer size 30-fold, while the drug combination reduced the number of microscopic tumor foci 2-fold compared to RAD001 alone.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology Upstate Medical University, Syracuse, N.Y., USA.

ABSTRACT
The Mirk/dyrk1B gene is upregulated and sometimes amplified in pancreatic ductal carcinomas. In poor microenvironmental conditions Mirk mediates cell survival by maintaining cancer cells in a largely quiescent, noncycling state and by decreasing toxic ROS levels through maintaining expression of a series of antioxidant genes. Premature entry into cycle, increased ROS levels, DNA damage, and apoptosis follow Mirk kinase depletion or inhibition. Mirk kinase inhibitor EHT5372 treated Panc1 spheroids lost quiescence markers coincident with an increase in cyclin A showing entry into cycle, and exhibited DNA damage, apoptosis and smaller size. EHT5372 treatment in vivo led to an increased fraction of Ki67 positive, cycling cells in Panc1 xenografts whose size was reduced. Pdx-1-cre LSL/KrasG12D/Ink4a/Arf B6 mice always develop pancreatic cancer, allowing only 30% survival by 8 weeks, while each of the Mirk kinase inhibitor treated mice survived 8 weeks. Mirk inhibition led to a roughly four-fold increase in tumor αSMA-positive fibroblasts and large stromal collagen-rich infiltrates in the pancreas that can restrain tumor growth. The mTOR inhibitor RAD001 alone, or together with EHT5372, reduced pancreatic cancer size 30-fold, while the drug combination reduced the number of microscopic tumor foci 2-fold compared to RAD001 alone.

No MeSH data available.


Related in: MedlinePlus

Mirk kinase inhibitor treatment in vitro or in vivo leads to increased entry into cell cycling, and DNA damage and apoptosisA. Panc1 spheroids were treated 3 or 7 days with the Mirk kinase inhibitor 0.5-10μM EHT5372 or 10μM RAD001 before analysis by western blotting of quiescence proteins p130/Rb2 and p27, cyclin A as a measure of entry into cycle, histone H2AX phosphorylation as a measure of DNA breaks, the apoptotic marker cleaved PARP and cleaved caspase 3, and for blotting controls actin and a cross-reacting band to confirm equal loading. B. 4 week old J:NU athymic mice (Jackson Labs) were injected subcutaneously under the backskin with 1 million viable Panc1 cells, 5 mice per group. After 3 weeks, palpable tumors were detected, and mice were subjected to twice weekly 0.1ml intraperitoneal injection with Mirk/dyrk1B inhibitor EHT5372 to give a final concentration of 2, 5 or 10μM (4mg/kg), or diluent over a two week period. Tumors from each group of mice were fixed, and stained for the proliferation marker Ki67, a DNA polymerase subunit. Sections containing all of the tumors in each group were photographed with the same contrast, and printed. Each print was “gridded”, and all of the cells within 4 alternate grid boxes were counted to eliminate counter bias. 454+/−21 cells were counted per treatment. Comparison of Ki67 percents by unpaired two-tailed t-tests for control vs. 10μM EHT5372 was statistically significant, p=0.0082.
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Figure 3: Mirk kinase inhibitor treatment in vitro or in vivo leads to increased entry into cell cycling, and DNA damage and apoptosisA. Panc1 spheroids were treated 3 or 7 days with the Mirk kinase inhibitor 0.5-10μM EHT5372 or 10μM RAD001 before analysis by western blotting of quiescence proteins p130/Rb2 and p27, cyclin A as a measure of entry into cycle, histone H2AX phosphorylation as a measure of DNA breaks, the apoptotic marker cleaved PARP and cleaved caspase 3, and for blotting controls actin and a cross-reacting band to confirm equal loading. B. 4 week old J:NU athymic mice (Jackson Labs) were injected subcutaneously under the backskin with 1 million viable Panc1 cells, 5 mice per group. After 3 weeks, palpable tumors were detected, and mice were subjected to twice weekly 0.1ml intraperitoneal injection with Mirk/dyrk1B inhibitor EHT5372 to give a final concentration of 2, 5 or 10μM (4mg/kg), or diluent over a two week period. Tumors from each group of mice were fixed, and stained for the proliferation marker Ki67, a DNA polymerase subunit. Sections containing all of the tumors in each group were photographed with the same contrast, and printed. Each print was “gridded”, and all of the cells within 4 alternate grid boxes were counted to eliminate counter bias. 454+/−21 cells were counted per treatment. Comparison of Ki67 percents by unpaired two-tailed t-tests for control vs. 10μM EHT5372 was statistically significant, p=0.0082.

Mentions: Earlier studies showed that spheroids formed from ovarian cancer cells are largely in a quiescent, dormant state, expressing elevated levels of the quiescence markers p130/Rb2 and the CDKI p27 compared to cycling adherent cells, and about 85% of the spheroid cells were in G0/G1 by flow cytometry [28]. Likewise, Panc1 spheroids exhibited the quiescence marker p130/Rb2 and the CDK inhibitor p27, which serves to block cell cycling (Fig.3A). Blocking the Mirk contribution to cell quiescence allows some cancer cells to enter cycle although remaining in poor culture conditions [10], [29]. In prior studies, Mirk kinase inhibition by RO5454948 forced more SW620 colon cancer cells to enter cycle as assayed by an increase in BrdU-incorporating cells [10], and Mirk kinase depletion enabled serum-starved HD6 colon cancer cells to leave quiescence and traverse G1 to the G0/G1 boundary with S phase [13]. Likewise, inhibition of Mirk kinase reduced the quiescence marker p130/Rb2 about 5-fold in Panc1 spheroid cells (Fig.2B, Fig.3A). The DREAM complex component p130/Rb2 sequesters transcription factors necessary for cell cycling [30], [31]. Significantly, EHT5372 at 5 and 10μM strongly reduced p27 levels, while at the same time increased levels of cyclin A, a marker of cells in S phase (Fig.3A), showing movement into cycle. Thus EHT5372 inhibition of Mirk kinase enabled more Panc1 cells grown in three-dimensional culture as spheroids to leave the quiescent state and enter cycle although they remained in serum-limited culture conditions. The loss of quiescence proteins and increase in cyclin A by EHT5372 at 5 and 10μM paralleled an increase in DNA damage as shown by increased levels of γH2AX, and the apoptosis markers cleaved caspase3 and cleaved PARP (Fig.3A), showing that inappropriate entry into cycle correlated with DNA damage, apoptosis and the resulting cell loss in these Panc1 spheroids.


Mirk kinase inhibition blocks the in vivo growth of pancreatic cancer cells.

Deng X, Friedman E - Genes Cancer (2014)

Mirk kinase inhibitor treatment in vitro or in vivo leads to increased entry into cell cycling, and DNA damage and apoptosisA. Panc1 spheroids were treated 3 or 7 days with the Mirk kinase inhibitor 0.5-10μM EHT5372 or 10μM RAD001 before analysis by western blotting of quiescence proteins p130/Rb2 and p27, cyclin A as a measure of entry into cycle, histone H2AX phosphorylation as a measure of DNA breaks, the apoptotic marker cleaved PARP and cleaved caspase 3, and for blotting controls actin and a cross-reacting band to confirm equal loading. B. 4 week old J:NU athymic mice (Jackson Labs) were injected subcutaneously under the backskin with 1 million viable Panc1 cells, 5 mice per group. After 3 weeks, palpable tumors were detected, and mice were subjected to twice weekly 0.1ml intraperitoneal injection with Mirk/dyrk1B inhibitor EHT5372 to give a final concentration of 2, 5 or 10μM (4mg/kg), or diluent over a two week period. Tumors from each group of mice were fixed, and stained for the proliferation marker Ki67, a DNA polymerase subunit. Sections containing all of the tumors in each group were photographed with the same contrast, and printed. Each print was “gridded”, and all of the cells within 4 alternate grid boxes were counted to eliminate counter bias. 454+/−21 cells were counted per treatment. Comparison of Ki67 percents by unpaired two-tailed t-tests for control vs. 10μM EHT5372 was statistically significant, p=0.0082.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4209603&req=5

Figure 3: Mirk kinase inhibitor treatment in vitro or in vivo leads to increased entry into cell cycling, and DNA damage and apoptosisA. Panc1 spheroids were treated 3 or 7 days with the Mirk kinase inhibitor 0.5-10μM EHT5372 or 10μM RAD001 before analysis by western blotting of quiescence proteins p130/Rb2 and p27, cyclin A as a measure of entry into cycle, histone H2AX phosphorylation as a measure of DNA breaks, the apoptotic marker cleaved PARP and cleaved caspase 3, and for blotting controls actin and a cross-reacting band to confirm equal loading. B. 4 week old J:NU athymic mice (Jackson Labs) were injected subcutaneously under the backskin with 1 million viable Panc1 cells, 5 mice per group. After 3 weeks, palpable tumors were detected, and mice were subjected to twice weekly 0.1ml intraperitoneal injection with Mirk/dyrk1B inhibitor EHT5372 to give a final concentration of 2, 5 or 10μM (4mg/kg), or diluent over a two week period. Tumors from each group of mice were fixed, and stained for the proliferation marker Ki67, a DNA polymerase subunit. Sections containing all of the tumors in each group were photographed with the same contrast, and printed. Each print was “gridded”, and all of the cells within 4 alternate grid boxes were counted to eliminate counter bias. 454+/−21 cells were counted per treatment. Comparison of Ki67 percents by unpaired two-tailed t-tests for control vs. 10μM EHT5372 was statistically significant, p=0.0082.
Mentions: Earlier studies showed that spheroids formed from ovarian cancer cells are largely in a quiescent, dormant state, expressing elevated levels of the quiescence markers p130/Rb2 and the CDKI p27 compared to cycling adherent cells, and about 85% of the spheroid cells were in G0/G1 by flow cytometry [28]. Likewise, Panc1 spheroids exhibited the quiescence marker p130/Rb2 and the CDK inhibitor p27, which serves to block cell cycling (Fig.3A). Blocking the Mirk contribution to cell quiescence allows some cancer cells to enter cycle although remaining in poor culture conditions [10], [29]. In prior studies, Mirk kinase inhibition by RO5454948 forced more SW620 colon cancer cells to enter cycle as assayed by an increase in BrdU-incorporating cells [10], and Mirk kinase depletion enabled serum-starved HD6 colon cancer cells to leave quiescence and traverse G1 to the G0/G1 boundary with S phase [13]. Likewise, inhibition of Mirk kinase reduced the quiescence marker p130/Rb2 about 5-fold in Panc1 spheroid cells (Fig.2B, Fig.3A). The DREAM complex component p130/Rb2 sequesters transcription factors necessary for cell cycling [30], [31]. Significantly, EHT5372 at 5 and 10μM strongly reduced p27 levels, while at the same time increased levels of cyclin A, a marker of cells in S phase (Fig.3A), showing movement into cycle. Thus EHT5372 inhibition of Mirk kinase enabled more Panc1 cells grown in three-dimensional culture as spheroids to leave the quiescent state and enter cycle although they remained in serum-limited culture conditions. The loss of quiescence proteins and increase in cyclin A by EHT5372 at 5 and 10μM paralleled an increase in DNA damage as shown by increased levels of γH2AX, and the apoptosis markers cleaved caspase3 and cleaved PARP (Fig.3A), showing that inappropriate entry into cycle correlated with DNA damage, apoptosis and the resulting cell loss in these Panc1 spheroids.

Bottom Line: EHT5372 treatment in vivo led to an increased fraction of Ki67 positive, cycling cells in Panc1 xenografts whose size was reduced.Mirk inhibition led to a roughly four-fold increase in tumor αSMA-positive fibroblasts and large stromal collagen-rich infiltrates in the pancreas that can restrain tumor growth.The mTOR inhibitor RAD001 alone, or together with EHT5372, reduced pancreatic cancer size 30-fold, while the drug combination reduced the number of microscopic tumor foci 2-fold compared to RAD001 alone.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology Upstate Medical University, Syracuse, N.Y., USA.

ABSTRACT
The Mirk/dyrk1B gene is upregulated and sometimes amplified in pancreatic ductal carcinomas. In poor microenvironmental conditions Mirk mediates cell survival by maintaining cancer cells in a largely quiescent, noncycling state and by decreasing toxic ROS levels through maintaining expression of a series of antioxidant genes. Premature entry into cycle, increased ROS levels, DNA damage, and apoptosis follow Mirk kinase depletion or inhibition. Mirk kinase inhibitor EHT5372 treated Panc1 spheroids lost quiescence markers coincident with an increase in cyclin A showing entry into cycle, and exhibited DNA damage, apoptosis and smaller size. EHT5372 treatment in vivo led to an increased fraction of Ki67 positive, cycling cells in Panc1 xenografts whose size was reduced. Pdx-1-cre LSL/KrasG12D/Ink4a/Arf B6 mice always develop pancreatic cancer, allowing only 30% survival by 8 weeks, while each of the Mirk kinase inhibitor treated mice survived 8 weeks. Mirk inhibition led to a roughly four-fold increase in tumor αSMA-positive fibroblasts and large stromal collagen-rich infiltrates in the pancreas that can restrain tumor growth. The mTOR inhibitor RAD001 alone, or together with EHT5372, reduced pancreatic cancer size 30-fold, while the drug combination reduced the number of microscopic tumor foci 2-fold compared to RAD001 alone.

No MeSH data available.


Related in: MedlinePlus