Targeted Disruption of the JAK2/STAT3 Pathway in Combination with Systemic Administration of Paclitaxel Inhibits the Priming of Ovarian Cancer Stem Cells Leading to a Reduced Tumor Burden

Abstract

Chemotherapy resistance associated with recurrent disease is the major cause of poor survival of ovarian cancer patients. We have recently demonstrated activation of the JAK2/STAT3 pathway and the enhancement of a cancer stem cell (CSC)-like phenotype in ovarian cancer cells treated in vitro with chemotherapeutic agents. To elucidate further these mechanisms in vivo, we used a two-tiered paclitaxel treatment approach in nude mice inoculated with ovarian cancer cells. In the first approach, we demonstrate that a single intraperitoneal administration of paclitaxel in mice 7 days after subcutaneous transplantation of the HEY ovarian cancer cell line resulted in a significant increase in the expression of CA125, Oct4, and CD117 in mice xenografts compared to control mice xenografts which did not receive paclitaxel. In the second approach, mice were administered once weekly with paclitaxel and/or a daily dose of the JAK2-specific inhibitor, CYT387, over 4 weeks. Mice receiving paclitaxel only demonstrated a significant decrease in tumor volume compared to control mice. At the molecular level, mouse tumors remaining after paclitaxel administration showed a significant increase in the expression of Oct4 and CD117 coinciding with a significant activation of the JAK2/STAT3 pathway compared to control tumors. The addition of CYT387 with paclitaxel resulted in the suppression of JAK2/STAT3 activation and abrogation of Oct4 and CD117 expression in mouse xenografts. This coincided with significantly smaller tumors in mice administered CYT387 in addition to paclitaxel, compared to the control group and the group of mice receiving paclitaxel only. These data suggest that the systemic administration of paclitaxel enhances Oct4- and CD117-associated CSC-like marker expression in surviving cancer cells in vivo, which can be suppressed by the addition of the JAK2-specific inhibitor CYT387, leading to a significantly smaller tumor burden. These novel findings have the potential for the development of CSC-targeted therapy to improve the treatment outcomes of ovarian cancer patients.

Keywords: JAK2/STAT3 pathway; cancer stem cells; chemoresistance; metastasis; ovarian carcinoma; recurrence.

Figure 1

HEY xenograft volume in mice administered with or without paclitaxel, CYT387 or a combination of paclitaxel and CYT387 (pac + CYT387). Average fold change in subcutaneous tumor volume. Tumors were measured at day 0 (prior to treatment) and at the end of the experiment (day 28). Data have been obtained from n = 5 mice in each group. Significant intergroup variations are indicated by *p < 0.05, **p < 0.01.

Figure 2

Immunohistochemistry images of phosphorylated P-JAK2 and T-JAK2 staining in xenografts generated from subcutaneous transplantation of HEY cells into mice administered with or without paclitaxel, CYT387, or pac + CYT387. Tumor sections were stained with antibodies specific for P-JAK2 and T-JAK2 as described in the Section “Materials and Methods.” Magnification 200×, scale bar = 10 μM. Average DAB intensity and proportion of staining of P-JAK2 or T-JAK2 in xenografts was standardized to a negative control. The quantification was derived from the staining of five independent xenografts in each group. Significant intergroup variations are indicted by *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3

Immunohistochemistry images of P-STAT3 and T-STAT3 staining in xenografts generated from subcutaneous transplantation of HEY cells into mice administered with or without paclitaxel, CYT387, or pac + CYT387. Tumor sections were stained with antibodies specific for P-STAT3 and T-STAT3 as described in the Section “Materials and Methods.” Magnification 200×, scale bar = 10 μM. Average DAB intensity and proportion of staining of P-STAT3 or T-STAT3 in xenografts was standardized to a negative control. The quantification was derived from the staining of five independent xenografts in each group. Significant intergroup variations are indicted by **p < 0.01 and ***p < 0.001.

Figure 4

Immunohistochemistry images of Ki67 and CA125 staining in xenografts generated from subcutaneous transplantation of HEY cells into mice administered with or without paclitaxel, CYT387, or pac + CYT387. Tumor sections were stained with antibodies specific for Ki67 and CA125 as described in the Section “Materials and Methods.” Magnification 200×, scale bar = 10 μM. Average DAB intensity and proportion of staining of Ki67 or CA125 in xenografts was standardized to negative control. The experiments were performed using five independent samples in each group. Significant intergroup variations are indicted by *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5

Immunohistochemistry images of CD117 and Oct4 staining in xenografts generated from subcutaneous transplantation of HEY cells into mice administered with or without paclitaxel, CYT387, or pac + CYT387. Tumor sections were stained with antibodies specific for CD117 and Oct4 as described in the Section “Materials and Methods.” Magnification 200×, scale bar = 10 μM. Average DAB intensity and proportion of staining of CD117 or Oct4 in xenografts was standardized to a negative control. The experiments were performed using five independent samples in each group. Significant intergroup variations are indicted by ***p < 0.001.

Figure 6

Immunohistochemistry images of CD34 staining in mice xenografts from subcutaneous transplantation of HEY cells into mice administered with or without paclitaxel, CYT387, or pac + CYT387. Tumor sections were stained with antibodies specific for CD34 as described in the Section “Materials and Methods.” Magnification 200×, scale bar = 10 μM. Average DAB intensity and proportion of staining of CD34 in xenografts was standardized to a negative control. The experiments were performed using five independent samples in each group. Significant intergroup variations are indicted by **p < 0.01 and ***p < 0.001.

Figure 7

The mRNA expression of embryonic stem cell marker Oct4 in xenografts generated from subcutaneous transplantation of HEY cells into mice administered with or without paclitaxel, CYT387, or a combination of paclitaxel and CYT387 (pac + CYT). mRNA from xenografts generated from the control group and treatment groups was extracted, cDNA was prepared, and q-PCR for Oct4 was performed as described in the Section “Material and Methods.” The resultant mRNA levels were normalized to 18S mRNA. The experiments were performed using five independent samples in triplicate. Significant intergroup variations are indicated by **p < 0.01 and***p < 0.001.

Figure 8

The mRNA expression of IL-6, IL-6R, gp130, CXCR4, MMP-2, and MMP-9 in xenografts generated from subcutaneous transplantation of HEY cells into mice administered with or without paclitaxel, CYT387, or pac + CYT. mRNA from xenografts generated from the control group and treatment groups was extracted, cDNA was prepared, and q-PCR for IL-6, IL-6R, gp130, MMP-2, MMP-9, and CXCR4 was performed as described in the Section “Material and Methods.” The resultant mRNA levels were normalized to 18S mRNA. The experiments were performed using four independent samples in triplicate. Significant intergroup variations are indicated by *p < 0.05 and **p < 0.01.