Inhibition of ROCK1 kinase modulates both tumor cells and stromal fibroblasts in pancreatic cancer

<div><p>ROCK, or Rho-associated coiled coil-containing protein kinase, is a member of the AGC kinase family and has been shown to play a role in cell migration, ECM synthesis, stress-fiber assembly, and cell contraction. Increased ROCK expression has been reported in multiple pathological conditions, including cancer. Here, we report increased expression of ROCK 1 in pancreatic tumor epithelial cells as well as in cancer associated fibroblasts (CAF). In our analysis, 62% of tumor samples exhibited ≥2+ in staining intensity by IHC analysis, versus 40% of adjacent normal tissue samples (P<0.0001). Thus, we hypothesized that ROCKs may play a significant role in pancreatic cancer progression, and may serve as a suitable target for treatment. We report a low frequency (4/34) amplification of the ROCK1 gene locus at chromosome 18q11.1 in pancreatic ductal adenocarcinoma (PDAC) patient tissue samples by aCGH analysis. Inhibition of ROCK kinase activity by a small molecule inhibitor (fasudil) resulted in moderate (IC₅₀s of 6–71 μM) inhibition of PDAC cell proliferation, migration, and activation of co-cultured stellate cells. In the KPC mouse model for pancreatic cancer, fasudil decreased tumor collagen deposition. This translated to an enhanced overall survival of the mice and an increase in gemcitabine uptake. Though fasudil may target both the tumor epithelial cells and the CAFs, our findings are consistent with the hypothesis that inhibition of tumor stroma enhances drug penetration and efficacy in PDAC. Overall, our data suggests that ROCK1 may serve as a potential therapeutic target to enhance current treatment regimens for pancreatic cancer.</p></div

Knockdown of ROCK1 by siRNA in pancreatic cancer cell inhibits cell proliferation.

<p>A) ROCK1 was detected in pancreatic cancer cell lines (PANC-1, Mia PaCa-2, SU.86.86, BxPC3, AsPC-1, and HS766T), the immortalized normal pancreatic ductal epithelial cell line (HPDE6), and the cancer associated fibroblasts (CW-1) by Western blotting. B) Western blotting analysis of ROCK1 knockdown by siRNA over the course of 72 hours. (C) Western blotting analysis of ROCK1 knockdown by siRNA (72 hour treatment) in two cell lines, SU.86.86 and PANC-1. (D) Growth curves of pancreatic cancer cells (PANC-1 and SU.86.86) treated with siRNA to ROCK1. * P < 0.001 (compared to the untreated control). UT, untreated; tR, transfection reagent only; NT, non-targeting; ASD, cell death (positive) control; R1, ROCK1 siRNA1; R2, ROCK1 siRNA 2.</p

Effects of fasudil treatment on tumor stroma in KPC mice.

<p>A) Pancreatic tumor tissues from vehicle, gemcitabine, or combination of gemcitabine plus fasudil treated KPC mice were harvested and stained for various stromal markers. Representative images are shown of H&E staining, α-SMA, Desmin, CD31, Collagen I, and Movat's pentachrome staining.</p

Summary of immunohistochemical staining of ROCK1 in PDAC tissue microarrays

<p>Summary of immunohistochemical staining of ROCK1 in PDAC tissue microarrays</p

Effects of the ROCK1 inhibition on pancreatic cancer cells and cancer-associated fibroblasts.

<p>A) Fasudil dose response curves in pancreatic cancer cells treated for 72 hours. B) Tumor cell migration in ROCK1 siRNA treated cells. C) Fluorescence microscopic analysis of fasudil treated, co-cultured pancreatic cancer cells and cancer-associated fibroblasts. Cells were treated with fasudil for 48 hours and then were stained for α-SMA (red), Collagen I (green), and DNA (blue). D) Fluorescence microscopic analysis of fasudil treated CAFs. E) Fasudil treated, mono- and co-cultured pancreatic cancer cells and cancer-associated fibroblasts were harvested and analyzed by immunoblotting (dot blot) for Collagen I expression under non-denaturing conditions.</p

Effects of gemcitabine and fasudil treatment in the KPC model for PDAC.

<p>A) Pancreatic tissues harvested and stained for CD31 were analyzed for positive staining per 20x field of view. B) Mouse liver sections were cut at multiple depths to assess the presence or absence of metastatic lesions in vehicle, gemcitabine, and gemcitabine plus fasudil treated tissues. Fasudil also enhanced the tumor growth inhibitory activity of gemcitabine in KPC mice. Three-dimensional volume measurements were acquired by ultrasonography of tumor bearing KPC mice before and after one (C) or two (D) 12-day treatment cycles. Percentage change is shown from baseline. IHC analysis of the proliferation marker Ki67 (E) and apoptosis marker cleaved-caspase 3 (CC3) (F) in epithelial tumor cells are also shown for mice treated in the various treatment cohorts. Fasudil enhanced survival of tumor bearing KPC mice. G) Kaplan-Meier curves of KPC mice treated with vehicle, gemcitabine, or the combination of gemcitabine plus fasudil. The combination treatment yields a significant improvement in overall survival compared to gemcitabine only group (Log-rank P value = 0.038). H) Pancreatic tissue from mice treated for three days with either vehicle or fasudil prior to a gemcitabine injection were analyzed for gemcitabine monophosphate concentrations in the tumor tissues. * P < 0.05. ** P < 0.01.</p

The aCGH results were validated by qPCR using the flow-sorted DNA.

<p>Primers were designed for <i>PKP4</i> and <i>pre-miR-651</i> genomic sequences. Primers were also designed using sequences outside the deleted regions for <i>PKP4</i> and <i>pre-miR-651</i> and were called <i>PKP4-</i>control and <i>pre-miR-651-</i>control respectively. Actin was used as the universal control. qPCR was performed and fold change was calculated and plotted. The left lower lung tumor sample validated as expected for <i>pre-miR-651</i> and <i>PKP4</i> deletion and the brain right frontal lobe sample did not show any of these deletions validating the aCGH results. The jejunum sample did not validate for these results.</p

Clonal analyses of the autopsy samples from the maxillary sinus cancer patient.

<p>A) DAPI-based DNA content analysis detected a 3.8N clonal population in brain right frontal pole brain left cerebellar and lung left lower lobe samples, while a 2.4N clonal population was seen in the jejunum sample. The diploid and aneuploid populations were sorted for subsequent aCGH studies. <b>B</b>) Zoomed in chromosome view showing the <i>PKP4</i> gene locus in the above four samples. <b>C</b>) Zoomed in chromosome view showing the <i>pre-miR-651 (labeled miR-651)</i> gene locus status in the above four samples.</p

Effect of YAP1 targeted siRNAs on the proliferation and apoptosis of pancreatic cancer cell lines.

<p>The cell growth curves of A) BxPC-3 and B) PANC-1 transfected with YAP1 siRNA oligonucleotides. The independent two-sample <i>t</i> test (two-tailed) was utilized to calculate the statistical significance at the 96-hour time point compared to Neg siRNA. * denotes p<0.05, and ** denotes p<0.01. C) The Caspase-Glo 3/7 Assay (Promega) was used to determine the level of apoptosis in pancreatic cancer cells transfected with YAP1 siRNA oligonucleotides after 72 hours. ** denotes an independent two-sample <i>t</i> test (two-tailed) p-value of <0.01 when compared to the negative siRNA control (Neg siRNA).</p

Nuclear YAP1 expression level and its correlation with histopathological parameters in pancreatic cancer.

<p>Abbreviation: pN, pathological nodal status; 0 = negative, 1 = metastasis present.</p