A Novel Interplay between Rap1 and PKA Regulates Induction of Angiogenesis in Prostate Cancer

<div><p>Angiogenesis inhibition is an important therapeutic strategy for advanced stage prostate cancer. Previous work from our laboratory showed that sustained stimulation of Rap1 by 8-pCPT-2'-O-Me-cAMP (8CPT) via activation of Epac, a Rap1 GEF, or by expression of a constitutively active Rap1 mutant (cRap1) suppresses endothelial cell chemotaxis and subsequent angiogenesis. When we tested this model in the context of a prostate tumor xenograft, we found that 8CPT had no significant effect on prostate tumor growth alone. However, in cells harboring cRap1, 8CPT dramatically inhibited not only prostate tumor growth but also VEGF expression and angiogenesis within the tumor microenvironment. Subsequent analysis of the mechanism revealed that, in prostate tumor epithelial cells, 8CPT acted via stimulation of PKA rather than Epac/Rap1. PKA antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis. Together these findings provide evidence for a novel interplay between Rap1, Epac, and PKA that regulates tumor-stromal induction of angiogenesis.</p> </div

Schematic depicting interplay between Rap1 and PKA in the tumor microenvironment.

<p>Schematic depicting interplay between Rap1 and PKA in the tumor microenvironment.</p

Effect of SDF-1α on secreted VEGF in prostate cancer cells under hypoxic-like conditions.

<p>Cells were treated as indicated and VEGF levels were measured by ELISA as described in Methods. (A and B) PC3 or PC3-cRap1 cells; *p<0.05, n = 3. (C and D) LNCaP or LNCaP-cRap1 cells; *p<0.05, n = 2.</p

Effect of PKA inhibitor on tumor growth and angiogenesis in PC3-cRap1 xenografts.

<p>(A and B) The PKA inhibitor, H-89 (2.5 µmol), reversed tumor growth inhibition and tumor weight reduction mediated by 8CPT in PC3-cRap1 xenografts. PC3-cRap1 tumors from mice infused with PBS, 8CPT, or H-89 were measured and tumor volume determined as described in Methods; * p<0.05; n = 8 for each treatment group. (C) CD31 and VEGF immunoreactivity in PC3-cRap1 tumors treated as indicated. Immunostaining was measured as described in Methods. <i>Upper Panels</i>: Representative photomicrographs. <i>Lower Panels</i>: Quantification of immunohistochemical staining of tumor slices; *p<0.05; n = 8 for each treatment group; a.u. = arbitrary units.</p

Effect of 8CPT treatment on HIF-1α and VEGF in PC3 and PC3-cRap1 cells under hypoxic-like conditions.

<p>(A) VEGF levels were measured by ELISA in PC3 and PC3-cRap1 cells under hypoxic-like conditions (CoCl₂) as described in Methods; *p<0.05, (n = 3). (B) Immunoblot analysis of nuclear extracts from PC3 and PC3-cRap1 cells treated as indicated. HIF-1α protein levels were determined by immunoblotting with specific antibody; (C) Analysis of VEGF mRNA levels by qPCR in PC3 and PC3-cRap1 cells treated as indicated. Data were normalized to GAPDH levels; *p<0.05 (<i>n</i> = 3).</p

Effect of constitutive Rap1 expression (cRap1) and/or 8CPT treatment on PC3 xenograft growth.

<p>(A and B) <i>Left Panel</i>: Tumor volume (expressed in mm³) of human PC3 and PC3-cRap1 xenografts treated as indicated was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049893#s2" target="_blank">Methods</a>. Day 0 represents the first day of treatment; *p<i><</i>0.05, n = 7 for each group. <i>Right Panel</i>: Images of a representative tumor from each treatment group at the end of the experiment (Day 28). (C) Tumor volume (expressed in mm³) of human PC3 and PC3-cRap1 xenografts treated as indicated was measured as in A; *p<i><</i>0.05, n = 7 for each group. (D) Tumors from panel C were weighed at the end of the experiment (Day 28) and expressed in grams; *p<0.05; (<i>n</i> = 7).</p

Resistance to ROS1 Inhibition Mediated by EGFR Pathway Activation in Non-Small Cell Lung Cancer

<div><p>The targeting of oncogenic ‘driver’ kinases with small molecule inhibitors has proven to be a highly effective therapeutic strategy in selected non-small cell lung cancer (NSCLC) patients. However, acquired resistance to targeted therapies invariably arises and is a major limitation to patient care. ROS1 fusion proteins are a recently described class of oncogenic driver, and NSCLC patients that express these fusions generally respond well to ROS1-targeted therapy. In this study, we sought to determine mechanisms of acquired resistance to ROS1 inhibition. To accomplish this, we analyzed tumor samples from a patient who initially responded to the ROS1 inhibitor crizotinib but eventually developed acquired resistance. In addition, we generated a ROS1 inhibition-resistant derivative of the initially sensitive NSCLC cell line HCC78. Previously described mechanisms of acquired resistance to tyrosine kinase inhibitors including target kinase-domain mutation, target copy number gain, epithelial-mesenchymal transition, and conversion to small cell lung cancer histology were found to not underlie resistance in the patient sample or resistant cell line. However, we did observe a switch in the control of growth and survival signaling pathways from ROS1 to EGFR in the resistant cell line. As a result of this switch, ROS1 inhibition-resistant HCC78 cells became sensitive to EGFR inhibition, an effect that was enhanced by co-treatment with a ROS1 inhibitor. Our results suggest that co-inhibition of ROS1 and EGFR may be an effective strategy to combat resistance to targeted therapy in some ROS1 fusion-positive NSCLC patients.</p></div

Crizotinib-resistant patient sample does not indicate <i>ROS1</i> gene amplification or histologic change.

<p>(A) Pre-treatment and post-resistance patient samples analyzed by break-apart FISH assay for <i>ROS1</i>. Red probes are to the 5′ region of ROS1 and green probes to the 3′ region. Values represent the mean number of signals per cell. The single 3′ signal (values underlined) is indicative of the <i>ROS1</i> fusion gene copy number. (B) RT-PCR, using primers to <i>SDC4</i> and <i>ROS1</i> that span the fusion point, performed on pre-treatment and post-resistance tumor samples. SD2;R32 is the ‘long’ variant (fusion of <i>SDC4</i> exon 2 to <i>ROS1</i> exon 32) and SD2;R34 is the ‘short’ variant (fusion of <i>SDC4</i> exon 2 to <i>ROS1</i> exon 34). (C) Hematoxylin and eosin staining of pre-treatment and post-resistance patient samples.</p

EGF stimulation desensitizes parental HCC78 cells and CUTO-2 cells to ROS1 inhibition.

<p>(A) Parental HCC78 and HCC78-TR cells were treated with TAE684 for 3 days with or without the addition of 100<b> </b>ng/mL EGF and then analyzed by MTS assay. Values represent the mean ± SEM (n = 3). Calculated IC₅₀ values for TAE684: parental+vehicle = 0.18 µM, parental+EGF = 0.57 µM, HCC78-TR+vehicle = 1.39 µM, and HCC78-TR+EGF = 1.45 µM. EGF significantly desensitized parental HCC78 but not HCC78-TR cells to TAE684 as determined by student’s paired t-test (p<0.05). (B) Parental HCC78 cells were treated with TAE684 for 4 hours, EGF for 10 minutes, or a combination of both. Lysates of the cells were then analyzed by Western blot using the indicated antibodies. (C) CUTO-2 cells were treated with TAE684 for 4 days with or without the addition of 100<b> </b>ng/mL EGF and then analyzed by MTS assay. Values represent the mean ± SEM (n = 3). Calculated IC₅₀ values for TAE684: +vehicle = 0.2 µM and +EGF = 0.81 µM. EGF significantly desensitized CUTO-2 cells to TAE684 as determined by student’s paired t-test (p<0.01). (D) CUTO-2 cells were treated with TAE684 for 4 hours, EGF for 10 minutes, or a combination of both. Lysates of the cells were then analyzed by Western blot using the indicated antibodies. Phosphorylated ROS1 bands were below the limit of detection and were therefore not included.</p

HCC78-TR cells are resistant to ROS1 inhibition.

<p>Cells were treated with TAE684 (A), crizotinib (B), or pemetrexed (C) as single-agents for 3 days and then analyzed by MTS assay. Values represent the mean ± SEM (n = 3–7). Calculated IC₅₀ values for TAE684: parental HCC78 = 0.14 µM, HCC78-TR = 1.09 µM, H322 = 1.42 µM, and HCC4006 = 1.15 µM. Calculated IC₅₀ values for crizotinib: parental HCC78 = 0.79 µM, HCC78-TR = 1.95 µM, H322 = 4.13 µM, and HCC4006 = 3.03 µM. Calculated IC₅₀ values for pemetrexed: parental HCC78 = 11<b> </b>nM and HCC78-TR = 14<b> </b>nM. HCC78-TR cells were significantly less sensitive than parental HCC78 cells to TAE684 (p<0.000005) and crizotinib (p<0.05) but not pemetrexed (p>0.05) as determined by student’s paired t-test.</p