Agent-based modeling predicts RAC1 is critical for ovarian cancer metastasis.

Experimental and computational studies pinpoint rate-limiting step(s) in metastasis governed by Rac1. Using ovarian cancer cell and animal models, Rac1 expression was manipulated, and quantitative measurements of cell-cell and cell-substrate adhesion, cell invasion, mesothelial clearance, and peritoneal tumor growth discriminated the tumor behaviors most highly influenced by Rac1. The experimental data were used to parameterize an agent-based computational model simulating peritoneal niche colonization, intravasation, and hematogenous metastasis to distant organs. Increased ovarian cancer cell survival afforded by the more rapid adhesion and intravasation upon Rac1 overexpression is predicted to increase the numbers of and the rates at which tumor cells are disseminated to distant sites. Surprisingly, crowding of cancer cells along the blood vessel was found to decrease the numbers of cells reaching a distant niche irrespective of Rac1 overexpression or knockdown, suggesting that sites for tumor cell intravasation are rate limiting and become accessible if cells intravasate rapidly or are displaced due to diminished viability. Modeling predictions were confirmed through animal studies of Rac1-dependent metastasis to the lung. Collectively, the experimental and modeling approaches identify cell adhesion, rapid intravasation, and survival in the blood as parameters in the ovarian metastatic cascade that are most critically dependent on Rac1.

The R-enantiomer of ketorolac reduces ovarian cancer tumor burden in vivo.

BACKGROUND: Rho-family GTPases, including Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), are important modulators of cancer-relevant cell functions and are viewed as promising therapeutic targets. Based on high-throughput screening and cheminformatics we identified the R-enantiomer of an FDA-approved drug (ketorolac) as an inhibitor of Rac1 and Cdc42. The corresponding S-enantiomer is a non-steroidal anti-inflammatory drug (NSAID) with selective activity against cyclooxygenases. We reported previously that R-ketorolac, but not the S-enantiomer, inhibited Rac1 and Cdc42-dependent downstream signaling, growth factor stimulated actin cytoskeleton rearrangements, cell adhesion, migration and invasion in ovarian cancer cell lines and patient-derived tumor cells. METHODS: In this study we treated mice with R-ketorolac and measured engraftment of tumor cells to the omentum, tumor burden, and target GTPase activity. In order to gain insights into the actions of R-ketorolac, we also performed global RNA-sequencing (RNA-seq) analysis on tumor samples. RESULTS: Treatment of mice with R-ketorolac decreased omental engraftment of ovarian tumor cells at 18 h post tumor cell injection and tumor burden after 2 weeks of tumor growth. R-ketorolac treatment inhibited tumor Rac1 and Cdc42 activity with little impact on mRNA or protein expression of these GTPase targets. RNA-seq analysis revealed that R-ketorolac decreased expression of genes in the HIF-1 signaling pathway. R-ketorolac treatment also reduced expression of additional genes associated with poor prognosis in ovarian cancer. CONCLUSION: These findings suggest that R-ketorolac may represent a novel therapeutic approach for ovarian cancer based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor. R-ketorolac modulates relevant pathways and genes associated with disease progression and worse outcome.

Distinct nodule and leaf functions of two different sucrose phosphate synthases in alfalfa.

MAIN CONCLUSION: In alfalfa, the B form of Sucrose phosphate synthase synthesizes sucrose in the leaves while the A form participates in regulatory cycles of synthesis/breakdown of sucrose/starch in the root nodules. Sucrose (Suc) is the major stable product of photosynthesis that is transported to all heterotrophic organs as a source of energy and carbon. The enzyme sucrose phosphate synthase (SPS) catalyzes the synthesis of Suc. Besides the leaves, SPS is also found in heterotrophic organs. There are two isoforms of SPS in alfalfa (Medicago sativa): SPSA and SPSB. While SPSA is expressed in the vasculature of all the organs and in the N2-fixing zone in the nodules, SPSB is exclusively expressed in the photosynthetic cells. Two classes of alfalfa transformants were produced, one with a gene construct consisting of the alfalfa SPSA promoter and the other with the SPSB promoter-both driving the maize SPS coding region-referred to as SPSA-ZmSPS and SPSB-ZmSPS, respectively. Both classes of transformants showed increased growth compared to control plants. The SPSB-ZmSPS transformants showed increased SPS protein levels and activity along with a significant increase in the Suc levels in the leaves. The SPSA-ZmSPS transformants showed an increase in the SPS protein level and enzyme activity both in the leaves and the nodules with no increase in Suc content in the leaves but a substantial increase in the nodules. Both SPSA and SPSB have unique roles in the nodules (sink) and leaves (source). SPSB is responsible for the synthesis of Suc in the photosynthetic cells and SPSA participates in a regulatory cycle in which Suc is simultaneously degraded and re-synthesized; both these functions contribute to plant growth in rhizobia nodulated alfalfa plants.

Dual Actions of Ketorolac in Metastatic Ovarian Cancer.

Cytoreductive surgery and chemotherapy are cornerstones of ovarian cancer treatment, yet disease recurrence remains a significant clinical issue. Surgery can release cancer cells into the circulation, suppress anti-tumor immunity, and induce inflammatory responses that support the growth of residual disease. Intervention within the peri-operative window is an under-explored opportunity to mitigate these consequences of surgery and influence the course of metastatic disease to improve patient outcomes. One drug associated with improved survival in cancer patients is ketorolac. Ketorolac is a chiral molecule administered as a 1:1 racemic mixture of the S- and R-enantiomers. The S-enantiomer is considered the active component for its FDA indication in pain management with selective activity against cyclooxygenase (COX) enzymes. The R-enantiomer has a previously unrecognized activity as an inhibitor of Rac1 (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42) GTPases. Therefore, ketorolac differs from other non-steroidal anti-inflammatory drugs (NSAIDs) by functioning as two distinct pharmacologic entities due to the independent actions of each enantiomer. In this review, we summarize evidence supporting the benefits of ketorolac administration for ovarian cancer patients. We also discuss how simultaneous inhibition of these two distinct classes of targets, COX enzymes and Rac1/Cdc42, by S-ketorolac and R-ketorolac respectively, could each contribute to anti-cancer activity.

The R-Enantiomer of Ketorolac Delays Mammary Tumor Development in Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) Mice.

Epidemiologic studies report improved breast cancer survival in women who receive ketorolac (Toradol) for postoperative pain relief compared with other analgesic agents. Ketorolac is a racemic drug. The S-enantiomer inhibits cyclooxygenases; R-ketorolac is a selective inhibitor of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), which are signaling molecules up-regulated during breast cancer progression and metastasis. The goal of this study was to determine whether R-ketorolac altered breast cancer development in the mouse mammary tumor virus-polyoma middle T-antigen model. Mice were administered ketorolac orally at 1 mg/kg twice daily to approximate the typical human dose. Mammary glands were analyzed for tumor number and immunohistochemical markers of proliferation and differentiation. R-ketorolac treatment significantly reduced mammary epithelial proliferation, based on Ki67 staining, and suppressed tumor development. Proliferative mammary epithelium from R-ketorolac-treated mice displayed greater differentiation, based on significantly higher total E-cadherin and decreased keratin 5 staining than epithelium of placebo-treated mice. No differences were detected in estrogen receptor, progesterone receptor, ß-catenin, or vimentin expression between placebo and R-ketorolac treatment groups. These findings indicate that R-ketorolac treatment slows tumor progression in an aggressive model of breast cancer. R-ketorolac may thus represent a novel therapeutic approach for breast cancer prevention or treatment based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor.