Versatile microRNAs choke off cancer blood supply, suppress metastasis

(Press-News.org) HOUSTON – A family of microRNAs (miR-200) blocks cancer progression and metastasis by stifling a tumor's ability to weave new blood vessels to support itself, researchers at The University of Texas MD Anderson Cancer Center report today in Nature Communications.

Patients with lung, ovarian, kidney or triple-negative breast cancers live longer if they have high levels of miR-200 expression, the researchers found.

Subsequent experiments showed for the first time that miR-200 hinders new blood vessel development, or angiogenesis, and does so by targeting cytokines interleukin-8 (IL-8) and CXCL1.

"Nanoparticle delivery of miR-200 blocked new blood vessel development, reduced cancer burden and inhibited metastasis in mouse models of all four cancers," said Anil Sood, M.D., professor of Gynecologic Oncology, senior author of the study.

The team's findings highlight the therapeutic potential of nanoparticle-delivered miR-200 and of IL-8 as a possible biomarker for identifying patients who might benefit from treatment. Sood said safety studies will need to be completed before clinical development can begin.

Micro RNAs do not code for genes like their cousins, the messenger RNAs. They regulate gene activation and expression.

"We initially looked at miR-200 because we have an approach for targeting and delivering these molecules with nanoparticles and miR-200 is known to inhibit EMT, a cellular transition associated with cancer progression and metastasis," said Sood, who also holds the Bettyann Asche Murray Distinguished Professorship in Ovarian Cancer Research.

First author Chad Pecot, M.D., a fellow in Cancer Medicine, said initial research provided a new perspective. "Cautionary tales emerged from the literature about poor outcomes in hormone-positive breast cancer, so we decided to delve more deeply into understanding the mechanisms involved."

miR-200 effect differs by breast cancer type

Sood and colleagues analyzed hundreds of annotated ovarian, renal, breast and non-small cell lung cancer samples from The Cancer Genome Atlas for expression of all five miR-200 family members. Low expression of miR-200 was associated with poor survival in lung, ovarian and renal cancers, but improved survival for breast cancer.

However, they found a striking difference when they analyzed breast cancers by those that are hormone-receptor positive (luminal) and those that lack hormone receptors or the HER2 protein, called triple-negative breast cancer. High expression for miR-200 was associated with improved survival for triple-negative disease, which is more difficult to treat due to its lack of therapeutic targets.

Gene expression analysis of ovarian and lung cancer cell lines pointed to an angiogenesis network involving both IL-8 and CXCL1. By mining public miRNA and messenger RNA databases, the researchers found:

An inverse relationship between expression of four of the five members of the miR-200 family and IL-8. Lung, ovarian, kidney and triple-negative breast cancer all have elevated IL-8 and CXCL1 expression compared to hormone-positive breast cancers. Elevated IL-8 associated with poor overall survival in lung, ovarian, renal and triple-negative breast cancer cases.

Treating cancer cell lines with miR-200 decreased levels of IL-8 and CXCL1, and the team also identified binding sites for these genes, meaning they are direct miR-200 targets.

Mice treated with miR-200 family members delivered in a fatty nanoparticle developed by Sood and Gabriel Lopez, M.D., professor of Experimental Therapeutics, had steep reductions in lung cancer tumor volume, tumor size and the density of small blood vessels compared to controls. Results were repeated with kidney, ovarian and triple-negative breast cancers.

miR-200 nanoparticles stymie metastasis In mouse models of lung and triple-negative breast cancers prone to spread to other organs, treatment with the miR-200 nanoliposomes significantly reduced the volume of the primary tumor and the number and size of metastases in other organs compared to controls. Similar results were observed in an ovarian cancer model, accompanied by sharp reductions in IL-8 levels and blood vessel formation.

Additional experiments showed that these therapeutic effects were due to blocking of IL-8 levels by miR-200. In tumors that had high amounts of synthetically produced IL-8 (designed so that miR-200 could not block it) the cancer burden was no longer reduced. Circulating IL-8 levels in the blood strongly correlated with tumor burden, Pecot said, suggesting it may serve as a possible biomarker for miR-200 treatment.

Treatment of blood vessels cuts metastases by 92 percent

The team then used a chitosan nanoparticle – derived from chitin in the shells of crustaceans – to deliver miR-200 straight to blood vessels. Combination delivery of two types of miR-200 reduced ovarian cancer metastases by 92 percent over controls. Targeting a second ovarian cancer line with the chitosan nanoparticles also developed by Sood and colleagues, resulted in decreased primary and metastatic tumor burden and reduced blood vessel formation with no apparent toxicity observed in treated mice.

### Co-authors with Sood, Pecot and Lopez are Rajesha Rupaimoole, Ph.D., Cristina Ivan, Ph.D., Chunhua Lu, Sherry Wu, Hee-Dong Han, Justin Bottsford-Miller, M.D., Behrouz Zand, M.D., Myrthala Moreno-Smith, Ph.D., Lingegowda Mangala, Ph.D., Ph.D., Morgan Taylor, Ph.D., Healther Dalton, Ph.D.,Yunfei Wen, Ph.D., and Yu Kang, M.D., all of the Gynecologic Oncology; Da Yang, Ph.D., Yuexin Liu, Ph.D., and Wei Zhang, Ph.D., of Pathology; Rehan Akbani, Ph.D., Anna Unruh, and Keith Baggerly, Ph.D., of Bioinformatics and Computational Biology; Maitri Shah, Cristian Rodriguez-Villasana, Ph.D., Vianey Gonzalez-Villasana, Ph.D., and George Calin, M.D., Ph.D., of Experimental Therapeutics; Sang Bae Kim, Vasudha Sehgal, Ph.D., Ju-Seog Lee, Ph.D., Prahlad Ram, Ph.D., and Ana-Maria Gonzalez-Angulo, M.D., of Breast Medical Oncology; Murali Ravoori and Vikas Kundra, M.D., Ph.D., of Experimental Diagnostic Imaging; Li Huang and Xinna Zhang of Cancer Biology; Rouba Ali-Fehmi, M.D., of Wayne State University School of Medicine, Karmanos Cancer Institute, Detroit; and Pierre Massion, M.D., of Vanderbilt Ingram Cancer Center, Vanderbilt University, Nashville, Tenn.

Sood, Lopez, Xinna Zhang, Wei Zhang, Calin, Mangala and Ivan are also with MD Anderson's Center for RNA Interference and Non-Coding RNA. Sang Bae Kim and Anna Unruh are students in The University of Texas Graduate School of Biomedical Sciences, a joint operation of MD Anderson and The University of Texas Health Science Center at Houston.

This research was funded by grants from the National Cancer Institute of the National Institutes of Health (CA109298, P50 CA083639, P50 CA098258, CA128797, RC2GM092599, U54 CA151668 and U24CA143835, CA009666, CA90949, CA143883, T32 CA101642, and U24CA143835); the Cancer Prevention and Research Institute of Texas, the Ovarian Cancer Research Fund, Inc., The U.S. Department of Defense, The Marcus Foundation, Inc., Laura Lee Blanton Ovarian Cancer Endowed Fund, the Vanderbilt SPORE in lung cancer ; the 2011 Conquer Cancer Foundation ASCO Young Investigator Award, MD Anderson's Division of Cancer Medicine Advanced Scholar Program, The Cancer Genome Atlas MD Anderson Data Analysis Center, an MD Anderson Odyssey Fellowship, Diane Denson Tobola Fellowship for Ovarian Cancer Research and the Harold C. and Mary L. Dailey Endowment Fellowships.