New Research Identifies the Brain-Derived Growth Factor TrkB, FOXO4 Protein, and SSeCKS/AKAP12 Gene As Lung And Prostate Cancer Metastasis Blockers.

Researchers at Harvard Medical School and its associated clinical institutions have now discovered an approach to blocking metastasis in the most common type of lung cancer, adenocarcinoma, that could be added to chemotherapy treatments aimed at shrinking the primary tumor.

A Harvard Medical School news release authored by Nancy Fliesler, based on an article that first appeared in Vector, Boston Children’s Hospital’s science and clinical innovation blog, reports that Kerstin Sinkevicius, Ph.D, a Research Fellow in Genetics at Boston Childrens Hospital, started with this question: “Is there anything in a lung tumors environment that makes it metastasize?” She sampled tissue from human lymph nodes — the first place cancers typically spread — to see if cells there were secreting anything that might lure cancer cells to migrate, and one chemical stood out: a growth factor called brain-derived neurotrophic factor, or BDNF. BDNF is secreted near maturing neurons and best known for its role in stimulating the developing nervous system.

Was BDNF the factor she was looking for? Ms. Fliesler notes that when Dr. Sinkevicius took BDNF out of the equation, migration of adenocarcinoma cells in cultures dropped nearly twofold, suggesting that cancer cells take advantage of the same signals neurons use, explaining that oftentimes cancer cells use built in mechanisms.

Based on that initial observation, Dr. Sinkevicius and senior investigator Dr. Carla Kim, Harvard Medical School associate professor of genetics at Boston Childrens, then systematically searched for any agent in the primary lung tumor that might be attracted by BDNF’s signal, their objective to determine whether blocking such communication might prevent cancer cells from metasticizing.

Those investigations pointed to a cellular receptor molecule called TrkB, which, when they removed it from the equation, cell migration significantly decreased. When they deleted TrkB in a live mouse model of adenocarcinoma, only 17 percent of the mice got metastases, compared with the usual rate of 50 percent.

“The primary tumors were the same, so we think TrkB is specifically important for metastasis, Dr. Sinkevicius told Ms. Fliesler. The researchers’ findings were published in July in the Proceedings of the National Academy of Sciences.

The paper, “Neurotrophin receptor TrkB promotes lung adenocarcinoma metastasis” (Proc Natl Acad Sci U S A. 2014 Jul 15;111(28):10299-304. doi: 10.1073/peas.1404399111. Epub 2014 Jun 30), is coauthored by Sinkevicius KW, Kriegel C, Bellaria KJ, Lee J, Lau AN, Leeman KT, Zhou P, Beede AM, Fillmore CM, Caswell D, Barrios J, Wong KK, Sholl LM, Schlaeger TM, Bronson RT, Chirieac LR, Winslow MM, Haigis MC, and Kim CF, variously of the Boston Children’s Hospital Stem Cell Program, Harvard Stem Cell Institute, Medical School Department of Genetics, Department of Cell Biology and Paul F. Glenn Laboratories for the Biological Mechanisms of Aging at Harvard Medical School, Boston; the Department of Pediatrics, Division of Newborn Medicine at Boston Children’s Hospital, the Department of Pediatric Oncology and Cancer Biology at Dana-Farber Cancer Institute in Boston, the Harvard Medical School Department of Neurobiology; the Stanford University School of Medicine Cancer Biology Program at Stanford, CA, the Dana-Farber Cancer Institute Department of Medical Oncology and Belfer Institute for Applied Cancer Science; Harvard Medical School’s Department of Genetics, and Department of Medicine; and the Department of Pathology at Brigham and Women’s Hospital in Boston.

The researchers observe that lung cancer is notorious for its ability to metastasize, but the pathways regulating lung cancer metastasis are largely unknown. However, an in vitro system designed to discover factors critical for lung cancer cell migration identified brain-derived neurotrophic factor, which stimulates cell migration through activation of tropomyosin-related kinase B (TrkB; also called NTRK2).

The scientists note that knockdown of TrkB in human lung cancer cell lines significantly decreased their migratory and metastatic ability in vitro and in vivo, and in an autochthonous lung adenocarcinoma model driven by activated oncogenic Kras and p53 loss, TrkB deficiency significantly reduced metastasis. Hypoxia-inducible factor-1 directly regulated TrkB expression, and, in turn, TrkB activated Akt signaling in metastatic lung cancer cells. They summarize that TrkB expression was correlated with metastasis in patient samples, and TrkB was detected more often in tumors that did not have Kras or epidermal growth factor receptor mutations, leading them to conclude that their findings identify TrkB as an important therapeutic target in metastatic lung adenocarcinoma.

“When adenocarcinoma cells were cultured in low-oxygen conditions under which lung cancers are more apt to metastasize, TrkB was especially abundant. Oxygen-deprived tumor cells are stressed out and may be looking to go somewhere else,” Dr. Sinkevicius notes in the release. “And TrkB is their ticket.”

Dr. Sinkevicius is a member of the Carla Kim Lab at Children’s Hospital Boston and Harvard Medical school Boston where the broad focus is on characterization of the biology of stem cells in normal lung and lung cancer. The Lab profile notes that numerous lung diseases such as cystic fibrosis or chronic obstructive pulmonary disease involve injured or depleted bronchiolar or alveolar epithelium, and bronchiolar and alveolar cells are also affected in adenocarcinoma, the most common form of lung cancer.

Moreover, it is likely that lung stem cells are critically affected in patients with these devastating diseases, and the lab’s long-term goal is to elucidate the role of stem cells in lung homeostasis as a prerequisite to development of therapeutic strategies that can be used to prevent or attenuate lung disease. Dr. Kim and her colleagues isolated the first stem cell population from the adult murine lung, termed bronchioalveolar stem cells (BASCs). BASCs are critically affected by an oncogenic K-ras mutation and may be the cell-of-origin of lung adenocarcinomas. They hypothesize that BASCs are the stem cells that maintain bronchiolar and alveolar cell homeostasis in vivo. They use a combination of mouse genetics, cell biology and genomics approaches to elucidate the biology of these cells during homeostasis and tumorigenesis.

Traditionally, even though ninety percent of lung cancer deaths are caused by spread of the cancer from the original site to other organs, Ms. Fliesler notes that metastasis has been an understudied area in cancer, with pharmaceutical companies preferring to seek out agents that shrink the primary tumor metric that’s fairly short term and easily measurable. Antimetastatic agents are a less attractive investment, since metastasis is variable and can take place over years.

However, compared with previously identified targets for preventing metastasis, TrkB may be easier to hit, she cites Dr. Kim observing that since chemical TrkB inhibitors are already available and being tested for depression and neurologic disorders such as epilepsy, there may be cancer patients for whom TrkB inhibition is particularly useful, since certain subsets of adenocarcinoma have higher levels of TrkB.

In their study, Dr. Kim and Dr. Sinkevicius found that targeting TrkB was especially effective in the roughly 60 percent of adenocarcinomas that lack two commonly seen mutations, in the genes Kras and EGFR, and it is hoped that other metastasis-inhibiting compounds could emerge from this work. Dr. Kim hopes to do gene-expression studies to see if there are changes in gene activity when TrkB is inhibited. These could help find additional drug targets.

Previously Unknown Metastasis Suppressor: FOXO4 And Prostate Cancer

In another metastasis study published in the journal PLOS 1 on July 1, a research team led by Irwin H. Gelman, Ph.D. , of Roswell Park Cancer Institute (RPCI) in Buffalo, NY, has identified a new suppressor of cancer metastasis that may point the way toward development of more effective treatments for prostate cancers and other malignant solid tumors.

Activation of the PI3K/AKT signal pathway is a known driver of the progression of prostate cancer to the castrate-resistant stage, the most lethal form of prostate cancer. Using a genome-wide genetic screen, Dr. Gelman and colleagues identified a previously unknown metastasis suppressor, the FOXO4 protein, which belongs to a family of genes that are produced by all human cells.

“Evidence from several publicly available cancer genomic databanks indicates that FOXO4 is typically turned off in metastatic prostate cancer compared to primary prostate tumors. Our research showed that the FOXO4 gene normally turns off genes that control specific metastatic behavior in malignant tumor cells, such as the ability to invade tissues and then to survive and multiply there,” says Dr. Gelman, the John & Santa Palisano Chair in Cancer Genetics at RPCI. In demonstrating that FOXO4 works to prevent the spread of cancerous tumors by binding to and inhibiting the protein RUNX2, the team identified a circuit that controls metastatic progression in prostate cancer.

“Our findings underline the importance of RUNX2 in promoting metastasis and suggest that drugs that inhibit its function would prevent or treat prostate cancer metastasis,” notes Dr. Gelman. “Given the devastating impact of cancer metastasis and the dire need for therapies to combat tumor spread, we’re highly encouraged by these findings and excited about the therapeutic possibilities they open up.” Dr. Gelman’s lab is now working with collaborators at the University of Maryland to test the effectiveness of the experimental agent CADD522, an inhibitor of RUNX2 function, in preventing or impeding prostate cancer metastasis.

The research article, entitled “A Genome-Wide RNAi Screen Identifies FOXO4 as a Metastasis-Suppressor through Counteracting PI3K/AKT Signal Pathway in Prostate Cancer” (Published: July 01, 2014 PLoS ONE 9(7): e101411. doi:10.1371/journal.pone.0101411) is coauthored by Bing Su, Lingqiu Gao, Catherine Baranowski, Bryan Gillard, Jianmin Wang, Ryan Ransom, Hyun-Kyung Ko, and Roswell Park Cancer Institute Irwin H. Gelman, and edited by Natasha Kyprianou of the University of Kentucky College of Medicine.

The coauthors observe that activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. In their study, the scientists identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis, and note that although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, they observe that forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells yet did not alter RUNX2 protein levels, and it also decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients.

They conclude that their data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness, and that the current study identifies FOXO4 as a potentially novel metastasis suppressor among several candidate genes identified using a genomic shRNA screen for increased LNCaP invasiveness. Furthermore, they say FOXO4 likely fulfills the currently accepted definition of a metastasis suppressor in that it is downregulated in clinical metastases compared to primary-site CaP lesion, its downregulation correlates with significantly decreased time-to-onset of clinical metastasis, its expression levels do no grossly affect primary tumor growth, yet its downregulation promotes metastatic invasiveness in vitro and metastatic formation in vivo.

Although redundant functionality between FOXO family members is known, only FOXO4 exhibited critical regulatory ability for invasiveness, namely that FOXO4 knockdown resulted in increased invasiveness whereas its overexpression suppressed invasiveness. Given this unique function for FOXO4, and that their evidence suggests that FOXO4 regulates metastasis by suppressing the ability of RUNX2 to induce pro-metastasis genes (discussed below), the researchers suggest it is likely that CaP metastasis is promoted by a non-redundant FOXO4 cistrome and/or by unique interactions between the repressor function of FOXO4 and pro-metastasis transcription factors.

The coauthors further report that the known role of FOXO family members, including FOXO4, as transcriptional repressors led them to analyze FOXO4-regulated gene expression changes shared by cultured LNCaP, primary tumors and LN metastases, on the assumption that a gene signature arising after FOXO4 knockdown would identify functions that contributed to increased invasiveness in vitro, and that if this signature was maintained through primary tumor cells and into metastases, these functions would likely also control increased metastatic potential.

Of the initial 19 genes identified by gene expression microarrays that fulfilled this definition, a 4-gene signature, PIP, PGC, CAMK2N1 and PLA2G16, passed qRT-PCR validation and, as well, showed evidence in the literature of involvement with metastatic processes. The notion that FOXO4 might directly repress expression of this gene group was ruled out because 1) only two gene promoters, those of PIP and PLA2G16, encode FOXO binding sites, and 2) overexpressed FOXO4 did not bind these sites in ChIP-qPCR experiments.

However, they say bioinformatics analysis of these genes and promoters indicated that they were all RUNX2-targets and -regulated genes, and indeed, RUNX2 knockdown could blunt the enhanced invasiveness induced by the loss of FOXO4 in LNCaP cells. Moreover, we showed that RUNX2 and FOXO4 interacted in cells, and that altering FOXO4 levels had an inverse effect on the ability of RUNX2 to bind its cognate site on the PIP promoter yet did not change RUNX2 expression levels. This finding parallels previous research by Zhang et al. who showed that FOXO1 binding to RUNX2 suppressed invasiveness by preventing RUNX2 access to the promoters of pro-metastasis genes such as OP, IL8, VEGF and MMP13.

The SSeCKS/AKAP12 Gene And Prostate Cancer Metastasis

In February, researchers at the Roswell Park Cancer Institute published advance findings on key gene related to cancer metastasis in Cancer Research, a scientific journal published by the American Association for Cancer Research, that lends support to the hypothesis that the SSeCKS/AKAP12 gene is a key inhibitor of prostate cancer metastasis. The data are some of the first to demonstrate this dynamic in transgenic animal models, with promising implications for development of targeted therapies for prostate cancer and perhaps for other solid-tumor cancers.

A team also led by Irwin H. Gelman, PhD, noted that aggressive prostate cancers in humans typically turn off or delete two major regulatory genes, SSeCKS/AKAP12 and Rb. To explore this dynamic, the researchers developed a transgenic animal model to study the effects on prostate cancer progression of deleting these two genes.

The study, entitled “A Transgenic Mouse Model for Early Prostate Metastasis to Lymph Nodes” (Cancer Res February 1, 2014 74:945-953; DOI: 10.1158/0008-5472.CAN-13-1157) is coauthored by Hyun-Kyung Ko, Shin Akakura, Jennifer Peresie, David W. Goodrich, Barbara A. Foster, and Irwin H. Gelman of the Departments of Cancer Genetics and 2Pharmacology and Therapeutics, at Roswell Park Cancer Institute.

The researchers observe that emergence of recurrent, metastatic prostate cancer following the failure of androgen-deprivation therapy represents the lethal phenotype of this disease, but little is known regarding the genes and pathways that regulate this metastatic process, and moreover, it is unclear whether metastasis is an early or late event.

They note that the individual genetic loss of the metastasis suppressor, SSeCKS/Gravin/AKAP12 or Rb, genes that are downregulated or deleted in human prostate cancer, results in prostatic hyperplasia, and in this study they show that the combined loss of Akap12 and Rb results in prostatic intraepithelial neoplasia (PIN) that fails to progress to malignancy after 18 months.

Strikingly, the researchers report that 83 percent of mice with PIN lesions exhibited metastases to draining lymph nodes, marked by relatively differentiated tumor cells expressing markers of basal (p63, cytokeratin 14) and luminal (cytokeratin 8 and androgen receptor) epithelial cells, although none expressed the basal marker, cytokeratin 5. The finding that PIN lesions contain increased numbers of p63/AR-positive, cytokeratin 5-negative basal cells compared with WT or Akap12/ prostate lobes suggests that these transitional cells may be the source of the lymph node metastases. Taken together, these data suggest that in the context of Rb loss, Akap12 suppresses the oncogenic proliferation and early metastatic spread of basal-luminal prostate tumor cells.

“This correlates with our earlier finding that SSeCKS/AKAP12 inhibits the chemotaxis of metastatic prostate tumor cells that is, their ability to move on to another environment in response to chemical attractants,” says Dr. Gelman, the John & Santa Palisano Chair in Cancer Genetics at RPCI, in a release. “Thus, our data suggest that SSeCKS plays a role in preventing the early dissemination of prostate cancer cells to metastatic sites. Importantly, we show that humans whose prostate cancers have turned off or deleted the SSeCKS/AKAP12 gene have significantly higher rates of metastasis formation compared to cases where SSeCKS/AKAP12 levels are sustained.”

While the SSeCKS/AKAP12 gene is deleted in about a third of metastatic prostate cancers, precluding benefit from targeted therapies exploiting this vulnerability, the remaining two-thirds of such tumors may be treatable with drugs that induce the reactivation of SSeCKS/AKAP12 production. Dr. Gelman and colleagues are now looking to identify the genomic signatures controlled by SSeCKS/AKAP12 in the suppression of metastasis pathways Children’sat the level of the tumor cells themselves and in the cells that form the metastatic microenvironment.

“At least 93 percent of cancer patients die because of complications due to metastatic cancers, yet the vast majority of pathways studied and therapies developed address the biology of primary cancers,” Dr. Gelman notes. “This current research is important in that it addresses specific mechanisms of cancer metastasis, with the result that genetic tests and therapies derived from such studies will have a higher chance of affecting cancer patient survival.”

 

The Roswell Park Cancer Institute is among the first and only upstate New York facilities to hold the National Cancer Institute designation of “comprehensive cancer center” and to serve as a member of the prestigious National Comprehensive Cancer Network. Many RPCI faculty serve on the NCCN panels that create the Clinical Practice Guidelines in Oncology the internationally recognized standards for clinical policy in oncology, and the most comprehensive, most frequently updated clinical practice guidelines available in any area of medicine.

Sources:
Harvard Medical School
Boston Children’s Hospital
Roswell Park Cancer Institute
PlOS 1
Cancer Research

Image Credits:
Carla Kim Lab
Roswell Park Cancer Institute