A team of researchers led by scientists at Baylor College of Medicine has new insights into the function of neurofibromin, a tumor suppressor produced by the NF1 gene.

Neurofibromin keeps cancer growth in check by repressing the activity of a cancer driver called Ras, which is part of a family of related proteins belonging to a class of protein called small GTPase. Ras is involved in transmitting signals within cells, known as cellular signal transduction. Ras is the prototypical member of the Ras superfamily of proteins, which regulate diverse cell behaviors.

Previously unknown functionThe team of researchers discovered a previously unknown function of neurofibromin directly repressing gene expression controlled by the estrogen receptor- (ER). Thus, when neurofibromin is lost, Ras and ER functions are both activated, causing treatment resistance and metastasis for ER+ breast cancer.

The findings, published in Cancer Cell, suggest that a therapeutic approach must combine two different drugs, a SERD (e.g., fulvestrant) to degrade ER and a MEK inhibitor (e.g., selumetinib or binimetinib) to inhibit Ras downstream signaling, in order to effectively treat neurofibromin-depleted ER+ breast cancer. When this combination therapy was tested in animal models, the result was tumor regression. The next step is to begin clinical trials of the effectiveness of this therapeutic approach in patients.[1]

Driving tamoxifen and aromatase inhibitor resistanceThis researchers first studied the importance of neurofibromin loss in a study they published in 2018 in which tumor DNA was sequenced to seek mutations that can promote resistance to tamoxifen, which is commonly used to prevent relapses from ER+ breast cancer.[2]

When we examined the mutational patterns in NF1, we observed that poor patient outcomes only occurred when neurofibromin was lost, not through mutations that selectively affect Ras regulation. This suggested to us that neurofibromin may have more than one function, explained Eric C. Chang, Ph.D. one of two senior authors on the paper, an associate professor in the Department of Molecular and Cellular Biology and a member in the Dan L Duncan Comprehensive Cancer Centers Lester and Sue Smith Breast Center.

This thought triggered studies, spearheaded by Zeyi Zheng a lab member in Changs laboratory, into the function of neurofibromin in ER+ breast cancer cells.

One of his early experiments showed that when the expression of NF1 is inhibited, a process designed to mimic neurofibromin loss in tumors, the resulting ER+ breast cancer cells were instead stimulated by tamoxifen and, not as usual, inhibited.

The researchers also noted that these neurofibromin-depleted cells became sensitive to a very low concentration of estradiol, a form of estrogen.

The clinical relevance of these findings was immediately apparent because it suggested that tamoxifen or aromatase inhibitors, which lower estrogen levels available to the cancer cells, would be the wrong choice for treatment when neurofibromin is lost by the tumor, said co-senior author Matthew J. Ellis, MB, BChir, BSc., Ph.D., FRCP, professor and director of the Lester and Sue Smith Breast Center and a McNair Scholar at Baylor.

Linking neurofibromin to ERFollow-up gene expression studies all strongly suggest that neurofibromin behaves like a classic ER co-repressor.

A co-repressor must bind ER directly, but the group hesitated to conduct such an experiment without more evidence because it is not trivial to do so, Chang noted.

A breakthrough came when Charles E. Foulds, Ph.D., a primary faculty member and associate professor at the Center for Precision Environmental Health/Molecular & Cellular Biology and Associate Member of the Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine Houston, Texas, United States, a co-author on the paper, searched the Epicome, a massive proteomic database created by Anna Malovannaya, Ph.D. and Jun Qin, Ph.D. at Baylor, as part of an effort by Bert OMalley, MD, a chancellor, and professor of molecular and cellular biology at Baylor, to comprehensively document all the proteins associated with ER.

Foulds found neurofibromin in the database, which encouraged the team to ultimately demonstrate that ER and neurofibromin interact directly. However, to seriously consider NF1 as an ER co-repressor, there was still another missing piece of the puzzle.

One day Charles casually asked me whether neurofibromin has a region rich in the amino acids leucine and isoleucine, because co-repressors use these motifs to bind ER, and it dawned on me that neurofibromin indeed does, Chang said.

In fact, neurofibromin has two such motifs that mediate ER binding in a cooperative manner. These motifs are frequently mutated in cancers but are not required for Ras regulation, he added.

Treating neurofibromin-deficient ER+ breast cancerSince tamoxifen or aromatase inhibitors were found to be ineffective for neurofibromin-deficient ER+ breast cancer tumors, preclinical models were used to show that the ER-degrading drug fulvestrant was still effective. However, fulvestrant only temporarily inhibited tumor growth because secondary Ras-dependent fulvestrant resistance was induced by neurofibromin-loss. This Ras-dependent growth phase could be inhibited with the addition of a MEK inhibitor, which shuts off a key signaling pathway downstream of Ras.

The researchers validated this combination treatment strategy using a patient-derived xenograft (PDX) mouse model which can maintain the genomics and drug response of the original human tumor from which it was derived. [3]

In this case, this PDX was derived from a patient who failed several lines of endocrine therapy and already developed fulvestrant resistance.

The results of the combination therapy were encouraging the tumor shrunk to almost undetectable levels, Chang said. Our next goal is to test this combination therapy in clinical trials in order to determine its therapeutic potential in the clinic.

Neurofibromin loss is present in at least 10% of metastatic ER+ tumors. As a result of these new data, we are now working on a clinical trial that combines a MEK inhibitor, with fulvestrant, said Ellis, Susan G. Komen Scholar and associate director of Precision Medicine at the Dan L Duncan Comprehensive Cancer Center at Baylor.

Interestingly, MEK inhibitors are also being used to control peripheral nerve tumors in patients with neurofibromatosis, where a damaged NF1 gene is inherited. Our findings contribute to an understanding of why female neurofibromatosis patients also have a much higher incidence of breast cancer.

Reference[1] Zheng ZY, Anurag M, Lei JT, Foulds CE, Ellis MJ, Chang EC, et al. Neurofibromin Is an Estrogen Receptor- Transcriptional Co-repressor in Breast Cance. Cancer cell. March 05, 2020 DOI:https://doi.org/10.1016/j.ccell.2020.02.003 [Article][2] Griffith OL, Spies NC, Anurag M, Griffith M, Luo J, Tu D, Yeo B, Kunisaki J, Miller CA, et al. The prognostic effects of somatic mutations in ER-positive breast cancer. Nat Commun. 2018 Sep 4;9(1):3476. doi: 10.1038/s41467-018-05914-x. Erratum in: Nat Commun. 2018 Nov 14;9(1):4850. PMID: 30181556; PMCID: PMC6123466. [Article][3] Li S, Shen D, Shao J, Crowder R, Liu W, Prat A, He X, Liu S, Hoog J, et al. Endocrine-therapy-resistant ESR1 variants revealed by genomic characterization of breast-cancer-derived xenografts. Cell Rep. 2013 Sep 26;4(6):1116-30. doi: 10.1016/j.celrep.2013.08.022. Epub 2013 Sep 19. PMID: 24055055; PMCID: PMC3881975. [Article]

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