Targeting Prolactin as a Novel Treatment for Breast Cancer

Institution: University of California, Riverside
Investigator(s): Ameae Walker, Ph.D. -
Award Cycle: 2011 (Cycle 17) Grant #: 17IB-0053 Award: $150,000
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure



Initial Award Abstract (2011)

A number of growth factor and hormone receptors and signaling molecules have natural “dominant negative” varieties, formed by “alternative splicing” of a single gene transcript. Genes are transcribed to produce pre-mRNAs, which are then spliced to create the mature mRNAs translated into protein. In the last few years, deep sequencing and microarray analyses have shown greater than 90% of human pre-mRNAs undergo alternative splicing, thereby amplifying the potential protein products from each gene. Alternatively spliced forms of pre-mRNA may code for proteins with related, distinct, or even opposing functions (dominant negatives). To our knowledge no one has previously tapped the potential of natural dominant negatives to combat disease in general, and breast cancer in particular. Researchin my laboratory is concerned with the growth factor activities of the hormone prolactin. These growth factor activities occur in many tissues including the pituitary, breast, endocrine pancreas, liver, prostate, and cells of the immune system. Although most prolactin is produced by the pituitary, some is produced in these other tissues where it may act as an autocrine (hormone or chemical messenger binding to the same cell) or paracrine (nearby cell) growth factor.

There is evidence that “upregulation” of the dominant negative version of prolactin will affect survival of cancer stem cells and prevent tumor angiogenesis. We will begin by examining the effects of our designed “splice modulating oligomers” (i.e., short, 20 nucleotide chains that bind to pre-mRNA and cause changes in splicing) on human and mouse breast cancer cells grown in culture. We will compare the effects of oligomers targeting the human and mouse receptors for their effects on cell proliferation, migration, invasion, cell death and the number of stem cells. If the effects are comparable in human and mouse cells, we will examine effects of the oligomers on tumors of mouse cells in normal mice. In this way, we can examine effects in an animal with a normal immune system using a model system that accurately recapitulates progression of the human disease. If the human and mouse cells do not respond equivalently, we will instead examine the effects of the oligomers on human tumors grown in mice without a normal immune system. It is necessary for mice in this instance to be immuno-compromised so that they will allow tumors of human cells to grow. At the same time in the animal experiments, we will examine the mice for potential unexpected side effects and toxicities.

In addition to the prolactin receptor, potential targets of this class of oligomer drugs include receptors for estrogen and growth hormone, each of which has been shown to have a dominant negative variety formed by alternative splicing. Other targets exist, and as we learn more about the basic biology of breast cancer, it is likely that many more targets will appear. An attractive feature of the splice modulating oligomers is that they can be combined to target more than one receptor or signaling molecule or transcription factor at a time, thereby potentially increasing the impact of treatment and reducing the possibility that cells can become resistant.




Final Report (2013)

This work has focused on the prolactin receptor as a target because 1) elevated serum prolactin levels are associated with an increased incidence of breast cancer equivalent to that seen with estrogen; 2) prolactin receptors are expressed at higher levels in cancerous lesions versus normal tissue; 3) high circulating prolactin is correlated with high breast density, itself associated with a higher incidence of breast cancer; 4) prolactin is a survival and growth-promoting factor in breast cancer cells also produced by mammary stromal cells; 5) increased prolactin expression in ductal epithelium leads to development of both estrogen receptor positive and estrogen receptor negative cancers; and 6) knockout of total prolactin receptors markedly slows the development of tumors induced by viral oncogene overexpression.

Up to 95 percent of primary tumors express the prolactin receptor. Given that ~70% express the estrogen receptor, therapies targeting the prolactin receptor could have greater utility than those targeting the estrogen receptor. We have dubbed the ~10kDa molecule designed to change prolactin receptor expression, Antimaia. We have achieved all of our project aims: Analyses using cells in culture confirmed the expected activity on receptor expression in both mouse and human breast cancer cell lines. Treatment with Antimaia reduced cell number and human cells were far more sensitive than the mouse cells. Because tumor stem cells have been implicated in the development of metastases, and metastases are the cause of death from the disease, we examined the effect of Antimaia on tumor-initiating cells in the population. Antimaia caused a slow, but progressive loss of tumor-initiating cells in the population, as judged by both stem cell-specific surface markers and the ability of the treated cells to form mammospheres. In order to test Antimaia in a mouse model with an intact immune system, we began our in vivo studies using mouse breast cancer cells. These cells are very aggressive and metastatic, and recapitulate the human disease in terms of sites of metastatic spread Analysis after injection of cells into the mammary fat pad showed that Antimaia treatment (2.4 nmoles /mouse/24h) reduced the number of proliferating cells, resulted in massive central tumor death (3 fold versus control), and reduced metastatic spread to the lungs by 75%, with no metastases at all in 3 of 8 animals. In addition, analysis of immune cells extracted from livers of control-treated and Antimaia-treated animals showed that treatment of the mice with Antimaia stimulated a tumor-specific immune response. Using the Her2-overexpressing human cell line in immune deficient mice and the same dose of Antimaia for 25 days, lung metastases were again reduced by 75%. Analysis of liver enzymes and immune cells showed no evidence of toxicity up to 5 weeks of treatment.

These results suggest that Antimaia treatment has the potential to clear residual tumor cells from the body, thereby effecting a true cure. This possibility will be directly tested if a new grant application is successful. This application will be to continue in vivo analyses to determine disease-free survival after longer treatments, and to perform rigorous preclinical testing to assess the possibility of moving to phase I clinical trials.

Publications:
Blockade of estrogen-stimulated proliferation by a constitutively-active prolactin receptor having lower expression in invasive ductal carcinoma
Prolactin inhibits a major tumor-suppressive function of wild type BRCA1



Blockade of estrogen-stimulated proliferation by a constitutively-active prolactin receptor having lower expression in invasive ductal carcinoma
Periodical:Cancer Letters
Index Medicus: Cancer Lett
Authors: Kuang-Tzu Huang, Dunyong Tan, Kuan-Hui E. Chen, Ameae M. Walker
Yr: 2015 Vol: 358 Nbr: 2 Abs: Pg:152-160