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In this study, we further investigated the mechanisms by which pseudophosphorylated prolactin (S179D PRL) inhibits the growth of human prostate cancer cells. When treated with S179D PRL for 3 days, LnCAP cells responded by increasing expression of the vitamin D receptor (VDR) and the cell cycle regulatory molecule, p21, whereas PC3 and DU145 cells did not. After 5 days of treatment, both PC3 and DU145 cells responded. Untreated LnCAP cells express the short 1b form (SF1b) of the human prolactin receptor, but DU145 and PC3 cells express only low amounts of this receptor until elevated by treatment with S179D PRL. DU145 and PC3 cells become sensitive to the negative effects of S179D PRL on cell number after induction of the SF1b. Transfection of either SF1b or SF1a into PC3 or DU145 cells made them sensitive to S179D PRL in the 3-day time frame, a finding that was not duplicated by transfection with the long form of the receptor. Treatment of LnCAP cells with S179D PRL increased long-term activation of extracellular signal-regulated kinase 1/2 (ERK1/2). This did not occur in PC3 and DU145 cells until transfection with SF1a/SF1b. Blockade of ERK signaling eliminated S179D PRL-stimulated expression of the VDR and p21 in LnCAP cells and transfected PC3 and DU145 cells. We conclude that initiation of alternative splicing to produce SF1b, and subsequent altered signaling, contribute to the growth inhibitory mechanisms of S179D PRL. This is the first indication of a role for short prolactin receptors in the regulation of cell proliferation.

The clinical relevance and prognostic significance of tumor-infiltrating Tregs in different types of cancers makes them appealing therapeutic targets. However, targeting all Tregs initiates autoimmune disease. In the current study, we have found that knockdown of the long form of the prolactin receptor (LFPRLR) inhibits the function of tumor Tregs with no functional change in peripheral Tregs. Knockdown of the LFPRLR was by splice modulating oligomer (SMO), tested in the syngeneic 4T1-Balb/c immune-competent breast cancer model. Similar effects would be predicted to occur in prostate and ovarian cancer. Knockdown reduced tumor output of CCL17, a chemokine regulated by the major LFPRLR signaling Jak2/Stat5 pathway. Reduced CCL17 output in turn resulted in impaired Treg recruitment into tumors, allowing enhanced CD4+ and CD8+ T cell activities within the tumor and reducing metastasis to distal organs. Also observed was a reduction in Tregs at metastatic sites. RNAseq transcriptomic analysis of LFPRLR SMO-treated tumor Tregs revealed that among immune-suppressive cytokines, expression of IL-10 and TGF-β was not affected, but EBI3, a component of IL-35, became undetectable. Of cytokines known to promote metastasis, IL-6, IL23a and IL34 were reduced. When tumor Tregs, isolated from LFPRLR SMO-treated animals, were co-cultured with 4T1 breast cancer cells, they failed to drive epithelial to mesenchymal transition, while tumor Tregs isolated from control SMO-treated animals did transform 4T1 cells into a more mesenchymal phenotype. Although tumor T regs were reduced by treatment, other major components of the tumor microenvironment, including B cells, CD8+ T cells, macrophages, NK T cells, dendritic cells, myeloid-derived suppressor cells, and fibroblasts were not changed by LFPRLR knockdown, while effector CD4+ T cells were increased and tumor endothelial cells were decreased. Although the population of CD8+ T cells was not changed, their activity was enhanced, as determined by their production of IL-2 and IFN-γ when stimulated. Examination of splenic Tregs from animals treated with LFPRLR- or control SMO for 28 days showed normal suppressive activity of anti-CD3-stimulated T cell proliferation. In conclusion, systemic LFPRLR knockdown has direct and indirect effects on tumor Tregs that modify their cytokine profile and reduce their suppressive capability and ability to drive epithelial to mesenchymal transition of tumor cells. Citation Format: Kuan-Hui E. Chen, Ameae Walker. Phenotypic change specific to tumor-infiltrating regulatory T cells by LFPRLR knockdown [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-211. doi:10.1158/1538-7445.AM2017-LB-211

Tumor infiltration by T regulatory cells (Tregs) occurs in a wide variety of cancers. Although there is evidence that tumor Tregs decrease immune responses to tumor cells and increase metastasis, there is much to be learned about the mechanisms involved. Tregs constitutively express the prolactin receptor (PRLR), suggesting that PRL is important to function. Essentially all Treg PRLR are the long isoform. To investigate the role that PRL might play in tumor Treg recruitment and function, we used the syngeneic 4T1 orthotopic breast cancer model. Mice were treated systemically with a splice modulating oligomer (SMO) that specifically knocks down expression of only the long isoform (LF) of the PRLR. Treatment with this SMO resulted in decreased tumor Tregs (0.65% of gated tumor cells in PRLRLF knockdown compared to 3.31% in control SMO-treated mice), demonstrating that prolactin (PRL) signaling through the PRLRLF directly or indirectly recruited Tregs to this kind of tumor. Further investigation in vitro and in vivo revealed two different ways that PRL assists in recruitment: 1) PRL directly stimulated tumor cell production of CCL17, a chemokine that attracts Tregs. This was inhibited when PRLRLF was knocked down. Furthermore, the PRLRLF antagonist, S179DPRL, inhibited basal CCL17, demonstrating that tumor cell autocrine PRL could stimulate CCL17 output. 2) PRL signaling is required for CCR4 expression on tumor Tregs. CCR4 is normally constitutively expressed on Tregs and is the receptor that responds to CCL17. Analysis of tumor cell chemokine production in response to estrogen or progesterone in vitro showed estrogen induced CCL22, another ligand for Treg CCR4, while progesterone induced CCL17. All three hormones therefore could promote Treg recruitment, but the ability of PRLRLF knockdown, which has little effect on levels of estrogen and progesterone, to inhibit recruitment suggests the effects of the other two hormones are mediated through PRL. PRL treatment of tumor cells in vitro showed a small decrease in mesenchymal markers consistent with reports in other breast cancer cells. Importantly however, in the presence of Tregs, PRL markedly increased expression of twist-1, snail-2, vimentin and fibronectin. Furthermore, if PRLRLF was knocked down in Tregs, there was no increase in mesenchymal markers in the tumor cells in response to PRL. Cancer stem cells are considered important players in metastatic spread. Analysis of isolated stem cells showed the same PRL and Treg interactions as for bulk tumor cells in terms of mesenchymal markers. Tregs with PRLRLF knockdown showed no lack of suppressive ability in a splenocyte response to lipopolysaccharide test. Taken together, results show that PRLRLF signaling in Tregs is essential for Treg-mediated EMT, but is dispensable for general immune suppressive ability. These effects contributed to an 80% inhibition of metastatic spread coincident with a marked reduction in inflammatory damage from responses to metastatic cells after 40days of PRLRLF knockdown. Citation Format: KuanHui E. Chen, Mrinal K. Ghosh, Tomohiro Yonezawa, Ameae M. Walker. Prolactin drives the recruitment of tumor Tregs and Treg-mediated metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-255. doi:10.1158/1538-7445.AM2014-LB-255

Traditional testing of candidate therapeutics has examined the ability of drugs to shrink pre-formed, primary xenografts in immunocompromised animals. While shrinkage of primary tumors can be crucial for inoperable tumors, the vast majority of primary tumors are surgically excised. Moreover, patient death results from cells that escaped the primary tumor. In new environments, these metastatic escapees may respond entirely differently to the drug, especially in the presence of an intact immune system. To test a novel anti-breast cancer drug, we have therefore used a highly metastatic, syngeneic model (4T1) that develops in an immunologically and endocrinologically normal animal. The drug, for brevity named Antimaia, is ∼10kDa, readily synthesized, and designed to inhibit expression of the long form (LF) of the prolactin receptor (PRLR). Studies in vivo demonstrated inhibited expression of LF PRLR without any effect on expression of the short forms of the PRLR or the growth hormone receptor in normal liver and ovary and the primary tumor. Treatment of 4T1 cells in vitro inhibited the ability of cells within the population to form mammospheres (100/well in controls and 0/well in Antimaia-treated, when defining a mammosphere as a colony >50μm) indicative of an effect on stem-like cells within the population. This anti-stem cell effect was duplicated when stem cells were identified on the basis of cell markers (Lin-CD29hiCD24+ AldH+), but stem cell elimination was still ongoing at 15 days. With the 4T1 model, orthotopic injection of 1x106 cells produces multiple, grossly-visible metastases in the lung and liver by 14 days. Antimaia treatment (100 pmoles/h/mouse via Alzet minipump) at the time of orthotopic injection reduced both gross and microscopic metastases without effect on primary tumor weight. However, histological examination of the primary tumors nevertheless showed only a rim of viable cells in the Antimaia-treated group. Morphometric analysis quantified the area of central tumor death as 3 fold that seen in the two control groups. Since in vitro stem cell death was still ongoing at 15 days, the next goal was to examine the effect of longer term treatment in vivo. To do this, it was necessary to initiate primary tumors with fewer cells to be compliant with standards of animal care. When 1x 103 cells were orthotopically injected and treatment continued for 29 days, 4 of 6 primary tumors in the Antimaia group (and 0 of 6 in the control group) showed a flattened growth curve after initial tumor formation. Micrometastasis histological analyses are ongoing, but ex vivo analysis of immune cells from the livers has shown an increase in tumor-specific IFNγ+ CD4+ cells that correlates with effects of treatment on final tumor weight. Analysis of liver enzymes at 14 and 29 days showed no evidence of toxicity. We conclude that Antimaia holds great promise as a breast cancer therapeutic. CBCRP 171B-0053. Citation Format: Tomohiro Yonezawa, KaunHui E. Chen, David Duong, Lisa Ma, Mrinal Ghosh, Mitsumori Kawaminami, Ameae M. Walker. Anticancer effects of antimaia in a highly metastatic, orthotopic, immunologically competent model of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2444. doi:10.1158/1538-7445.AM2013-2444

The literature supports a role for prolactin (PRL) in the development of some cancers through PRL's promotion of the cell cycle and/or inhibition of apoptosis. The selective PRL receptor modulator, S179D PRL, on the other hand, inhibits cancer cell proliferation and promotes differentiation and/or apoptosis. BRCA1 is an important tumor suppressor, shown to play a role in breast (B), ovarian (O), and prostate (P) cancer. The purpose of the current study was to determine the effect of the functionally distinct ligands for the PRL receptor on aspects of BRCA1 biology. Contrary to expectation, both PRL and S179D PRL (at 500 ng/ml) elevated levels of BRCA1 protein (∼3 fold in 24h and up to 10 fold in 48h) in all cancer types examined (B, O, &P). The promoter for the cell cycle inhibitory molecule, p21, has a BRCA1 binding site, but only S179D PRL increased levels of p21 protein as a result of BRCA1 elevation. Using deletion analysis of a p21 promoter-luciferase construct, we confirmed that the BRCA1 binding site (−84 to −129) was crucial to p21 expression in response to S179D PRL. An important question therefore was how elevated BRCA1 in response to each ligand had a different end result in terms of p21 expression. Examination of nuclear translocation of BRCA1 in response to cell stimulation with each ligand showed that both promoted movement into the nucleus. Moreover, both ligands similarly increased BRCA1 interaction with the important region (−84 to −129) of the endogenous promoter of the p21 gene (ChIP assay). A major difference in the signaling resulting from receptor engagement by PRL versus S179D PRL is that only PRL activates Stat5. With PRL, but not S179D PRL, BRCA1 formed a complex with phospho-Stat5 and this complex was present in the nucleus. Since both ligands increase BRCA1 interaction with the p21 promoter, but only PRL results in a BRCA1-phospho-Stat5 complex, it appears that binding of phospho-Stat5 to BRCA1, while not inhibiting its ability to bind to the promoter, negates the ability of BRCA1 to transactivate the p21 promoter. For already synthesized p21, additional studies demonstrated that stimulation of cells with PRL, but not S179D PRL, resulted in phosphorylation of p21, a posttranslational modification that inhibits nuclear translocation and therefore the ability of p21 to inhibit the cell cycle. This study has uncovered two ways in which p21 function is hampered by PRL and hence additional ways in which PRL promotes cell proliferation. At the same time, since S179D PRL inhibits Stat5 activation in response to PRL in addition to itself signaling to p21 elevation, this work also furthers our understanding of the anti-cancer activities of this selective PRL receptor modulator. Supported by California Breast Cancer Research program # 10PB-0127 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 700.