PI3K/mTOR inhibition can impair tumor invasion and metastasis in vivo despite a lack of antiproliferative action in vitro: implications for targeted therapy

Oncogenic PI3K/mTOR activation is frequently observed in human cancers and activates cell motility via p27 phosphorylations at T157 and T198. Here we explored the potential for a novel PI3K/mTOR inhibitor to inhibit tumor invasion and metastasis. An MDA-MB-231 breast cancer line variant, MDA-MB-231-1833, with high metastatic bone tropism, was treated with a novel catalytic PI3K/mTOR inhibitor, PF-04691502, at nM doses that did not impair proliferation. Effects on tumor cell motility, invasion, p27 phosphorylation, localization, and bone metastatic outgrowth were assayed. MDA-MB-231-1833 showed increased PI3K/mTOR activation, high levels of cytoplasmic p27pT157pT198 and increased cell motility and invasion in vitro versus parental. PF-04691502 treatment, at a dose that did not affect proliferation, reduced total and cytoplasmic p27, decreased p27pT157pT198 and restored cell motility and invasion to levels seen in MDA-MB-231. p27 knockdown in MDA-MB-231-1833 phenocopied PI3K/mTOR inhibition, whilst overexpression of the phosphomimetic mutant p27T157DT198D caused resistance to the anti-invasive effects of PF-04691502. Pre-treatment of MDA-MB-231-1833 with PF-04691502 significantly impaired metastatic tumor formation in vivo, despite lack of antiproliferative effects in culture and little effect on primary orthotopic tumor growth. A further link between cytoplasmic p27 and metastasis was provided by a study of primary human breast cancers which showed cytoplasmic p27 is associated with increased lymph nodal metastasis and reduced survival. Novel PI3K/mTOR inhibitors may oppose tumor metastasis independent of their growth inhibitory effects, providing a rationale for clinical investigation of PI3K/mTOR inhibitors in settings to prevent micrometastasis. In primary human breast cancers, cytoplasmic p27 is associated with worse outcomes and increased nodal metastasis, and may prove useful as a marker of both PI3K/mTOR activation and PI3K/mTOR inhibitor efficacy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10549-012-2389-6) contains supplementary material, which is available to authorized users

Exploring the Oncogenic Role of p27 in Regulating Tumor Progression

p27 is a cell cycle regulator and an atypical tumor suppressor. However, p27 gene deletions or mutations are rarely observed in human cancers. Instead, p27 is frequently degraded or mislocalized to the cytoplasm in aggressive human cancers. Phosphorylation at T157 or T198 by different PI3K effector kinases leads to p27 cytoplasmic accumulation. However, the functional contribution of phosphorylated and cytoplasmic p27 to cancer progression remains unknown. By introducing a cell cycle defective (CK-) and double phosphomimetic p27 mutant (T157D/T198D or DD), we found that phosphorylation of p27 at T157 or T198 by PI3K/mTOR directly regulated tumor cell migration and invasion. Targeted inhibition of PI3K/mTOR impaired tumor cell motility and metastasis via modulation of p27. Furthermore, we uncovered a novel oncogenic function of p27 in regulating tumor progression. p27CK-DD induced epithelial-mesenchymal transition (EMT) and transformation of human mammary epithelial cells and enhanced the mesenchymal characteristics and metastatic potential of cancer cell lines. Knockdown of p27 in mesenchymal cell lines with enriched p27T157/T198 reverted EMT and impaired metastatic potential. Mechanistically, p27CK-DD activated STAT3 and facilitated its transactivation of TWIST1 to induce EMT. Pharmacological inhibition of STAT3 or dominant negative STAT3 (STAT3DN) decreased TWIST1 and reversed p27CK-DD-mediated EMT and tumor progression, while constitutive active STAT3 (STAT3CA) rescued EMT phenotype and metastatic potential in p27 knockdown cells. We also identified a potential signaling feedforward loop containing AKT activation, p27 phosphorylation, STAT3 activation and further AKT activation that might contribute to tumor progression. These findings reveal a novel oncogenic function of p27 to regulate tumor progression through EMT that involves STAT3-mediated TWIST1 regulation. Combined inhibition of both AKT and STAT3 in PI3K/mTOR activated, p27pT157/pT198 enriched human cancers may ultimately have therapeutic potential to limit p27-mediated EMT and cancer metastasis.</p

p27: a barometer of signaling deregulation and potential predictor of response to targeted therapies

Phosphorylation of the cyclin-dependent kinase inhibitor p27 by upstream mitogenic signaling pathways regulates its stability, localization, and biological function. In human cancers, loss of the antiproliferative action of p27 can arise through reduced protein levels and/or cytoplasmic mislocalization, leading to increased cell proliferation and/or cell migration, respectively. Reduced p27 expression levels and p27 mislocalization have potential prognostic and therapeutic implications in various types of human cancers. This review highlights mechanisms of functional deregulation of p27 by oncogenic signaling that provide an important molecular rationale for pathway targeting in cancer treatment

Molecularly targeted therapies for p53-mutant cancers

The tumor suppressor p53 is lost or mutated in approximately half of human cancers. Mutant p53 not only loses its anti-tumor transcriptional activity, but also often acquires oncogenic functions to promote tumor proliferation, invasion, and drug resistance. Traditional strategies have been taken to directly target p53 mutants through identifying small molecular compounds to deplete mutant p53, or to restore its tumor suppressive function. Accumulating evidence suggest that cancer cells with mutated p53 often exhibit specific functional dependencies on secondary genes or pathways to survive, providing alternative targets to indirectly treat p53-mutant cancers. Targeting these genes or pathways, critical for survival in the presence of p53 mutations, holds great promise for cancer treatment. In addition, mutant p53 often exhibits novel gain-of-functions to promote tumor growth and metastasis. Here, we review and discuss strategies targeting mutant p53, with focus on targeting the mutant p53 protein directly, and on the progress of identifying genes and pathways required in p53-mutant cells

Interleukin-21 Potently Induces Direct and Indirect Cytotoxicity of Mantle Cell Lymphoma

Abstract Mantle cell lymphoma (MCL) is a distinct subtype of non-Hodgkin lymphomacharacterized by overexpression of cyclin D1 (CCND1) in 95% of patients due to the t(11;14)(q13;q32) chromosomal translocation. This incurable lymphoma is highly chemoresistant, with short response duration, frequent relapses and eventual death, even with the most aggressive chemotherapeutic regimens. Interleukin 21 (IL-21), a member of the IL-2 cytokine family, possesses potent anti-tumor activity against a variety of cancers not expressing IL-21 receptor (IL-21R) through activation of the immune system. Previously, we established that apart from its immuno-stimulatory effects, IL-21 exerts direct cytotoxicity on IL-21R-expressing diffuse large B cell lymphoma (DLBCL) cells (Sarosiek KA et al.Blood, 2010). Herein we carried out a comprehensive study to delineate the effects of IL-21 on MCL cell lines and primary tumors. Flow-cytometric analysis revealed that all MCL cell lines (Mino, HBL2, Jeko1, G519, IRM2, SP53, Z138, UPN1 and L128) as well as primary tumors expressed surface IL-21R at variable levels. Treatment of Mino, HBL2 and SP53 cells with IL-21 (100ng/mL) led to a marked time-dependent decrease in cell proliferation and increased cell death. In contrast, Jeko1, IRM2, L128, Z138, UPN1 and G519 cells exhibited resistance to IL-21 treatment. Similarly, primary MCL tumors treated with IL-21 in vitro exhibited significant cell death in 4 of 5 cases expressing IL-21R. To decipher the mechanism of IL-21-induced direct cytotoxicity, responsive and resistant cell lines as well as primary tumors were utilized. Similarly to our previous study in DLBCL, IL-21 stimulation resulted in dramatic phosphorylation of STAT1 and STAT3 in IL-21 responsive cell lines (Mino, HBL-2, SP53) and a primary tumor, while minimal STAT5 phosphorylation was observed only in Mino. We have previously demonstrated that IL-21-induced cell death in DLBCL is mediated by STAT3-induced upregulation of c-Myc expression. Correspondingly, IL-21 treatment led to c-Myc upregulation only in IL-21-sensitive MCL cell lines and primary tumors but not in the resistant cell lines and primary tumors, independent of the IL-21R expression levels. Knockdown of c-Myc prevented IL-21-induced Mino cell death, whereas c-Myc overexpression in resistant MCL cell lines facilitated IL-21-induced cytotoxicity. Furthermore, IL-21 resulted in upregulation of the pro-apoptotic protein Bax and downregulation of the anti-apoptotic proteins Bcl-XL and Bcl-2, as previously observed in DLBCL. Knockdown of STAT3 or Bax using specific siRNAs in Mino cells resulted in abrogation of the IL-21-induced cell death. In contrast to a previous report (Gelebart P et al. Leukemia, 2009), knockdown of STAT1 or overexpression of dominant negative STAT1 failed to prevent IL-21-induced Mino cell death. We also discovered that apart from its direct cytotoxic effects, IL-21 also leads to NK-cell dependent lysis of MCL cell lines resistant to direct cytotoxicity. In vivo treatment with IL-21 resulted in complete FC-muMCL1 tumor regression in syngeneic mice (p<0.0001). To understand the contribution of immune system components to the IL-21 induced anti-tumor activity, we carried out cell subset depletion studies. In vivo depletion of NK cells together with CD4+ T cells abrogated IL-21 induced tumor regression of the FC-muMCL1 in xenograft mice, suggesting that both NK and CD4+ cells contribute to the indirect immune mediated cytotoxicity of MCL. Collectively, our data indicate that IL-21 has potent anti-tumor activity against MCL cells via direct (IL-21R mediated) and indirect (immune cell mediated) effects, and should be evaluated in clinic for treatment of patients with MCL. Disclosures No relevant conflicts of interest to declare

Abstract 2333: The atypical tumor suppressor p27 regulates cellular proliferation, invasion, and metastasis via subcellular localization in distinct microenvironments

Abstract While mutational loss of the p27 cell cycle regulatory protein is rare, oncogenic deregulation of p27 via accelerated degradation or mislocalization to the cytoplasm is commonly observed in human tumors. A growing body of evidence indicates that, when mislocalized to the cytoplasm, p27 promotes cell motility via RhoA-ROCK signaling inhibition. The present data explores the role of p27 in the regulation of both orthotopic mammary tumor growth and ectopic pulmonary metastasis in the MDA-MB-231-4175 model of malignant human breast cancer. The MDA-MB-231-4175 cell line (hereafter 4175) shows much greater metastasis to the lungs in murine xenograft models. Relative to the parental MDA-MB-231 cells, the metastatic 4175 derivative demonstrates activation of PI3K/mTOR signaling. These cells have elevated pmTOR, pAKTS473, pSGK1, and pRSK1. This results in increased total p27 and, when equal levels of total p27 are titrated, the highly metastatic 4175 cell line demonstrates extensive phosphorylation of p27 at the T157 and T198 sites. These sites have previously been implicated in the modulation of p27 localization and in the binding of p27 to RhoA. Thus, in the 4175 line, we demonstrate increased cytoplasmic p27, increased p27:RhoA binding, and impaired RhoA-ROCK signaling. This attenuation of RhoA activity correlates with increased migration (as measured by scratch assay) and increased invasion (as measured by modified transwell assay). Importantly, this hypermotility was abrogated by lentiviral knockdown of p27. Knockdown of p27 did not alter in vitro cell cycle profiles of either cell line as assessed by flow cytometry. The enhanced pulmonary tropism of the 4175 cell line was also abrogated in vivo following p27 knockdown: shRNAp27 reverts the lung metastatic phenotype of these cells back toward parental levels following tail vein injection in Balb/c nude female mice as assessed by IVIS imaging. Interestingly, orthotopic injection of these same lines, treated with the same lentivirus, into the mammary fat pad of Balb/c nude female mice produced a dramatically different result: shRNAp27 resulted in a significant increase in parental MDA-MB-231 tumor growth, while the metastatic derivative 4175 cells did not demonstrate a statistically meaningful change in orthotopic growth. These results indicate that, within the same cellular background, and in the same murine species, loss of p27 results in substantially different phenotypes depending upon the microenvironmental context: after tail vein injection cytoplasmic p27 plays a crucial role in promoting extravasation and colonization in the lung tissue; loss of p27 in this context impairs the metastatic process. Conversely, in the orthotopic environment, p27's pro-invasive role is not necessary to establish productive microenvironmental interactions and loss of p27 does not attenuate tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2333. doi:10.1158/1538-7445.AM2011-2333</jats:p

Abstract 3335: Self-renewal, tumorigenicity and metastatic potential of CD44+ cells in ER-negative lines and dissociated primary human breast cancers is increased in CD24+ subsets

Abstract Primary human breast cancer subpopulations with a CD44+ CD24NEG/LOW ESA+ phenotype exhibit self-renewal and generate xenograft tumors with as few as 100 cells in immunodeficient mice. While CD44 expression is strongly associated with self renewal in several cancers, the relative contributions of CD24 negative versus low subpopulations to stemness is poorly defined. Early reports have not distinguished negative from low CD24 expression in human lines and primary tumors, and both, together with CD44+ status have been linked to breast cancer stem cell phenotypes. In contrast, certain reports have associated CD24 expression with tumor progression and metastasis. Here, we show increased mitogenic kinase activation, expression of ESC and EMT genes in the CD24LOW subpopulation of CD44+ cells in both triple negative MDA-MB-231 breast cancer cells and primary dissociated breast cancers (DTs) compared to the CD24NEG cells. Moreover, tumorigenicity was increased and metastasis arose exclusively from orthotopic xeno-implantation of the CD44+CD24LOW cells. MDA-MB-231 and two different primary DTs were live sorted into CD44+CD24NEG and CD44+CD24LOW enriched subpopulations. Cells expressing other putative stem cell markers, ALDH1 and ESA were almost entirely CD44+CD24LOW. CD44+CD24LOWcells showed higher PI3-kinase/Akt, MAPK, and Src activites compared to CD44+ CD24NEG cells. While both subpopulations formed soft agar colonies and/or mammospheres, those arising from CD44+CD24LOW were greater in number and size than from CD44+CD24NEG cells. When cultured in 2D after sorting, CD44+ CD24LOW cells gave rise to both CD44+ CD24LOW and CD44+ CD24NEG progeny while CD44+ CD24NEG yielded only CD44+ CD24NEG progeny. CD44+CD24LOW expressed higher levels of ESC genes and EMT-associated genes than CD44+CD24NEG cells and uniquely expressed stem cell-associated miRNAs. On orthotopic injection into BalbC nude mice, both subpopulations initiated tumors with as few as 100 cells, however tumors arising from CD44+CD24LOW cells had a shorter latency and grew more rapidly than those arising from CD44+CD24NEG cells. In vivo imaging of luciferase positive cells showed CD44+CD24LOW cells yield metastasis while CD44+CD24NEG cells did not. Our data demonstrate, for the first time, distinct biological properties between the CD44+CD24LOW and CD44+CD24NEG enriched subpopulations of MDA-MB-231 and primary human breast cancer DTs. Low surface expression of CD24 is associated with ALDH1 and ESA+ status, increased self-renewal as shown by mammosphere and colony formation, tumorigenicity and metastatic potential of CD44+ cells. The ability to isolate and characterize such TSC-enriched subpopulations will be necessary for development of therapeutic strategies that preferentially target breast cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3335. doi:10.1158/1538-7445.AM2011-3335</jats:p