Targeting cholesterol-rich microdomains to circumvent tamoxifen-resistant breast cancer

Adjuvant treatment with tamoxifen substantially improves survival of women with estrogen-receptor positive (ER+) tumors. Tamoxifen resistance (TAMR) limits clinical benefit. RRR alpha tocopherol ether-linked acetic acid analogue (alpha-TEA) is a small bioactive lipid with potent anticancer activity. We evaluated the ability of alpha-TEA in the presence of tamoxifen to circumvent TAMR in human breast cancer cell lines. Methods: Two genotypically matched sets of TAM-sensitive (TAMS) and TAM-resistant (TAMR) human breast cancer cell lines were assessed for signal-transduction events with Western blotting, apoptosis induction with Annexin V-FITC/PI assays, and characterization of cholesterol-rich microdomains with fluorescence staining. Critical involvement of selected mediators was determined by using RNA interference and chemical inhibitors. Results: Growth-factor receptors (total and phosphorylated forms of HER-1 and HER-2), their downstream prosurvival mediators pAkt, pmTOR, and pERK1/2, phosphorylated form of estrogen receptor-alpha (pER-alpha at Ser-167 and Ser-118, and cholesterol-rich lipid microdomains were highly amplified in TAMR cell lines and enhanced by treatment with TAM. alpha-TEA disrupted cholesterol-rich microdomains, acted cooperatively with TAM to reduce prosurvival mediators, and induced DR5-mediated mitochondria-dependent apoptosis via an endoplasmic reticulum stress-triggered pro-death pJNK/CHOP/DR5 amplification loop. Furthermore, methyl-beta-cyclodextrin (M beta CD), a chemical disruptor of cholesterol rich microdomains, acted cooperatively with TAM to reduce prosurvival mediators and to induce apoptosis. Conclusions: Data for the first time document that targeting cholesterol-rich lipid microdomains is a potential strategy to circumvent TAMR, and the combination of alpha-TEA + TAM can circumvent TAMR by suppression of prosurvival signaling via disruption of cholesterol-rich lipid microdomains and activation of apoptotic pathways via induction of endoplasmic reticulum stress.Clayton Foundation for ResearchCenter for Molecular and Cellular Toxicology at the University of TexasNIEHS/NIH T32 ES07247Nutritional Science

PDK-1/AKT pathway as a novel therapeutic target in rhabdomyosarcoma cells using OSU-03012 compound

Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma including two major subtypes, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). Increasing evidence suggests that oncogenesis of RMS involves multistages of signalling protein dysregulation which may include prolonged activation of serine/threonine kinases such as phosphoinositide-dependant kinase-1 (PDK-1) and AKT. To date, whether PDK-1/AKT pathway is activated in RMS is unknown. This study was to examine phosphorylation status of AKT and to evaluate a novel small molecular inhibitor, OSU-03012 targeting PDK-1 in RMS. We examined phosphorylation levels of AKT using ARMS and ERMS tissue microarray and immunohistochemistry staining. Our results showed phospho-AKTThr308 level is elevated 42 and 35% in ARMS and ERMS, respectively. Phospho-AKTSer473 level is also increased 43% in ARMS and 55% in ERMS. Furthermore, we showed that OSU-03012 inhibits cell viability and induces apoptosis in ARMS and ERMS cell lines (RH30, SMS-CTR), which express elevated phospho-AKT levels. Normal cells are much less sensitive to OSU-03012 and in which no detectable apoptosis was observed. This study showed, for the first time, that PDK-1/AKT pathway is activated in RMS and may play an important role in survival of RMS. PDK-1/AKT pathway may be an attractive therapeutic target for cancer intervention in RMS using OSU-03012

Dual EGFR and mTOR targeting in squamous cell carcinoma models, and development of early markers of efficacy

The epidermal growth factor receptor (EGFR) is a validated target in squamous cell carcinoma (SCC) of the head and neck. Most patients, however, do not respond or develop resistance to this agent. Mammalian target of rapamycin (mTOR) is involved in the pathogenesis of SCC of the head and neck (SCCHN). This study aimed to determine if targeting mTOR in combination with EGFR is effective in SCC, and to develop early pharmacodynamic markers of efficacy. Two SCC cell lines, one resistant (HEP2) and one of intermediate susceptibility (Detroit 562) to EGFR inhibitors, were xenografted in vivo and treated with an mTOR inhibitor (temsirolimus), an EGFR inhibitor (erlotinib) or a combination of both. Temsirolimus exerted superior growth arrest in both cell lines than erlotinib. The combined treatment resulted in synergistic antitumor effects in the Detroit 562 cell line. Immunohistochemical assessment of pharmacodynamic effects in fine-needle aspiration (FNA) biopsies early after treatment using phospho MAPK, Phospho-P70 and Ki67 as end points demonstrated pathway abrogation in the Detroit 562 tumours treated with the combination, the only group where regressions were seen. In conclusion, an mTOR inhibitor showed antitumor activity in EGFR-resistant SCC cell lines. Marked antitumor effects were associated with dual pathway inhibition, which were detected by early FNA biopsies

Constitutively Nuclear FOXO3a Localization Predicts Poor Survival and Promotes Akt Phosphorylation in Breast Cancer

Background: The PI3K-Akt signal pathway plays a key role in tumorigenesis and the development of drug-resistance. Cytotoxic chemotherapy resistance is linked to limited therapeutic options and poor prognosis. Methodology/Principal Findings: Examination of FOXO3a and phosphorylated-Akt (P-Akt) expression in breast cancer tissue microarrays showed nuclear FOXO3a was associated with lymph node positivity (p = 0.052), poor prognosis (p = 0.014), and P-Akt expression in invasive ductal carcinoma. Using tamoxifen and doxorubicin-sensitive and -resistant breast cancer cell lines as models, we found that doxorubicin- but not tamoxifen-resistance is associated with nuclear accumulation of FOXO3a, consistent with the finding that sustained nuclear FOXO3a is associated with poor prognosis. We also established that doxorubicin treatment induces proliferation arrest and FOXO3a nuclear relocation in sensitive breast cancer cells. Induction of FOXO3a activity in doxorubicin-sensitive MCF-7 cells was sufficient to promote Akt phosphorylation and arrest cell proliferation. Conversely, knockdown of endogenous FOXO3a expression reduced PI3K/Akt activity. Using MDA-MB-231 cells, in which FOXO3a activity can be induced by 4-hydroxytamoxifen, we showed that FOXO3a induction up-regulates PI3K-Akt activity and enhanced doxorubicin resistance. However FOXO3a induction has little effect on cell proliferation, indicating that FOXO3a or its downstream activity is deregulated in the cytotoxic drug resistant breast cancer cells. Thus, our results suggest that sustained FOXO3a activation can enhance hyperactivation of the PI3K/Akt pathway. Conclusions/Significance: Together these data suggest that lymph node metastasis and poor survival in invasive ductal breast carcinoma are linked to an uncoupling of the Akt-FOXO3a signaling axis. In these breast cancers activated Akt fails to inactivate and re-localize FOXO3a to the cytoplasm, and nuclear-targeted FOXO3a does not induce cell death or cell cycle arrest. As such, sustained nuclear FOXO3a expression in breast cancer may culminate in cancer progression and the development of an aggressive phenotype similar to that observed in cytotoxic chemotherapy resistant breast cancer cell models. © 2010 Chen et al.published_or_final_versio

Key signalling nodes in mammary gland development and cancer. Signalling downstream of PI3 kinase in mammary epithelium: a play in 3 Akts

The protein serine/threonine kinase Akt, also known as protein kinase B (PKB), is arguably the most important signalling nexus in the cell. Akt integrates a plethora of extracellular signals to generate diverse outcomes, including proliferation, motility, growth, glucose homeostasis, survival, and cell death. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is the second most frequently mutated pathway in cancer, after p53, and mutations in components of this pathway are found in around 70% of breast cancers. Thus, understanding how Akt relays input signals to downstream effectors is critically important for the design of therapeutic strategies to combat breast cancer. In this review, we will discuss the various signals upstream of Akt that impact on its activity, how Akt integrates these signals and modulates the activity of downstream targets to control mammary gland development, and how mutations in components of the pathway result in breast cancer

Loss of PTEN function may account for reduced proliferation pathway sensitivity to LY294002 in human prostate and bladder cancer cells.

PURPOSE: Inhibition of phosphoinositide 3 (PI3)-kinase pathway is attractive for cancer treatment. To examine the role of the phosphatase and tensin homolog (PTEN) in the development of resistance to the treatment. METHODS: We cultured human prostate cancer cells (DU145 and PC-3 cells) and bladder cancer cells (EJ-1 and UM-UC-3 cells) with a PI3-kinase inhibitor, LY294002 for more than 6 weeks and cell proliferation was studied. Activation of Akt1 and ERK was examined by immunoblotting. We introduced the wild type PTEN in UM-UC-3 cells and their proliferation along with the signaling pathways was also examined. RESULTS: After 6 weeks, proliferation pathway sensitivity to LY294002 was reduced in cells expressing PTEN, but not in PTEN-null cells. PD98059, a MAPK/ERK kinase inhibitor, significantly inhibited proliferation of PTEN-expressing cells, but not PTEN-null cells. Stable PTEN expression in PTEN-null UM-UC-3 cells increased serum-induced ERK activation and sensitivity to PD98059-treatment, and reduced sensitivity to LY294002 after 6 weeks of exposure. CONCLUSIONS: Loss of PTEN function may protect against resistance to PI3-kinase inhibitors through an addiction to the PI3-kinase/Akt pathway

Targeting tumorigenesis: development and use of mTOR inhibitors in cancer therapy

The mammalian target of rapamycin (mTOR) is an intracellular serine/threonine protein kinase positioned at a central point in a variety of cellular signaling cascades. The established involvement of mTOR activity in the cellular processes that contribute to the development and progression of cancer has identified mTOR as a major link in tumorigenesis. Consequently, inhibitors of mTOR, including temsirolimus, everolimus, and ridaforolimus (formerly deforolimus) have been developed and assessed for their safety and efficacy in patients with cancer. Temsirolimus is an intravenously administered agent approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) for the treatment of advanced renal cell carcinoma (RCC). Everolimus is an oral agent that has recently obtained US FDA and EMEA approval for the treatment of advanced RCC after failure of treatment with sunitinib or sorafenib. Ridaforolimus is not yet approved for any indication. The use of mTOR inhibitors, either alone or in combination with other anticancer agents, has the potential to provide anticancer activity in numerous tumor types. Cancer types in which these agents are under evaluation include neuroendocrine tumors, breast cancer, leukemia, lymphoma, hepatocellular carcinoma, gastric cancer, pancreatic cancer, sarcoma, endometrial cancer, and non-small-cell lung cancer. The results of ongoing clinical trials with mTOR inhibitors, as single agents and in combination regimens, will better define their activity in cancer

Inhibitory effect of HGF on invasiveness of aggressive MDA-MB231 breast carcinoma cells, and role of HDACs

Hepatocyte growth factor (HGF), through Met receptor binding, fulfils numerous functions in invasive tumour growth (survival/proliferation, motility, apoptosis), but epigenetic control of gene expression in this process is poorly understood. In HGF-treated breast cancer cells we studied (a) the chemoinvasion towards CXCL12 (ligand of the chemokine-receptor CXCR4) and (b) the mechanistic basis, that is, the transduction pathways that regulate CXCR4-mediated invasion, and the role played by histone deacetylases (HDACs) after blockade with trichostatin A (TSA). In highly invasive and metastatic MDA-MB231 cells HGF had a dual inhibitory effect, reducing spontaneous migration and specific chemoinvasion towards CXCL12, the latter by decreasing CXCR4 transactivation and protein level. After HGF the levels of phosphorylated (therefore active) c-Src and Akt persistently increased, indicating a role of these signal transducers in the HGF-dependent cellular and molecular effects. c-Src wild-type expression vector (Srcwt) increased active c-Src and mimicked the HGF-dependent inhibition of CXCR4 transactivation. Our findings indicate that HDACs participated in the HGF-inhibitory effects. In fact, blockade of HDACs hindered the HGF- and Srcwt-dependent reductions of CXCR4 transactivation and invasiveness, while inhibition of endogenous c-Src was additive with HGF, further reducing specific chemoinvasion. In conclusion, in MDA-MB231 cells HDAC blockade with TSA partly counteracted the HGF-dependent effects through molecular events that included enhancement of the expression of the genes for invasiveness Met and CXCR4 (depending on serum conditions), reduction of endogenous phospho-c-Src/c-Src and phosphoAkt/Akt ratios and triggering of apoptosis. The potential therapeutic use of TSA should take into account the variable aggressiveness of breast carcinoma cells and microenvironment signals such as HGF at the secondary growth site of the tumour. It was interesting that HGF reduced motility and CXCR4 functionality only of MDA-MB231 cells, and not of low-invasive MCF-7 cells, suggesting a mechanism implicated in metastatic cell homing