Calcium/calmodulin-dependent protein kinase kinase 2 regulates hepatic fuel metabolism.

OBJECTIVE: The liver is the primary internal metabolic organ that coordinates whole body energy homeostasis in response to feeding and fasting. Genetic ablation or pharmacological inhibition of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) has been shown to significantly improve hepatic health and peripheral insulin sensitivity upon overnutrition with high fat diet. However, the precise molecular underpinnings that explain this metabolic protection have remained largely undefined. METHODS: To characterize the role of CaMKK2 in hepatic metabolism, we developed and challenged liver-specific CaMKK2 knockout (CaMKK2LKO) mice with high fat diet and performed glucose and insulin tolerance tests to evaluate peripheral insulin sensitivity. We used a combination of RNA-Sequencing, glucose and fatty acid istotopic tracer studies, a newly developed Seahorse assay for measuring the oxidative capacity of purified peroxisomes, and a degenerate peptide libarary to identify putative CaMKK2 substrates that mechanistically explain the protective effects of hepatic CaMKK2 ablation. RESULTS: Consistent with previous findings, we show that hepatic CaMKK2 ablation significantly improves indices of peripheral insulin sensitivity. Mechanistically, we found that CaMKK2 phosphorylates and regulates GAPDH to promote glucose metabolism and PEX3 to blunt peroxisomal fatty acid catabolism in the liver. CONCLUSION: CaMKK2 is a central metabolic fuel sensor in the liver that significantly contributes to whole body systems metabolism.

Differential gene expression in tamoxifen-resistant breast cancer cells revealed by a new analytical model of RNA-Seq data.

Resistance to tamoxifen (Tam), a widely used antagonist of the estrogen receptor (ER), is a common obstacle to successful breast cancer treatment. While adjuvant therapy with Tam has been shown to significantly decrease the rate of disease recurrence and mortality, recurrent disease occurs in one third of patients treated with Tam within 5 years of therapy. A better understanding of gene expression alterations associated with Tam resistance will facilitate circumventing this problem. Using a next generation sequencing approach and a new bioinformatics model, we compared the transcriptomes of Tam-sensitive and Tam-resistant breast cancer cells for identification of genes involved in the development of Tam resistance. We identified differential expression of 1215 mRNA and 513 small RNA transcripts clustered into ERα functions, cell cycle regulation, transcription/translation, and mitochondrial dysfunction. The extent of alterations found at multiple levels of gene regulation highlights the ability of the Tam-resistant cells to modulate global gene expression. Alterations of small nucleolar RNA, oxidative phosphorylation, and proliferation processes in Tam-resistant cells present areas for diagnostic and therapeutic tool development for combating resistance to this anti-estrogen agent.

Mislocalization of cell-cell adhesion complexes in tamoxifen-resistant breast cancer cells with elevated c-Src tyrosine kinase activity.

c-Src activation has been implicated in metastasis of tamoxifen-resistant breast cancer. Here we investigated how c-Src activity affects cell adhesion using a tamoxifen-resistant variant of MCF-7 cells (MTR-3) containing elevated c-Src activity. In MTR-3 cells, adhesion proteins beta-catenin and E-cadherin are mislocalized, forming novel structures perpendicular to cell-cell junctions. c-Src is associated with beta-catenin/E-cadherin complexes and beta-catenin tyrosine phosphorylation is enhanced. Blocking c-Src tyrosine kinase activity decreased beta-catenin tyrosine phosphorylation and restored localization of beta-catenin and E-cadherin at cell-cell junctions. These findings suggest that inhibition of c-Src signaling may prevent metastasis of tamoxifen-resistant breast cancer.

Anti-tumor effect of honokiol alone and in combination with other anti-cancer agents in breast cancer.

Honokiol, an active component isolated and purified from Chinese traditional herb magnolia, was demonstrated to inhibit growth and induce apoptosis of different cancer cell lines such as human leukaemia, colon, and lung cancer cell lines; to attenuate the angiogenic activities of human endothelial cells in vitro; and to efficiently suppress the growth of angiosarcoma in nude mice. In this study, we have demonstrated that treatment of different human breast cancer cell lines with honokiol resulted in a time- and concentration-dependent growth inhibition in both estrogen receptor-positive and -negative breast cancer cell lines, as well as in drug-resistant breast cancer cell lines such as adriamycin-resistant and tamoxifen-resistant cell lines. The inhibition of growth was associated with a G1-phase cell cycle arrest and induction of caspase-dependent apoptosis. The effects of honokiol might be reversely related to the expression level of human epidermal growth receptor 2, (HER-2, also known as erbB2, c-erbB2) since knockdown of her-2 expression by siRNA significantly enhanced the sensitivity of the her-2 over-expressed BT-474 cells to the honokiol-induced apoptosis. Furthermore, inhibition of HER-2 signalling by specific human epidermal growth receptor 1/HER-2 (EGFR/HER-2) kinase inhibitor lapatinib synergistically enhanced the anti-cancer effects of honokiol in her-2 over-expressed breast cancer cells. Finally, we showed that honokiol was able to attenuate the PI3K/Akt/mTOR (Phosphoinositide 3-kinases/Akt/mammalian target of rapamycin) signalling by down-regulation of Akt phosphorylation and upregulation of PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) expression. Combination of honokiol with the mTOR inhibitor rapamycin presented synergistic effects on induction of apoptosis of breast cancer cells. In conclusion, honokiol, either alone or in combination with other therapeutics, could serve as a new, promising approach for breast cancer treatment.

Prevention of age-related spontaneous mammary tumors in outbred rats by late ovariectomy.

BACKGROUND: Breast cancer prevention trials have shown that the antiestrogen tamoxifen inhibits development of estrogen receptor (ER)-positive tumors. In Sprague-Dawley rats, removal of ovarian function in young animals can reduce the incidence of spontaneous age-dependent mammary tumors. However, it is not known whether removal of ovaries late in life, before middle age onset, can still prevent mammary tumor development. METHODS: In this study we used Hsd:Sprague-Dawley SD (Hsd) rats to determine the effect of late ovariectomy on mammary tumor development. Intact, sham-ovariectomized and ovariectomized rats were followed until 110 weeks of age, or over their life span. In some experiments, palpable tumors were surgically removed upon presentation. RESULTS: Removal of ovaries before middle age onset ( approximately 5-7 months) inhibited development of spontaneous mammary tumors by 95%. Only one mammary tumor was observed in 19 late ovariectomized animals while 47 total tumors developed in 42 non-ovariectomized animals. Tumor incidence was reduced from 73.8 to 5.3% (relative risk=0.05, 95% CI=0.0072-0.354). The frequency of mammary carcinomas in non-ovariectomized virgin female rats was one in eight rats. Spontaneous rat carcinomas expressed ER and other biomarkers, such as cyclin D1. When palpable tumors were removed by surgical excision, tumor multiplicity increased from 0.76 to 1.61 tumors per rat. Surprisingly, ovariectomy increased the 110-week survival rate and maximum life span of Hsd rats. CONCLUSION: Late ovariectomy prevents spontaneous mammary tumor development in Hsd rats. This animal model may be useful for evaluating novel interventions in breast cancer prevention.

Estrogen-dependent regulation of Eg5 in breast cancer cells.

HsEg5 (Eg5) is a kinesin required for proper execution of mitosis. Several compounds that specifically block Eg5 are in clinical development and have the potential to be used in the treatment of breast cancer. In this study, we investigated the interaction between Eg5 and estrogen receptor signaling. We observed decreased Eg5 expression after treatment of estrogen receptor-positive human breast cancer MCF-7 cells with the estrogen receptor downregulator fulvestrant. Downregulation of Eg5 expression in response to fulvestrant was also observed in another estrogen receptor-positive cell line ZR-75, but not in the estrogen receptor-negative breast cancer cell line MDA-231. Moreover, in MCF-7 cells previously arrested in the G0/G1 phase of the cell cycle by fulvestrant, addition of estrogen increased Eg5 expression. This upregulation correlated with progression through S-phase. Nevertheless, the effect of fulvestrant in Eg5 expression could not be explained solely by cell cycle arrest, because treatments that blocked cell cycle progression did not consistently decrease Eg5 expression. Pharmacological inhibition of Eg5 function, with either S-trityl-L-cysteine or monastrol, prevented growth of estrogen-treated MCF-7 cells with an IC50 of 0.46 and 29.71 micromol/l, respectively. Simultaneous inhibition of estrogen receptor function with fulvestrant increased the IC50 for S-trityl-L-cysteine to 2.30 micromol/l and for monastrol to 112.69 micromol/l. Our results suggest that pharmacological inhibition of Eg5 may be an effective treatment for estrogen receptor-positive breast cancer, even without concomitant hormonal therapy.

Estrogen promotes reversible epithelial-to-mesenchymal-like transition and collective motility in MCF-7 breast cancer cells.

The role of estrogen in the motility and invasion of breast cancer cells is controversial. Although estrogen receptor (ER)-positive breast tumors are considered less aggressive and more differentiated they still undergo metastasis. In many types of epithelial cancers, the ability to undergo metastasis has been associated with a loss of epithelial features and acquisition of mesenchymal properties leading to migration of individual cells, a process known as epithelial-to-mesenchymal transition (EMT). In this report, we show that a subset of ER-positive breast cancer cells can acquire mesenchymal-like features and motility in a reversible manner. In MCF-7 breast cancer cells estrogen-promoted acquisition of mesenchymal-like features while antiestrogens, such as tamoxifen, prevented this transition. Moreover, pharmacological inhibition of Src family kinases decreased the ability of estrogen to promote epithelial-to-mesenchymal-like transition. In addition to mesenchymal-like motility, a subset of estrogen-treated cells also moved as cell clusters (collective motility). While membrane localization of E-cadherin/beta-catenin was decreased in fibroblast-like cells, enhanced levels of E-cadherin/beta-catenin were detected in motile cell clusters. Thus, during tumor progression, estrogen may foster motility and invasion of ER-positive breast cancer by promoting simultaneously reversible EMT-like changes and collective motility. These studies suggest that antiestrogen therapy and Src family kinase inhibitors may decrease development of metastases in ER-positive breast cancer by blocking estrogen-dependent migration of human breast cancer cells.

Synergistic inhibition of breast cancer cell lines with a dual inhibitor of EGFR-HER-2/neu and a Bcl-2 inhibitor.

The epidermal growth factor receptor (EGFR) (ErbB1) and HER-2/neu (ErbB2) are members of the ErbB family of receptor tyrosine kinases. These receptors are overexpressed in a variety of human tumors and overexpression generally correlates with poor prognosis and decreased survival. Lapatinib, a reversible inhibitor of both EGFR and HER-2/neu, has shown some success in achieving clinical responses in heavily pretreated advanced cancer patients. GW2974 is a reversible dual inhibitor similar to lapatinib, but GW2974 was not progressed to clinical trials due to pharmacokinetic issues. Bcl-2, an anti-apoptotic protein, is also overexpressed in a number of human tumors. Bcl-2 inhibitors induce apoptosis and sensitize cancer cells to other therapies. The purpose of this study was to assess the effects of combining ErbB and Bcl-2 inhibitors on the growth of human breast cancer cell lines. EGFR/HER-2/neu tyrosine kinase inhibitors (lapatinib and GW2974) were combined with Bcl-2 inhibitors (HA14-1 or GX15-070) and the anti-proliferative effects were determined by the MTT tetrazolium dye assay. Combinations were tested in MCF-7 human breast cancer cells, a HER-2/neu transfected MCF-7 cell line (MCF/18), and a tamoxifen-resistant MCF-7 cell line (MTR-3). A synergistic inhibitory effect was observed with the combination of inhibitors of EGFR-HER-2/neu (lapatinib or GW2974) and Bcl-2 (GX15-070 or HA14-1) on the growth of the MCF-7, MCF/18, and MTR-3 human breast cancer cell lines. This study suggests that simultaneously blocking the ErbB family of receptor tyrosine kinases and Bcl-2 family of proteins may be a benefit to breast cancer patients.

Overexpression of c-Myc and Bcl-2 during progression and distant metastasis of hormone-treated breast cancer.

The aim of this study was to identify molecules involved in the proliferation and survival of recurrent estrogen receptor (ER)-positive breast cancer at the site of metastasis. Most studies of biomarkers are done using the initial primary breast tumor whereas pathological studies of breast cancer lesions after distant recurrence are scarce. Here we evaluated the expression of the oncogenes c-Myc and Bcl-2, mediators of estrogen-dependent proliferation and survival, during breast cancer progression and relapse after adjuvant hormonal therapy. Using a preclinical model of tamoxifen-resistant growth, we found overexpression of c-Myc in all (3/3) and of Bcl-2 in most (2/3) tamoxifen resistant-breast cancer variants. To determine whether c-Myc and Bcl-2 are expressed during breast cancer progression in the clinics we identified breast cancer patients who had received adjuvant hormonal therapy for the treatment of their localized disease and had later experienced relapse. From 583 patients who had received adjuvant hormonal therapy a total of 82 experienced recurrence. Nevertheless, only 22 patients had had a biopsy of their metastatic lesion done after relapse. Twenty-one biopsies were useful for this biomarker study. These biopsies were obtained mostly (20) from breast cancer patients who had received tamoxifen as their adjuvant hormonal therapy. One patient had received an aromatase inhibitor instead. Our results showed that almost all (20) metastatic recurrences expressed ER. Expression of c-Myc was observed in 18 out of 19 metastatic lesions scored while expression of Bcl-2 was detected in 17 out of 21 metastatic tumors. A correlation between ER expression and Bcl-2, but not with c-Myc, was found in these recurrent metastatic lesions. In addition, c-Myc expression was correlated with the nuclear grade of the metastatic lesion. Thus, the frequent expression of c-Myc and Bcl-2 in metastatic breast cancer recurrences suggests that combining hormonal therapy with strategies to block c-Myc and Bcl-2 may prevent growth of ER-positive breast cancer at the site of metastasis.

Targeting c-Src kinase enhances tamoxifen's inhibitory effect on cell growth by modulating expression of cell cycle and survival proteins.

PURPOSE: Many studies have implicated the non-receptor tyrosine kinase c-Src in the development and metastatic progression of many types of cancer. In breast cancer, c-Src has been proposed to mediate the actions of estrogen in cell cycle progression. METHODS: In this study we investigated the interaction between c-Src inhibition and estrogen receptor (ER) function using the ER-positive and tamoxifen-sensitive MCF-7 breast cancer cells. RESULTS: Pharmacological inhibition of c-Src blocked estrogen-dependent proliferation in MCF-7 cells and enhanced the inhibitory effects of tamoxifen or estrogen-deprivation on cell growth. Maximum inhibition (95%) of cell growth was obtained when tamoxifen and c-Src blockade were combined. Inhibition of c-Src kinase decreased levels of the ER targets c-Myc and cyclin D1 expression but not of Bcl-2. Nevertheless, blocking c-Src kinase in tamoxifen-treated MCF-7 cells led to apoptosis. Inhibition of c-Src kinase altered the ratio of Mcl-1 isoforms in favor of cell death whereas expression of the proapoptotic molecules Bad, Bak, and Bax was not altered. Surprisingly, blocking ER function increased the levels of Bad phosphorylation at serine 112 (BadpS112), an inactive (nonapoptotic) form of Bad. This inactivation of Bad upon ER blockade seemed to depend on c-Src function as chemical inhibition of c-Src kinase reduced BadpS112 levels in cells with impaired ER function but not in estrogen-treated cells. CONCLUSION: These results indicate a crucial role for c-Src kinase in the survival of ER-positive breast cancer cells only when ER function is blocked. Therefore, this study suggests that targeting simultaneously c-Src and ER may effectively inhibit growth of ER-positive breast cancer.

Cyclin D1 is necessary for tamoxifen-induced cell cycle progression in human breast cancer cells.

Despite the success of tamoxifen in treating hormone-responsive breast cancer, its use is limited by the development of resistance to the drug. Understanding the pathways involved in the growth of tamoxifen-resistant cells may lead to new ways to treat tamoxifen-resistant breast cancer. Here, we investigate the role of cyclin D1, a mediator of estrogen-dependent proliferation, in growth of tamoxifen-resistant cells using a cell culture model of acquired resistance to tamoxifen. We show that tamoxifen and 4-hydroxytamoxifen (OHT) promoted cell cycle progression of tamoxifen-resistant cells after growth-arrest mediated by the estrogen receptor down-regulator ICI 182,780. Down-regulation of cyclin D1 with small interfering RNA blocked basal cell growth of tamoxifen-resistant cells and induction of cell proliferation by OHT. In addition, pharmacologic inhibition of phosphatidylinositol 3-kinase/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 pathways decreased basal cyclin D1 expression and impaired OHT-mediated cyclin D1 induction and cell cycle progression. These findings indicate that cyclin D1 expression is necessary for proliferation of tamoxifen-resistant cells and for tamoxifen-induced cell cycle progression. These results suggest that therapeutic strategies to block cyclin D1 expression or function may inhibit development and growth of tamoxifen-resistant tumors.

Estrogen induces death of tamoxifen-resistant MCF-7 cells: contrasting effect of the estrogen receptor downregulator fulvestrant.

A common problem in breast cancer therapy is resistance to the antiestrogen tamoxifen. However, tamoxifen-resistant breast tumors can still respond to other hormonal therapies. In animal models of tamoxifen-resistant breast cancer cells, physiological levels of estrogen can induce tumor regression. Recently, the estrogen receptor downregulator fulvestrant was shown to promote tumor growth of tamoxifen-resistant cells when added in combination with physiological levels of estrogen. Here, we show, using a cell culture model, that continuous exposure of tamoxifen-resistant cells to physiological levels of estrogen leads to cell death. Addition of the estrogen receptor downregulator fulvestrant prevents estrogen-induced death in a dose-dependent manner. Our data indicate that endogenous levels of estrogen affect the response of tamoxifen-resistant cells to fulvestrant. These results suggest that failure of fulvestrant to inhibit tumor growth in some tamoxifen-resistant patients may be due to endogenous estrogen levels. Moreover, these studies support short-term treatment with estrogen as a second-line hormonal therapy for tamoxifen-resistant breast cancer.

Role of c-Src and focal adhesion kinase in progression and metastasis of estrogen receptor-positive breast cancer.

The non-receptor tyrosine kinases c-Src and focal adhesion kinase (Fak) mediate signal transduction pathways that regulate cell proliferation, survival, invasion, and metastasis. Here, we investigated whether c-Src and Fak are activated during progression of hormone-dependent breast cancer. Maximally active c-Src was overexpressed in a subset of tamoxifen-resistant variants and in metastases of recurrent hormone-treated breast cancer. Active Fak was also frequently observed in these tumors. We also show that estrogen receptor (ER) can bind to Fak and that estrogen can modulate Fak autophosphorylation supporting a cross-talk between these two pathways. Inhibition of c-Src activity blocked proliferation of all tamoxifen-resistant variants, suggesting that inhibitors of c-Src-Fak activity may delay or prevent progression and metastasis of ER-positive tumors. These studies also raise the possibility that fully active forms of c-Src and Fak in breast tumors may be biomarkers to predict tamoxifen resistance and/or risk of recurrence in ER-positive breast cancer.

Cyclin D1 expression is dependent on estrogen receptor function in tamoxifen-resistant breast cancer cells.

The development of resistance to tamoxifen, the most common antiestrogen used in the treatment of breast cancer, is a frequent and severe clinical problem. Tamoxifen-resistant tumors are still capable of responding to other hormonal therapies such as those that downregulate estrogen receptor expression. Mechanisms leading to acquisition of tamoxifen-resistant but hormone-sensitive growth are not completely understood. In tamoxifen-sensitive breast cancer cells, tamoxifen inhibits, whereas estrogen induces, expression of cyclin D1, a key cell cycle regulatory protein. Ectopic expression of cyclin D1 can lead to antiestrogen resistance. Thus, to determine whether cyclin D1 is involved in the growth of tamoxifen-resistant cells, we developed several tamoxifen-resistant variants from MCF-7 cells. These variants grow in the absence of estrogen or in the presence of tamoxifen, but their growth is inhibited by estrogen receptor downregulators. We show here that cyclin D1 expression is maintained at comparable levels in all tamoxifen-resistant variants, whereas pS2, another estrogen-regulated protein, is not. The addition of physiological levels of estrogen further stimulates cyclin D1 expression and proliferation. In contrast, treatment with estrogen receptor downregulators decreases cyclin D1 expression and proliferation. Thus, changes in cyclin D1 expression upon second-line hormonal therapy may predict hormonal sensitivity of tamoxifen-resistant tumors. These studies suggest that estrogen receptor mediates cyclin D1 expression and growth of tamoxifen-resistant tumors.