Available online http://breast-cancer-research.com/content/6/5/219
`
`Review
`New targets for therapy in breast cancer
`Mammalian target of rapamycin (mTOR) antagonists
`Hetty Carraway and Manuel Hidalgo
`
`Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Hospital, Cancer Research Building, Baltimore, Maryland, USA
`
`Corresponding author: Hetty Carraway, hcarraw1@jhmi.edu
`
`Published: 12 August 2004
`
`Breast Cancer Res 2004, 6:219-224 (DOI 10.1186/bcr927)
`© 2004 BioMed Central Ltd (Print ISSN 1465-5411; Online ISSN 1465-542X)
`
`Abstract
`Mammalian target of rapamycin (mTOR) is a serine-threonine kinase member of the cellular
`phosphatidylinositol 3-kinase (PI3K) pathway, which is involved in multiple biologic functions such as
`transcriptional and translational control. mTOR is a downstream mediator in the PI3K/Akt signaling
`pathway and plays a critical role in cell survival. In breast cancer this pathway can be activated by
`membrane receptors, including the HER (or ErbB) family of growth factor receptors, the insulin-like
`growth factor receptor, and the estrogen receptor. There is evidence suggesting that Akt promotes
`breast cancer cell survival and resistance to chemotherapy, trastuzumab, and tamoxifen. Rapamycin is
`a specific mTOR antagonist that targets this pathway and blocks the downstream signaling elements,
`resulting in cell cycle arrest in the G1 phase. Targeting the Akt/PI3K pathway with mTOR antagonists
`may increase the therapeutic efficacy of breast cancer therapy.
`
`Keywords CCI-779, epidermal growth factor receptor, mammalian target of rapamycin, phosphatidylinositol 3-
`kinase pathway, PTEN
`
`Introduction
`Mammalian target of rapamycin (mTOR) is a serine-
`threonine kinase member of the cellular phosphatidyl-
`inositol 3-kinase (PI3K) pathway, which is involved in
`multiple functions such as transcriptional and translational
`control. Activation of mTOR as a consequence of nutrients
`and growth factors results in the phosphorylation and
`activation of the 40S ribosomal protein S6 kinase
`(p70S6K) and the eukaryotic initiation factor 4E-binding
`protein-1 (4EBP1; Fig. 1). These proteins play a key role in
`ribosomal biogenesis and cap-dependent translation,
`which result in increased translation of mRNAs that are
`important to the control and progression of the cell cycle.
`mTOR is a downstream mediator in the PI3K/Akt signaling
`pathway and plays a critical role in cell survival.
`
`It has been shown that Akt regulates mTOR through the
`tuberous sclerosis (TSC) complex [1]. Under non-
`stimulated conditions, the TSC complex acts as a negative
`
`regulator of mTOR. Phosphorylation of TSC2 (tuberin) by
`Akt inactivates the complex, releasing its inhibitory effects
`on mTOR and resulting in mTOR activation. In addition,
`TSC regulation of mTOR is mediated by the small G
`protein Rheb. When in its GTP state, Rheb is a potent
`activator of mTOR. Phosphorylated TSC shifts Rheb to
`the inactive GDP state [2].
`
`In breast cancer the PI3K/Akt pathway can be activated
`by membrane receptors, including the HER (or ErbB)
`family of growth factor receptors, the insulin-like growth
`factor (IGF) receptor, and the estrogen receptor (ER) [3].
`Stimulation of the PI3K/Akt pathway can also occur
`through oncogenic Ras. There is evidence suggesting that
`Akt promotes breast cancer cell survival and resistance to
`chemotherapy, trastuzumab, and tamoxifen [4–7]. This
`suggests that targeting the Akt/PI3K pathway with mTOR
`antagonists may increase the therapeutic efficacy of
`breast cancer therapy. Rapamycin and rapamycin analogs
`
`ER = estrogen receptor; IGF = insulin-like growth factor; mTOR = mammalian target of rapamycin; PI3K = phosphatidylinositol 3-kinase; TSC =
`tuberous sclerosis.
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`Breast Cancer Research Vol 6 No 5 Carraway and Hidalgo
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`Figure 1
`
`Rapamycin-sensitive signal transduction pathways. Both rapamycin
`and rapamycin analogs bind to the immunophilin FK506 binding
`protein-12 (FKBP-12). The rapamycin-FKBP12 complex binds to
`mammalian target of rapamycin (mTOR), inhibiting its kinase activity,
`which in turn inhibits the phosphorylation and activation of the
`downstream translational regulators 4EBP1/PHAS-1 and p70S6K.
`These downstream effects decrease the translational processing of
`mRNA for specific proteins that are essential for G1 to S phase
`transition. 4E-BP1, 4E binding protein-1; GF, growth factor; GPB,
`growth factor receptor bound; MAP, mitogen activated protein kinase;
`PI3K, phosphatidylinositol 3-kinase; PHAS, phosphorylated heat and
`acid stable protein; pRb, retinoblastoma protein; PTEN, phosphatase
`and tensin homologue deleted from chromosome 10; RAP, rapamycin;
`SOS, son-of-sevenless; TSC, tuberous sclerosis complex.
`
`(CCI-779, RAD001, AP23573) are specific mTOR
`antagonists that are used to target this pathway and block
`the downstream signaling elements and result in cell cycle
`arrest in the G1 phase. These agents have exhibited
`impressive growth inhibitory effects against a broad range
`of human cancers, including breast cancer, in preclinical
`and early clinical evaluations [8,9].
`
`lactone produced by
`is a macrolytic
`Rapamycin
`Streptomyces hygroscopicus, which has
`immuno-
`suppressive, antimicrobial, and antitumor properties.
`Rapamycin binds intracellularly to FK506 binding protein-
`12 (tacrolimus-binding protein) and targets a principal
`protein kinase that was named mTOR. Other names
`include FKBP-rapamycin associated protein
`(FRAP),
`rapamycin FKBP12 target (RAFT1), and rapamycin target
`(RAPT1). Inhibition of the phosphorylation of mTOR by
`rapamycin specifically blocks the activation of the 40S
`ribosomal protein S6 kinase and 4E-binding protein-1, and
`directly reduces the translation of mRNAs that encode
`essential components of the protein synthesis machinery,
`including growth factors, oncoproteins, and cell cycle
`regulators. Rapamycin treatment also results in prevention
`
`220
`
`inhibition of
`of cyclin-dependent kinase activation,
`phosphorylation of
`the
`retinoblastoma protein, and
`acceleration of the turnover of cyclin D1 mRNA and
`protein, leading to a deficiency of active cyclin-dependent
`kinase 4/cyclin D1 complexes. The combination of these
`events likely contributes to the prominent inhibitory effects
`of rapamycin at the G1/S boundary of the cell cycle,
`induction of apoptosis, and inhibition of angiogenesis in
`several preclinical cancer models [10].
`
`fungicide,
`to be a potent
`found
`Rapamycin was
`particularly against Candida albicans and other
`filamentous fungi. Later, another related derivative was
`identified and found to be a potent immunosuppressant
`(tacrolimus). In bone marrow transplant research, further
`evaluation of
`the
`immunosuppressive qualities of
`rapamycin revealed successful activity in reversing acute
`allograft rejection and enhancing long-term donor-specific
`allograft tolerance. Because this drug is a potent immuno-
`suppressant with negligible toxicity, it has regulatory
`approval for use in the prevention of allograft rejection
`after organ transplantation.
`
`Rapamycin was found to have antiproliferative actions in a
`diverse range of experimental tumors, including lymphoma,
`small cell lung cancer, and rhabdomyosarcoma [11–13].
`Antitumor actions of rapamycin have been principally
`attributed to its ability to modulate the synthesis of critical
`proteins that are required for ribosome biosynthesis,
`protein translation, and G1 to S cell cycle phase progres-
`sion. Rapamycin’s poor aqueous solubility and chemical
`stability limited its development as an anticancer agent,
`and consequently rapamycin analogs with more favorable
`pharmaceutical characteristics were developed, including
`CCI-779, RAD 001, and AP23573.
`
`Although not all of the relevant elements of rapamycin-
`sensitive signal
`transduction pathways have been
`elucidated, PI3K/Akt appears to be the key modulating
`element in the upstream pathway by which interactions
`between growth factors and growth factor receptors affect
`the phosphorylation state of mTOR. The enzyme PI3K,
`which plays a central role in cellular proliferation, motility,
`neovascularization, viability and senescence, is upregulated
`in many types of malignant cells. The best characterized
`effector of PI3K is the serine/threonine kinase Akt. Both
`PI3K and Akt are proto-oncogenes, because they have
`been demonstrated
`to possess
`cell-transforming
`properties. PI3K has other downstream effectors, but the
`Akt pathway is of particular interest because of its role in
`inhibiting apoptosis and promoting cell proliferation.
`
`With regard to breast cancer pathogenesis, elements of
`the PI3K/Akt pathway have been demonstrated to be
`activated by the ErbB family, IGF receptor, and oncogenic
`Ras [14–17]. Over-expression of IGF-I receptor and IGF-I
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`commonly occurs in breast cancers, and circulating IGF-I
`has been related to the risk for developing breast cancer
`[18–21]. Furthermore, elements of the IGF signaling
`pathway are found to be highly expressed in primary
`breast tumors, and overexpression of
`IGF pathway
`elements has been associated with poor prognosis [22].
`Upon ligand binding, IGF-I receptor activates insulin
`receptor substrates that activate elements that are
`involved in signaling through the PI3K/Akt pathway. Breast
`tumors can possess genetic alterations in the PI3K/Akt
`pathway and often exhibit high levels of constitutive Akt
`activity. Recently, a high frequency of mutations of the
`PIK3CA gene was reported in 32% of colon cancers and
`almost 10% of breast cancers [23]. This may be a useful
`tool to identify which patients may respond to rapamycin-
`directed therapy.
`
`ER activation can directly drive the PI3K/Akt pathway [24].
`Cross-talk between ErB1 and ER has been shown to
`activate the PI3K/Akt pathway, which has been associated
`with estrogen-independent transcriptional activity and anti-
`estrogen resistance [2–5]. In support of this hypothesis,
`breast cancer cell lines with constitutively active Akt can
`proliferate in the absence of exogenous estrogen and are
`resistant to the growth inhibitory and proapoptotic effects
`of tamoxifen in vitro and in vivo [25]. The role played by
`the PI3K/Akt pathway in the development of estrogen
`resistance is also supported by studies of IGF. For
`example, IGF-I has been demonstrated to downregulate
`the progesterone receptor via a transcriptional mechanism
`that involves the PI3K/Akt pathway and is independent of
`the ER. These data may provide an explanation for why
`progesterone receptor negative tumors are particularly
`insensitive to estrogen deprivation [26].
`
`Additional evidence points to a rationale for targeting the
`PI3K/Akt pathway in the treatment of breast cancer. Over-
`expression of D-type cyclin has been reported
`in
`approximately 50% of invasive breast cancers and was
`found to be associated with tumor progression [27,28].
`
`Upstream regulators of the PI3K/Akt pathway include the
`tumor suppressor gene PTEN (phosphatase and tensin
`homolog deleted from chromosome 10). PTEN inhibits the
`activity of PI3K. Thus, loss of PTEN suppressor gene
`function has been associated with Akt activation. A familial
`syndrome characterized by germline mutations in PTEN
`appear to be responsible for Cowden’s syndrome, which
`predisposes to the development of several types of
`malignant neoplasms, including breast cancer. Another
`familial syndrome is Bannayan–Zonana, which has similar
`features. It is important to note that fewer than 5% of
`breast tumors harbor a PTEN mutation. However, hemi-
`zygous deletions of the PTEN locus and subsequent lack
`of PTEN protein occurs in about 30–40% of patients with
`sporadic breast cancer. This heterozygosity of the PTEN
`
`Available online http://breast-cancer-research.com/content/6/5/219
`
`locus is associated with an increased activation of Akt
`[29–33]. The hyperactivation of PI3K/Akt signaling
`elements in PTEN-deficient malignancies suggests that
`these cancers are dependent on this pathway for growth
`and maintenance. Furthermore, experiments conducted in
`PTEN knockout mice
`that PTEN-deficient
`indicate
`cancers are extraordinarily sensitive
`to
`the growth
`inhibitory effects of rapamycin and rapamycin analogs
`[34]. Interestingly, treatment of doxorubicin-resistant and
`PTEN-defective prostate cancer cells with CCI-779 has
`been shown to reverse doxorubicin resistance [35].
`Transfection of those prostate cancer cells with functional
`PTEN produced a similar modulatory effect, suggesting
`that doxorubicin resistance in this model is mediated
`through the downstream activation of mTOR.
`
`In breast cancer cells, PI3K/Akt and mTOR pathways
`appear to be critical for the proliferative responses
`mediated by epidermal growth factor receptor, the IGF
`receptor and the ER. These findings suggest that it may
`be rational to pursue the use of pharmacologic inhibitors
`of mTOR
`in treating patients with breast cancers,
`particularly because these malignancies have evidence of
`hyperactive PI3K/Akt
`signaling
`elements
`and/or
`aberrations in tumor suppressor proteins such as PTEN.
`
`Clinical studies with rapamycin/rapamycin
`analogs
`The safety and efficacy of rapamycin in the prevention of
`organ
`rejection have been demonstrated
`in
`two
`randomized, double-blind, multicenter, controlled trials
`involving over 1000 adult patients according to the
`investigator brochure for CCI-779 formulation. Typical
`dosing was 2 mg or 5 mg administered daily. In these and
`most other trials, rapamycin has been administered with
`cyclosporine and corticosteroids, and limited pharmaco-
`kinetic PK data are available alone in this setting. Major
`side effects noted in these studies included thrombo-
`cytopenia, hypercholesterolemia, hypertriglyceridemia, and
`diarrhea. Renal function was not impaired.
`
`The water-soluble rapamycin ester CCI-779 was selected
`for clinical development through collaborative efforts
`between Wyeth-Ayerst Laboratories and the National
`Cancer Institute. In the US National Cancer Institute’s 60
`tumor type-specific cell line screening panel, CCI-779 and
`rapamycin had nearly identical growth inhibitory profiles
`and 50% inhibitory concentration (IC50) values that were
`frequently in the nanomolar range according to the
`investigator brochure for CCI-779 formulation. Cell lines
`derived from breast, prostate, and small cell lung cancer
`were among the most sensitive to CCI-779. In further
`laboratory-based studies by Wyeth, breast cancer cell lines
`(BT-474, SK BR-3, and MCF-7) exhibited extraordinary
`sensitivity to CCI-779, with IC50 values ranging from
`0.0006 to 0.001 µmol/l [34]. Most breast cancer cell lines
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`highly sensitive to CCI-779 were found to be estrogen
`dependent, over-expressed Erb-2, and/or had PTEN
`deletions [21]. The resistant breast cancer cell lines lacked
`these features. Furthermore, sensitive breast cancer cell
`lines generally had higher levels of activated Akt, perhaps
`leading
`to downstream activation of mTOR and
`subsequent sensitivity to mTOR inhibitors. Laboratory data
`have also supported the synergistic growth inhibitory
`effects of MCF-7 breast cancer cell lines and xenografts
`with combinations of CCI-779 and ER antagonists.
`Recently, both control MCF-7 and MCF-7/Aro (aromatase
`expressing) cells were found to be sensitive to treatment
`with the rapamycin analog RAD001 in vitro. RAD001 was
`found to almost completely inhibit estradiol induced
`proliferation in both control MCF-7 cells as well as estradiol
`and androstenedione induced proliferation in MCF-7/Aro
`cells, suggesting that mTOR signaling is required for
`estrogen proliferative response in MCF-7 cells. Further-
`more, combination therapy with letrozole and RAD001
`resulted in increased inhibition of cell proliferation as well
`as a synergistic effect, even at suboptimal levels of
`RAD001. No evidence of antagonism was observed [36].
`
`Human pharmacokinetics
`The pharmacokinetic properties of rapamycin have been
`studied in healthy individuals, pediatric dialysis patients,
`hepatically
`impaired adult patients, and adult renal
`transplant patients. Oral doses of both liquid and pill forms
`of rapamycin are rapidly but variably absorbed. Mean time
`to peak concentrations range from 1 hour in healthy
`individuals to 2 hours in renal transplant patients. Half-life
`is upwards of 2.5 days. Metabolism is by the intestinal and
`hepatic CYP3A4 enzyme
`family, and 91% of
`the
`elimination of the drug is via the gastrointestinal tract. The
`area under the curve correlates well with peak and trough
`concentrations.
`
`Patients who ingested the drug after a high fat breakfast
`did have a delayed time to maximum concentration
`(Cmax), and it is recommended that rapamycin be
`consistently taken with or without food. In a phase I
`pharmacokinetic study conducted in renal transplant
`patients, doses ranging from 0.5 to 6.5 mg/m2 were
`administered every 12 hours. Phase III studies to date
`have had concomitant use of cyclosporine or steroid, or
`both. At a dose of 2 mg/day the rapamycin trough
`concentration was 8.58 ± 4 ng/ml and at 5 mg/day the
`trough was 17.3 ± 7.4 ng/ml. Rapamycin concentrations
`in stable renal transplant patients are dose proportional
`between 3 and 12 mg/m2. Also, in this population a
`loading dose of three times the maintenance dose
`provided a near steady state concentration within 1 day in
`most patients. Stable renal transplant recipients have
`received single doses of up to 21 mg/m2. No toxicity has
`been observed in any of several single dosing studies with
`rapamycin doses ranging from 3 to 21 mg/m2.
`
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`
`Development of mammalian target of
`rapamycin inhibitors for cancer treatment
`Based on preclinical studies suggesting that inhibition of
`mTOR has antiproliferative effects in a variety of cancer
`models, clinical studies were initiated with two rapamycin
`analogs, namely RAD001 and CCI-779. Preliminary
`results from these studies indicate that these drugs are
`well tolerated and have promising anticancer activity [37].
`The principal toxicities of CCI-779 – the agent for which
`more clinical data are available – include thrombo-
`cytopenia, hyperlipidemia, skin toxicity, and elevation in
`liver function tests. These side effects were in general mild
`to moderate in intensity and not necessarily associated
`with administered dose of the drug. In addition, antitumor
`activity was demonstrated in a variety of tumor types,
`including breast cancer and renal cancer. A randomized
`phase II study of multiple dose levels of CCI-779 in
`patients with advanced refractory renal cell carcinoma has
`shown antitumor activity and encouraging survival as well
`as drug tolerability [38].
`
`Additionally, a multicenter European phase II study was
`conducted in 109 patients with advanced breast cancer
`[39]. Patients who had progressed on taxanes and
`anthracyclins were allowed
`to
`receive a weekly
`intravenous dose of CCI-779 at either 75 mg or 225 mg.
`Clinical benefit was observed
`in 37% of patients,
`including 10 partial responses and 26 patients with stable
`disease for longer than 8 weeks. Activity was seen at both
`dose levels, with a low toxicity profile. It is interesting to
`note that activity was even seen in patients with liver
`metastases at both dose levels but no response was seen
`in any of the 33 HER-2 negative patients. Further studies
`were
`initiated because of
`these promising results.
`Because of the association of hormone resistance and
`activation of the PI3K and mTOR pathways, development
`of clinical trials combining mTOR inhibitors and hormonal
`agents are logical. A randomized phase III, placebo-
`controlled, double-blind
`study of oral CCI-779
`administered in combination with letrozole versus letrozole
`alone in patients with estrogen-dependent breast cancer
`is ongoing. Combinations of mTOR
`inhibitors and
`cytotoxic agents are also expected.
`
`Conclusion
`The PI3K/Akt signaling pathway regulates many normal
`cellular processes including cellular proliferation, survival,
`growth and motility, all of which are critical processes for
`tumorigenesis. It is clear that alteration of this pathway
`occurs in many cancerous states, and perhaps targeted
`manipulation to optimize control of this pathway will
`mitigate its contribution to oncogenic activity. In several
`cancers, mTOR
`inhibitors have been shown to be
`promising agents in reducing tumor growth in vitro and in
`vivo, including renal cancer and breast cancer. Hopefully,
`with better understanding of these pathways and improved
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`This article is the second in a review series on
`New targets for therapy in breast cancer,
`edited by Stephen RD Johnston.
`
`Other articles in the series can be found at
`http://breast-cancer-research.com/articles/
`review-series.asp?series=bcr_NewTargets
`
`‘profiling’ of individual patients tumors, future clinical trials
`and treatment options will identify and target those patients
`who will benefit from these directed therapies.
`
`Competing interests
`HC declares that she has no competing interests. MH has
`received reimbursements, fees, and funding from Genen-
`tec, Astra-Zeneca, OSI pharmaceuticals, Weth, and
`Brystol Myers Squibb.
`
`Acknowledgements
`Dr Hetty Carraway would like to acknowledge The Stetler Fund for the
`support for her research at the Sidney Kimmel Comprehensive Cancer
`Center at Johns Hopkins Hospital.
`
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