		David M. Sabatini, MD. PhD, is a principal investigator at the Sabatini Lab at the Whitehead Institute for Biomedical Research and associate professor of biology at Massachusetts Institute of Technology (MIT). His research into the mTOR pathway has led to the identification of numerous components of this pathway, as well as its cellular and organismal functions. He is a recipient of the 2009 Paul Marks Prize for Cancer Research, among numerous other distinctions.

		What role does mTOR signaling play in normal cells?

		In normal cells, mTOR is essential for cells to generate and accumulate mass, and therefore grow (ie, increase in size). It is one of the central growth-promoting pathways in all cells.

		How important is it in cancer development?

		mTOR is also a key growth regulator in cancer cells. In addition, there are many cancer-causing mutations (in tumor suppressors and oncogenes) that lead to mTOR activation—so that it is estimated that 60% to 80% of tumors have hyperactive mTOR signaling.

		Are there any currently available mTOR-targeted treatments?

		Yes. The best-known mTOR inhibitor is a molecule called rapamycin, and there are also derivatives of rapamycin that are in clinical use. These molecules are used as immunosuppressants, blockers of coronary vessel restenosis in drug-eluting stents, and as treatments for cancer. They also have potential uses in neurodegenerative diseases.

		Which components of the mTOR pathway do they target?

		They all target the mTOR kinase, which is the central component of the pathway. They are allosteric inhibitors of mTOR.

		What is the future for mTOR signaling in cancer research?

		The key will be to identify which cancers are particularly sensitive to mTOR inhibitors. Currently, we know that many cancers exhibit mTOR hyperactivation, but not all respond equally to mTOR inhibitors. What is most needed is the identification of a cancer-specific mutation that causes cancers to be addicted to mTOR activity.

	The wealth of literature describing the mammalian target of rapamycin (mTOR) features a common analogy: that mTOR is a “master switch,” “central regulator,” or “key integrator” of cell growth and proliferation. Therefore, it is unsurprising to find that aberrant mTOR signaling is implicated in the pathogenesis of cancer and has attracted substantial interest from researchers since its discovery 20 years ago.

	mTOR is a serine/threonine kinase that exists in 2 multiprotein complexes, mTORC1 and mTORC2, defined by the different component proteins bound to mTOR. Both complexes consist of mTOR and the mLST8 and Deptor proteins. mTORC1 also contains the regulatory-associated protein of mTOR (Raptor) and the proline-rich Akt substrate of 40kDa (PRAS40) protein. mTORC2 contains the rapamycin-insensitive companion of mTOR (Rictor), mammalian stress-activated kinase-interacting protein 1 (mSin1), and the protein Protor.

	Rapamycin, available since the mid-1970s, is a naturally occurring inhibitor originally isolated from a strain of the soil bacterium Streptomyces hygroscopicus and was initially used as an immunosuppressive drug. The discovery of 2 yeast genes, TOR1 and TOR2, led to the identification and cloning of mTOR. And, once mTOR was implicated in cancer, researchers turned their attention to the role of rapamycin in cancer development.

	The exact mechanism of rapamycin inhibition remains unclear; it forms a complex with FKBP12 (FK506-binding protein of 12kDa) and mTOR. It is believed that only mTORC1 is infl uenced by rapamycin. However, recent evidence suggests that mTORC2 may also be susceptible to prolonged rapamycin exposure.

	Conserved from yeast to man, mTOR integrates incoming signals from nutrient and energy sensors on the cell surface with activation of cellular machinery to regulate growth and proliferation, ensuring that these processes happen only under appropriate conditions. mTOR receives signaling input from growth factors, hormones (eg, insulin), nutrients (eg, amino acids), cellular energy levels, and stress conditions.

	A principal signaling pathway that activates mTORC1 is the phosphatidylinositol 3-kinase/ protein kinase B (PI3K/Akt) pathway, which is well known for its central role in cell survival and proliferation. In the context of mTOR signaling, activated Akt phosphorylates and inhibits the tuberous sclerosis complex (TSC) and PRAS40, both of which are negative regulators of mTORC1, thereby activating mTORC1.

	Notably, mTORC1 can also be activated via AMPactivated serine/threonine protein kinase (AMPK), a sensor of cellular energy status. The activation of mTORC2 is understood to a far lesser degree, but it is believed to occur exclusively in response to growth factors.

	The best characterized targets of mTOR signaling are ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1), both of which regulate protein synthesis. However, mTOR activates a vast range of proteins that regulate many of the “hallmarks of cancer” described by Robert Weinberg in his landmark Cell article in 2000, including cell division, angiogenesis, and cell survival.

	Surprisingly, a direct involvement of mTOR in cancer development has yet to be proven. Researchers have been unable to identify any cancer-driving mutations in the TOR gene, and it is still unclear whether aberrant mTOR activation is required for cancer initiation.

	What is clear is the indirect involvement of mTOR signaling in a plethora of different cancers, via overexpression or loss of upstream activators, downstream targets, and regulators of mTOR activity. The main culprits leading to aberrant mTOR signaling include mutations in PI3K, overexpression of Akt, overexpression or mutation of the TSC complex, many receptor tyrosine kinases, and the mTOR target proteins S6K1 and 4EBP1. In addition, the loss of function of the phosphatase and homolog tensin gene (

) counteracts PI3K function; it is among the most commonly inactivated tumor-suppressor genes in human cancers.

	Overall, aberrant mTOR signaling is estimated to be activated in 60% to 80% of gastric adenocarcinomas, 25% to 45% of hepatocellular carcinomas, and numerous other tumor types, including HER2-positive breast cancer, non-Hodgkin lymphoma, colon cancer, and prostate cancer.

	mTOR Inhibition: Something Old, Something New

	Currently, there are 2 mTOR inhibitors approved by the FDA, both of which are analogs of rapamycin (dubbed rapalogs). They are everolimus (Afinitor), an oral agent dosed once per day, and temsirolimus (Torisel), administered intravenously once per week. Both are approved for the treatment of renal cell carcinoma (RCC): everolimus for refractory, advanced RCC after failed treatment with the receptor tyrosine kinase inhibitors sorafenib (Nexavar) and sunitinib, (Sutent) and temsirolimus for poorprognosis, untreated RCC. In Europe, temsirolimus is also approved for refractory mantle cell lymphoma (MCL) and a third rapalog, ridaforolimus, is approved for RCC.

	Clinical trials of rapalogs are ongoing in a variety of different tumors as single agents and in combination with other therapies. However, despite promising improvements in progression-free survival, overall survival, and overall response rate in RCC and MCL, rapalogs have proven only modestly successful in many clinical trials. In part this reflects the fact that rapalogs do not effectively inhibit mTORC2 and an incomplete understanding of the functions of mTOR and the complex feedback loops in its signaling network. Particularly relevant in respect to the latter is a recently identified negative feedback effect on the PI3K/Akt pathway, suggesting that inhibition of mTOR may inadvertently activate this pathway, thereby promoting growth and survival.

		This illustration depicts a simplified scheme of the mTOR signal transduction pathway

		Campa D, Hüsing A, Stein A, et al. Genetic variability of the mTOR pathway and prostate cancer risk in the European prospective investigation on cancer (EPIC). 

 6(2): e16914. doi:10.1371/journal.pone.0016914.

	In response to the deficiencies of the rapalogs, a second generation of smarter compounds is being developed. Among them are specifi c mTOR inhibitors that target the kinase activity of both mTORC1 and mTORC2, including WYE354 and WYE132 (Pfi zer), PP30 and PP242 (Intellikine), AZD8055 (AstraZeneca), OSI-027 (OSI Pharmaceuticals), and Torin1. These inhibitors have been shown to induce more potent and durable inhibition of mTOR.

	In order to circumvent the activation of feedback loops that may preclude tumor responsiveness to mTOR inhibition, dual inhibitors of mTOR/PI3K have also been developed, including BEZ235 (Novartis), XL765 (Exelixis), GDC-0980 (Genentech), PF-04691502 (Pfizer), and PF- 05212384 (Pfi zer). All of these second-generation agents are currently undergoing phase I and II clinical testing in solid tumors and a variety of other cancers.

	Finally, given the unpredictable response to rapalog treatment, the development and validation of molecular biomarkers will be crucial in determining which patients will benefi t. Since 2000, major efforts have been directed toward achieving this end. A number of putative predictors have been identified in preclinical models, but are not yet fully validated in the clinic.

			Borders EB, Bivona C, Medina PJ. Mammalian target of rapamycin: biological function and target for novel anticancer agents. 

			Dancey JE. Inhibitors of the mammalian target of rapamycin. 

			Efeyan A, Sabatini DM. mTOR and cancer: many loops in one pathway. 

			Faivre S, Kroemer G, Raymond E. Current development of mTOR inhibitors as anticancer agents. 

Guertin DA, Sabatini DM. Defi ning the role of mTOR in cancer. 

Guertin DA, Sabatini DM. The pharmacology of mTOR inhibition. 

Hay N, Sonenberg N. Upstream and downstream of mTOR. 

Vilar E, Perez-Garcia J, Tabernero J. Pushing the envelope in the mTOR pathway: the second generation of inhibitors. 

Wander SA, Hennessey BT, Slingerland JM. Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy. 

Yuan R, Kay A, Berg WJ, Lebwohl D. Targeting tumorigenesis: development and use of mTOR inhibitors in cancer therapy. 

Articles

The mammalian target of rapamycin (mTOR) is a master switch, central regulator, or key integrator of cell growth and proliferation

A Tale of 2 Complexes: The mTOR Signaling Pathway in Cancer Development

	In 1995, the Nobel Prize in Medicine was jointly awarded to Edward B. Lewis, Christiane Nüsslein- Volhard, and Eric F. Wieschaus for discoveries relating to the “genetic control of embryonic development” that began with a series of experiments in the fruit fly some 20 years earlier.

	The researchers were interested in understanding how the newly fertilized fruit fly egg develops into a segmented embryo and, after testing mutations in approximately 20,000 fly genes, identified 150 involved in early embryonic development, 15 of which, if mutated, caused defects in body segmentation.

	Among them was a gene that caused the fly embryo to develop as a spiny ball reminiscent of a hedgehog. Thus, the Hedgehog gene, 

	We now know that Hh is an intracellular signaling ligand that initiates the Hh pathway, which is important in the regulation of embryo development from flies to humans. In the embryo, Hh acts as a morphogen, generating different responses from cells, depending on its concentration. It drives developing cells to different fates, thereby controlling the formation of many organs, including the central nervous system, limbs, lungs, heart, and eyes, and the development of left-right asymmetry in the body.

	Although Hh activity is significantly reduced in adults, recent evidence suggests that it may also have important functions later on in life, in regulating the growth of some stem cells, including those in the adult brain, and in tissue regeneration.

	In human beings, there are 3 Hh proteins: sonic hedgehog (SHH), Indian hedgehog (IHH), and desert hedgehog (DHH). Essentially, the Hh signaling pathway involves the binding of Hh ligand to membrane receptors known as Patched (Ptch) on the surface of target cells, either the same cell (autocrine signaling) or different cells (paracrine signaling). There are 2 Ptch receptors in humans, Ptch-1 and Ptch-2.

	In the absence of the Hh ligand, Ptch suppresses the expression of the downstream protein Smoothened, Smo; therefore, when Hh binds Ptch, Smo becomes activated. Smo is subsequently free to activate the Gli family of transcription factors, which initiate Hh-target gene expression in the nucleus. In mammals, there are 3 Gli proteins: Gli1 and Gli2 activate target gene expression, while Gli3 represses it.

	The first link between the Hh pathway and cancer stemmed from the discovery that Gorlin syndrome, a condition in which people are prone to the development of numerous basal cell carcinomas (BCCs) and other forms of cancer, is caused by an inherited mutation in Ptch-1.

	Accumulating evidence suggests that aberrant Hh signaling, caused either by mutations in the molecular components of the pathway (ligandindependent signaling) or by overexpression of the Hh protein (ligand-dependent signaling), is involved in a variety of different cancers.

	The former is implicated in 2 main forms of cancer: BCC, the most common form of cancer among white people, and medulloblastoma, the most common malignant brain tumor in children, both of which have an unmet need for improved treatments.

	Ligand-independent signaling most commonly occurs as a result of inactivating mutations in Ptch- 1 (identified in the majority of cases of sporadic BCC and approximately 10% of medulloblastomas), or activating mutations of Smo (found in approximately 10% of sporadic BCCs). Both of these mutations lead to activation of the pathway even in the absence of ligand.

	The first case of ligand-dependent signaling was identified in small-cell lung carcinoma. Since then, Hh-overexpression has been demonstrated in numerous other solid tumors, including gastrointestinal, pancreatic, ovarian, and breast cancers. Overall, improper Hh signaling is thought to be involved in as many as one-third of all cancers and this number is growing with the identification of more Hh-overexpressing tumors.

	In 1889, the British surgeon Stephen Paget proposed his visionary “seed and soil” hypothesis that the normal cells, or microenvironment, surrounding the tumor are also capable of supporting tumor growth and metastasis. Since Hh ligands can also act in a paracrine fashion on surrounding cells (sometimes at a substantial distance from the Hh-producing cell), an Hh-overexpressing tumor cell may also promote tumor growth and metastasis via modifi cation of the microenvironment.

	Arrows show the sequence of events promoting

	the overexpression of the Hedgehog gene.

	Given the role of Hh signaling in cancer initiation and progression, much importance has been placed on the search for ways to block its effects. A Smo inhibitor called cyclopamine, a naturally occurring chemical isolated from the corn lily plant, has historically been used to elucidate the function of Hh signaling in vertebrates. This compound, however, is not a viable therapeutic agent given its structural complexity, scarcity, and poor solubility and stability. Thus, a search began for small-molecule inhibitors with improved therapeutic viability.

	While no Hh-targeted therapies are currently approved by the FDA, numerous Smo inhibitors have demonstrated significant antitumor activity and excellent pharmaceutical properties, including oral bioavailability, long plasma and tumor half-life, and dose-dependent inhibition of tumor growth, in a number of preclinical models and are now undergoing clinical trials.

	GDC-0449 (Genentech, Roche, and Curis) has undergone positive phase I studies in patients with metastatic BCC and medulloblastoma, with a response rate of 50% in patients with metastatic disease, and has now entered phase II trials.

	IPI-926 (Infinity Pharmaceuticals) is undergoing phase I trials for solid tumors. Results of a phase I trial for XL139 (Bristol- Myers Squibb and Exelixis) recently confirmed a partial response in Gorlin syndrome patients. Human clinical trials of LDE225 (Novartis) were initiated in 2009 and a phase I trial is currently ongoing in patients with advanced solid tumors and Gorlin syndrome. PF-04449913 (Pfizer) entered phase I clinical testing in 2010 in patients with select hematologic malignancies.

	These inhibitors may also improve the access of chemotherapeutic agents to tumor cells through their effects on the tumor microenvironment. Thus, several are also being tested as combination therapies. IPI-926 is undergoing phase II trials in combination with gemcitabine for pancreatic cancer and PF- 04449913 is undergoing phase I clinical testing in combination with dasatinib in patients with chronic myeloid leukemia.

	Finally, other proposed Hh pathway inhibitors targeting SHH (eg, robotnikinin) and the Gli family of proteins (eg, GANT58 and GANT61), as well as antibodies against components of the pathway, are also undergoing preclinical development.

Jane de Lartigue, PhD, is a freelance medical writer and editor based in the United Kingdom.

These articles were consulted to prepare this report.

			Bak M, Hansen C, Tommerup N, Larsen LA. The hedgehog signaling pathway—implications for drug targets in cancer and neurodegenerative disorders. 

			di Magliano, MP, Hebrok M. Hedgehog signalling in cancer formation and maintenance. 

			Evangelista M, Tian H, de Sauvage FJ. The hedgehog signaling pathway in cancer. 

			Jiang J, Hui CC. Hedgehog signaling in development and cancer. 

			Kelleher FC. Hedgehog signaling and therapeutics in pancreatic cancer. [Published online ahead of print December 24, 2010]. 

 2011;32(4):445-451. doi:10.1093/carcin/bgq280.

			Mullor JL, Sanchez P, Ruiz AA. Pathways and consequences: hedgehog signaling in human disease. 

			Peukert S, Miller-Moslin K. Small-molecule inhibitors of the hedgehog signaling pathway as cancer therapeutics. 

			Robarge KD, Brunton SA, Castanedo GM, et al. GDC- 0449—a potent inhibitor of the hedgehog pathway.[Epub ahead of print August 15, 2009]. 

			Rubin LL, de Sauvage FJ. Targeting the hedgehog pathway in cancer. 

The Hedgehog gene is an intracellular signaling ligand that initiates the Hh pathway, which is important in the regulation of embryo development from flies to humans

Jane de Lartigue, PhD

Articles

A Tale of 2 Complexes: The mTOR Signaling Pathway in Cancer Development

Ferreting Out the Hedgehog Pathway Emerges as Ripe Target for Cancer Drug Development