Date of Degree

2-2014

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor(s)

David A. Foster

Subject Categories

Biochemistry | Biology | Cell Biology

Keywords

Amino acid, Cancer, Cell Cycle, Glutamine, Metabolism, mTOR

Abstract

Growth factors (GFs) as well as nutrient sufficiency regulate cell division in metazoans. The vast majority of mutations that contribute to cancer are in genes that regulate progression through the G1 phase of the cell cycle. A key regulatory site in G1 is the growth factor-dependent Restriction Point (R), where cells get permissive signals to divide. In the absence of GF instructions, cells enter the quiescent G0 state. Despite fundamental differences between GF signaling and nutrient sensing, they both have been confusingly referred to as R and therefore by definition considered to be a singular event in G1. Autonomy from GF signaling is one of the hallmarks in cancer; however, cancer cells also have metabolic rewiring enabling them to engage in anabolic biosynthetic pathways. In the absence of GF instructions and nutrients, cells commonly undergo apoptotic cell death. Thus, it is of importance to elucidate the differences between GF and nutrient deregulation in cancer to develop novel strategies in targeting tumor cell proliferation and survival.

Here, we report that the GF-mediated mid-G1 restriction point (R) is distinct and distinguishable from a series of late-G1 metabolic checkpoints mediated by essential amino acids, conditionally essential amino acid - glutamine, and mTOR - the mammalian target of rapamycin. Our data indicate that the arrest sites mediated by various blocking conditions are in the order of GF -> EAA -> Q -> mTOR. We temporally mapped the EAA and glutamine checkpoints at 12 hr from G0 and mTOR mediated arrest occurring at 16 hr from G0. Distinct profiles for cell cycle regulator expression and phosphorylation was observed when released from restriction point relative to the metabolic checkpoints. These data are consistent with a mid-G1 R where cells decide whether they should divide, followed by late-G1 metabolic checkpoints where cells determine whether they have sufficient nutrients to divide. Since mTOR inhibition using rapamycin or Torin1 arrested the cells latest in G1, mTOR may serve as the final arbiter for nutrient sufficiency prior to replicating the genome. Significantly we also observed that in addition to GF autonomy, several cancer cells also have dysregulated nutritional sensing, and arrest in S- and G2/M phase upon essential amino acid and glutamine deprivation. We identified K-Ras mutation as the underlying genetic cause for this phenomenon. We found that treating cancer cells harboring K-Ras mutation with aminooxyacetate (AOA) - drug that interferes with glutamine utilization - causes them to arrest in S- and G2/M-phase, where synthetic lethality could be created to phase-specific cytotoxic drugs. Thus, besides addressing the long standing assumption of GF and nutrients regulating G1 cell cycle progression, our work provides rationale and proof of principle for targeting metabolic deregulations in cancer cells.

 
 

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