Browsing by Subject "RAS"

The RAS family is a group of small GTPases that can become constitutively activated by point mutations that are found in about 30% of all cancer patients. There are three well-characterized RAS family members: HRAS, NRAS, and KRAS, the latter of which is alternatively spliced at the C-terminus into KRAS4A and KRAS4B. The RAS proteins are all nearly identical at their N-termini and core effector binding domains, but have divergent C-terminal membrane-binding regions that impart different subcellular localization and subtle differences in signaling. Although the role of constitutively activated oncogenic RAS has been well established to play a role in cancer, recent work has suggested that wild-type RAS signaling may also be important in tumorigenesis. Wild-type RAS proteins have been shown to be activated in the presence of oncogenic KRAS. However, the consequences of this activation are context-dependent, as signaling through the wild-type RAS proteins has been shown to both suppress neoplastic growth and promote tumorigenesis under different circumstances.

I sought to investigate the role of the wild-type RAS proteins in two clinically –relevant models of cancer: pancreatic, the type of cancer most frequently associated with KRAS mutations, and lung cancer, the cancer in which KRAS mutations affect the highest number of patients. First, I tested whether a loss of wild type Hras altered tumorigenesis in a mouse model of pancreatic cancer driven by oncogenic Kras. Hras homozygous null mice (Hras-/- ) exhibited more precancerous lesions of the pancreas as well as more off-target skin papillomas compared to their wild type counterparts, indicating that Hras suppresses early Kras-driven pancreatic tumorigenesis. Loss of Hras also reduced the survival of mice engineered to develop aggressive pancreatic cancer by the additional disruption of one allele of the tumor suppressor p53 (Trp53R172H/+). However, this survival advantage was lost when both alleles of Trp53 were mutated, suggesting that wild-type HRas inhibits tumorigenesis in a p53-dependant manner.

Next, I investigated the role that wild-type Hras and Nras play in a chemical carcinogen-induced model of lung cancer. In mice treated with urethane, a carcinogen that induces Kras-mutation positive lung lesions, Hras-/ mice once again developed more tumors than wild-type mice. Interestingly, however, this effect was not observed in mice lacking wild-type Nras. Mice lacking both Hras and Nras alleles developed approximately the same number of tumors as Hras-/- mice, thus the additional loss of Nras does not appear to enhance the tumor-promoting effects of loss of Hras. In summary, signaling through wild-type Hras, but not Nras, suppresses tumorigenesis in a carcinogen-induced model of lung cancer.

The tumor-suppressive effects of wild-type Ras signaling were traced to the earliest stages of pancreatic tumorigenesis, suggesting that wild-type Ras signaling may suppress tumorigenesis as early as the time of initiation. These findings suggest that differences in expression of the wild-type Ras isoforms could potentially play a role in an individual’s predisposition for developing cancer upon oncogenic insult.

The Ras family of small GTPases, comprised of the KRAS, NRAS, and HRAS genes, are mutated to encode constitutively-active, GTP-bound, oncogenic proteins in upwards of one quarter or more of all human cancers, which is well established to promote tumorigenesis. Despite the prominent role these genes play in human cancer, the encoded proteins have proven difficult to pharmacologically inhibit. Therefore, it is important to understand how Ras proteins are activated.

RAS proteins cycle between a GDP-bound inactive state and a GTP-bound active state through guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). GEFs facilitate the GDP-to-GTP exchange of RAS and promote RAS activation. Similar to GEFs, reactive oxygen/nitrogen species can also promote RAS activation through reactions with the thiol residue of cysteine 118 (C118). This residue may therefore play a role in RAS activation in cancer. To test this possibility, I investigated the effect of mutating C118 to serine (C118S) in Kras on (1) carcinogen-induced lung tumorigenesis, and (2) xenograft tumor growth of HRAS12V-transformed cells.

To explore the impact of the C118S mutation in Kras on carcinogen-induced lung tumorigenesis, I introduced a C118S mutation into the endogenous murine Kras allele and exposed the resultant mice to the carcinogen urethane, which induces Kras mutation-positive lung tumors. Kras+/C118S and KrasC118S/C118S mice developed fewer and smaller lung tumors than Kras+/+ mice. Although the KrasC118S allele did not appear to affect tumorigenesis when the remaining Kras allele was conditionally oncogenic (KrasG12D), there was a moderate imbalance of oncogenic mutations favoring the native Kras allele in tumors from Kras+/C118S mice treated with urethane. Therefore, mutating C118 of Kras impedes urethane-induced lung tumorigenesis.

To explore the the impact of the C118S mutation in Kras on xenograft tumor growth of HRAS12V-transformed cells, I tested and found that redox-dependent reactions with cysteine 118 (C118) and activation of wild type KRAS are critical for oncogenic HRAS-driven tumorigenesis. Such redox-dependent activation of KRAS affected both PI3K-AKT and RAF-MEK-ERK pathways. These findings were confirmed in the endogenous mouse Kras gene. Speicfically, oncogenic HRAS-transformed KrasC118S/C118S MEFs grew in soft agar and as xenograft tumors more slowly than similarly transformed Kras+/+ MEFs, suggesting that redox-dependent reactions with C118 of Kras promotes transformation and tumorigenesis.

Taken together, I have demonstrated a critical role of redox-dependent reactions with Kras C118 in tumorigenesis.