TP53 and Let-7a micro-RNA Regulate K-Ras Activity in HCT116 Colorectal Cancer Cells

Recent reports have indicated that KRAS and TP53 mutations predict response to therapy in colorectal cancer. However, little is known about the relationship between these two common genetic alterations. Micro-RNAs (miRNAs), a class of noncoding RNA implicated in cellular processes, have been increasingly linked to KRAS and TP53. We hypothesized that lethal-7a (let-7a) miRNA regulates KRAS through TP53. To investigate the relationship between KRAS, TP53, and let-7a, we used HCT116 KRASmut human colorectal cancer cells with four different genotypic modifications in TP53 (TP53−/−, TP53+/−, TP53mut/+, and TP53mut/−). Using these cells we observed that K-Ras activity was higher in cells with mutant or knocked out TP53 alleles, suggesting that wild-type TP53 may suppress K-Ras activity. Let-7a was present in HCT116 KRASmut cells, though there was no correlation between let-7a level and TP53 genotype status. To explore how let-7a may regulate K-Ras in the different TP53 genotype cells we used let-7a inhibitor and demonstrated increased K-Ras activity across all TP53, thus corroborating prior reports that let-7a regulates K-Ras. To assess potential clinical implications of this regulatory network, we examined the influence of TP53 genotype and let-7a inhibition on colon cancer cell survival following chemoradiation therapy (CRT). We observed that cells with complete loss of wild-type TP53 alleles (−/− or −/mut) were resistant to CRT following treatment with 5-fluorouracil and radiation. Further increase in K-Ras activity with let-7a inhibition did not impact survival in these cells. In contrast, cells with single or double wild-type TP53 alleles were moderately responsive to CRT and exhibited resistance when let-7a was inhibited. In summary, our results show a complex regulatory system involving TP53, KRAS, and let-7a. Our results may provide clues to understand and target these interactions in colorectal cancer.


Introduction
Colorectal cancer (CRC) is the 4 th most common cancer and the 2 nd most common cause of cancer-related death in the US [1]. Recent advances that have improved outcomes in CRC have included the identification of accurate prognostic and predictive molecular biomarkers [2][3][4]. These have included genetic alterations in KRAS and TP53, which are frequently detected in patients with CRC. KRAS mutations are present in approximately 30-50% of CRCs, but also in 17-25% of all human tumors [2,[5][6][7]. Similarly, TP53 alterations are common in patients with CRC (nearly 50%) [8]. Both prognostic and predictive roles have been identified for both genes [9]. Our own group and others has recently shown that detection of concurrent KRAS and TP53 mutations, with an incidence of 5% to 20% in CRC patients, correlated with resistance to neoadjuvant chemoradiation therapy (CRT) in patients with rectal cancer [10][11][12]. Despite the frequency of these mutations in CRC, little is known about interactions between the two genes.
The link between these two frequently altered genes in CRC may lie in micro-RNA (miRNA), a class of non-coding RNA which function in transcriptional regulation and more specifically may influence the regulation of cell proliferation and apoptosis [13,14]. Recent reports have suggested that the tumor suppressor activity of miRNA lethal 7a (let-7a) may be due to its association with KRAS and that inhibition of tumor growth may occur by suppression of K-Ras expression by let-7a [15,16]. Emerging clinical data suggest that intra-tumor let-7a expression correlates with tumor response and overall survival in metastatic colorectal cancer patients receiving epidermal growth factor (EGFR) targeting agents in both KRAS wild-type and mutant populations [17]. Additional studies have speculated that the role of TP53 in DNA repair and apoptosis may in part be regulated by miRNAs, including let-7a [18,19]. Therefore, a complex regulatory network for TP53 and KRAS may be linked by let-7a.
To investigate potential interactions between TP53, KRAS, and let-7a, we analyzed a unique family of colorectal cancer cell lines with mutant KRAS and modifications in TP53 genotype. These cells enabled us to examine the changes in K-Ras expression and activity that corresponded with variations in TP53 genotype. Furthermore, we were able to better interrogate the role of let-7a in the setting of mutant KRAS and various TP53 genotypes. Our results indicate novel increases in K-Ras activity with different TP53 genotypes. However, we did not find a clear relationship between let-7a level and TP53 genotype. Nonetheless, changes in K-Ras activity were regulated by let-7a. This first report of TP53 and let-7a regulation of K-Ras activity provides clues to better understand the complex interaction between TP53 and KRAS.

Cell Culture
The human CRC cell line HCT116, harboring a single mutant KRAS allele and double wild-type TP53 (TP53 +/+ ) alleles, was modified into four stable cell lines with different TP53 genotypes (TP53 2/2 , TP53 +/2 , TP53 mut/+ , and TP53 mut/2 ). The parent and modified cell lines were kindly provided by Dr. Bert Vogelstein (Johns Hopkins University; Baltimore, MD) [20]. Mutant and knockout alleles were both used to assess potential differences between the two alleles, as suggested in prior reports [21]. All cell lines were assessed by DNA extraction, polymerase chain reaction (PCR), and sequencing for KRAS and TP53 gene mutations to verify genotypes. Cells were maintained in McCoy 5A medium (Irvine Scientific; Santa Ana, CA) with 10% fetal bovine serum (Omega Scientific; Tarzana, CA) and 1% penicillin-streptomycinglutamine (Invitrogen; Carlsbad, CA) at 5% CO 2 at 37uC.

Immunoblotting
Cell lysates were collected using RIPA buffer (Invitrogen; Carlsbad, CA) plus protease inhibitors (Thermo Scientific; Rockford, IL). Twenty micrograms of protein were separated on 12% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore; Bedford, MA). The membranes were blocked for 1 h with 5% non-fat dry milk and probed overnight with primary antibodies. After washing, membranes were labeled with horseradish peroxidase (HRP)-conjugated secondary antibodies (BioRad; Hercules, CA). Membranes were developed using a chemiluminescent substrate (Amersham Pharmacia; Piscataway, NJ) and imaged. Antibodies used were: anti-K-Ras (Millipore) and anti-GAPDH (Cell Signaling; Danvers, MA).

K-Ras Activity
K-Ras activity was measured using the Ras Activation ELISA Assay Kit (Millipore). In brief, cell lysates were incubated with Raf-1 Ras Binding Domain (RBD)-agarose. Raf-1-RBD was used to capture the active GTP-bound K-Ras protein, which was then detected by the addition of an anti-K-Ras-antibody (Millipore). An HRP-conjugated secondary antibody was then added. A luminometer was used to measure the signals after addition of a chemiluminescent reagent (Perkin-Elmer; Shelton, CT). Since assays were performed to assess the relative changes in K-Ras activity among the different TP53 genotypes, K-Ras activity from the parental TP53 +/+ line was set as 1. For assays with let-7a inhibitor, each genotype acted as its own control. Three independent assays were performed for each cell line, and the mean absorbance 6 the standard deviation (SD) was plotted for each line.

Reverse Transcription
RT-PCR was performed using the Taqman H miRNA assay (Invitrogen) following the manufacturer's protocol. Briefly, reverse transcription was performed using the provided Multiscribe RT TM Reagent master mix plus cell lysates and the let-7a specific primers (Invitrogen). Reactions were performed in a thermal cycler at 16uC for 30 min, 42uC for 30 min, and 85uC for 5 min.

Real Time-PCR
RT-PCR was performed using the TaqmanH miRNA Cells to CT TM Kit (Invitrogen) according to the manufacturer's protocol. Briefly, the RT product was added to the PCR cocktail (TaqmanH miRNA assay reagent plus provided TaqmanH master mix) and the RT-PCR reactions were run at 95uC for 10 min, followed by 40 cycles of 15 sec at 95uC and 1 min at 60uC in the Applied Biosystems 7500 fast RT-PCR system (Foster City, CA). Each sample was assayed in triplicate.

Let-7a Inhibitor
Let-7a was inhibited by miRIDIAN miRNA inhibitors (Dharmacon; Lafeyette, CO) following the manufacturer's protocol. Briefly, HCT116 cells (3610 5 ) were plated in 6-well plates and incubated overnight. They were then transfected with 100 nM of miRIDIAN miRNA inhibitor for 24 h. Cells were incubated for an additional 48 h prior to being utilized in assays.

Chemoradiation Therapy
HCT116 cells were treated with CRT according to an established protocol [22]. After a total of 72 h incubation with let-7a inhibitor, cells were plated in 6-well plates and incubated overnight. Cells were then treated with 4 nM 5-Fluorouracil (5-FU) (Sigma Aldrich; St. Louis, MO) for 16 h after which they were exposed to 4 Gy of radiation. Drug exposure was halted 6 h after radiation treatment by medium exchange. Cells that had not been treated with let-7a inhibitor were similarly treated with CRT.

Determination of Cell Viability
Cell viability was assessed using an ATP-based assay (Cell Titer-Glo; Promega; Madison, WI) as per manufacturer's protocol. Briefly, HCT116 cells (5610 3 ) were plated in 96-well plates and incubated overnight. Cell Titer-Glo reagent was added and cell lysis induced. Luminescence was recorded with a luminometer. Three independent experiments were performed. Data shown represent the average viability, as a percent of the viability of untreated cells.

Statistical Analysis
Statistical analysis was performed on Microsoft excel software (Microsoft; Redmond, WA). Each data point represents the average value of three independent experiments. Control and treatment conditions were compared by Student's t-test and differences among cell lines were compared by analysis of variance (ANOVA). P value ,0.05 was considered statistically significant.

K-Ras Protein Level is not Dependent on TP53 Mutation Status
To determine the effect of different TP53 genotypes on K-Ras expression, western blot assay was performed to compare protein levels. Our results show that K-Ras expression levels were not influenced by the different TP53 genotypes ( Figure 1A) suggesting that TP53 does not regulate K-Ras protein expression.

Effect of TP53 Mutation Status on K-Ras Activity
The effect of TP53 genotype on K-Ras activity was measured using a Raf pull-down assay. We discovered that K-Ras activity was lowest in TP53 +/+ cells. Increased K-Ras activity was most pronounced in TP53 mut/2 , followed by TP53 2/2 , TP53 +/mut , and TP53 2/+ (44%, 32%, 20%, and 10% increases, respectively, p,0.05) ( Figure 1B). These results show that K-Ras activity was lowest in cells with wild-type TP53 alleles; and the highest levels were identified in cells with homozygous loss of the wild-type TP53 alleles.

Let-7a is Expressed in All HCT116 Lines
Let-7a expression in HCT116 cell lines was measured by qPCR. Let-7a expression was detected in all cell lines (Figure 2A). There was no pattern of change in let-7a levels which corresponded to alterations in TP53 genotype.

Let-7a does not Regulate K-Ras Protein Levels
Let-7a gene expression was inhibited .90% by qPCR. K-Ras protein levels were assessed across the TP53 genotypes in the presence or absence of let-7 inhibition. K-Ras protein levels were not altered by let-7a inhibition ( Figure 2B).

Let-7a Negatively Regulates K-Ras Activity
The effect of let-7a inhibition on K-Ras activity was also determined. With let-7a inhibition, K-Ras activity increased across all TP53 genotypes (Figure 3). We found that K-Ras activity increased by 50% to 112% compared to let-7a expressing cells (p,0.05). The percent increase in K-Ras activity across all cell lines was not clearly associated with TP53 genotype and whether knockout or mutant alleles were present.
Cells with Wild-type TP53 Alleles are Sensitive to CRT and let-7a Inhibition Decreases Response to CRT Cells harboring different TP53 genotypes were treated with CRT and cell viability was measured ( Figure 4). Cells lacking any wild-type TP53 allele were resistant to CRT, whereas cells harboring a single wild-type TP53 allele exhibited approximately 50% cell death. CRT treatment was performed again with let-7a inhibition, which decreased CRT-induced cell death by nearly 20% in cells harboring at least one wild-type TP53 allele (p,0.05). Cells with homozygous loss of wild-type TP53 alleles ( 2/2 and mut/ 2 ), which correlated with highest K-Ras activity (see Figure 1B), exhibited no change in cell viability regardless of changes in let-7a expression levels.

Discussion
KRAS and TP53 are common somatic mutations with predictive and prognostic value in patients with CRC. The detection of select KRAS mutations has been associated with increased risk of disease relapse and death [23] and has predicted resistance to anti-EGFR therapies in CRC [9]. Similarly, select TP53 alterations have been associated with poor outcomes in CRC [8]. We have also reported that the concurrent detection of TP53 and KRAS mutations in patients receiving neoadjuvant CRT for rectal cancer predicted failure of pathologic complete response [10]. In this investigation, we sought to better understand a possible interaction between the two genes. We identified let-7a as a potential link between TP53 and KRAS. Prior characterization of let-7a has revealed its role in controlling cell cycle progression and division in human lung and colon cancers [24,25]. Moreover, Johnson et al. [15] discovered  TP53, let-7, and K-Ras in Colorectal Cancer Cells PLOS ONE | www.plosone.org that let-7 negatively controlled KRAS expression in lung cancer. In addition, Ruzzo et al. [17] reported an improved overall survival in patients with elevated let-7a within a KRAS mutant colorectal cancer population treated with anti-EGFR therapy, suggesting a tumor inhibitory role for let-7a in KRAS-activated tumors. TP53 has been known to transcriptionally regulate the expression of miRNAs [26,27]. Therefore, we theorized the existence of a series of regulatory steps from TP53 to let-7a to KRAS. We discovered that oncogenic K-Ras activity was regulated by TP53 genotype and let-7a; and changes in both influenced response to CRT.
In our study we utilized CRC cells harboring various TP53 genotypes. These cells were derived from a parental cell line harboring the gain of function KRAS mutation located on codon 13 [28,29]. There is rationale for utilizing cells with knockout and mutant TP53 alleles in our study. Despite the common loss of wildtype TP53 alleles, the knockout and mutant TP53 alleles are not alike. Complete loss of TP53 alleles results in corresponding loss of tumor suppressor activity while mutant TP53 alleles may express p53 proteins that lose tumor suppressor activity but also gain oncogenic properties [21]. However, in this investigation we were unable to find consistent differences between KO and mutant TP53 alleles with regards to K-Ras activity at baseline and in response to let-7a inhibition, nor in response to CRT.
As noted previously, we observed changes in K-Ras activity with inhibition of let-7a expression. However, we did not observe corresponding changes in K-Ras protein expression, which conflicts with prior reports demonstrating let-7 miRNA regulation of K-Ras expression [30,31]. Nevertheless, our findings are consistent with established regulatory mechanisms of miRNA. Indeed, miRNA can regulate genes through a variety of mechanisms from mRNA translation to stability in the cytoplasm to cell cycle activity [14,24]. The differences between our results and those of prior reports could also be due to differences between species or cell lines utilized. Thus, while K-Ras expression remained unchanged by let-7a inhibition, our experiments showed that K-Ras activity was affected by let-7a. In addition, cells harboring homozygous wild-type TP53 alleles expressed the lowest K-Ras activity, which suggests that wild-type TP53 alleles may suppress K-Ras activity. Furthermore, our results are consistent with a report in pancreatic cancer, showing an increase in K-Ras activity when mutant KRAS was paired with mutant TP53 [32]. Finally, K-Ras activity clearly increased when let-7a was inhibited regardless of TP53 genotype, further suggesting let-7a regulation of K-Ras activity.
Chemotherapy and radiation are important components of the multimodal management of surgical patients with rectal cancer; and neoadjuvant CRT has been associated with downstaging of disease and decreased local recurrence [33,34]. In our investigation, we discovered that cells lacking wild-type TP53 alleles were resistant to CRT and exhibited no cell death when treated with CRT. In contrast, cells with at least one wild-type allele (TP53 +/+ , TP53 mut/+ , and TP53 2/+ ) had approximately 50% cell death when treated with CRT. These results are consistent with the results from our prior clinical trials wherein patients with double mutations (KRAS and TP53) failed to demonstrate a pathologic complete response after treatment with neoadjuvant CRT [10]. When let-7a expression was inhibited (leading to increased K-Ras activity), cells lacking wild-type TP53 alleles (TP53 2/2 and TP53 mut/2 ) had 100% cell survival. However, in cells harboring at least one wild-type TP53 allele (previously showing some cell death), cell survival increased in response to let-7a inhibition. These results have two important implications: (1) TP53 genotype influences K-Ras activity and may predict response to CRT and (2) let-7a expression levels influence K-Ras activity and may alter response to CRT.
In summary, we provide insight into potential mechanisms linking KRAS, TP53, and let-7a. Although double mutants are relatively rare, the condition appears to have a poor phenotype with resistance to CRT. Our results contribute to a better understanding of the connection between KRAS and TP53. Knowledge of the pathways linking KRAS, TP53, and let-7a may provide greater insight into the mechanisms driving the poor phenotype observed in certain mutations in CRC. While we acknowledge that the limitations of our study include the lack of in vivo modeling and the limitations of one cell line, our focus was on the investigation of the potential cross-talk between let-7a and p53, which we clearly demonstrate in our model. It is feasible that this interaction may be cell line specific or may depend on other aberrant pathways in HCT-116 (MSI-H, PIK3 mutant). Future work will focus on investigating the potential mechanisms of interaction between let-7a and p53 across different colorectal cell lines and with different molecular profiles.