Targeting signaling pathways downstream of Ras
Given that patients taking PLX4032 and similar B-Raf inhibitors as a treatment for melanoma often develop SCCs, targeted therapeutics are needed in order to prevent this unfortunate side effect. Our mouse model provides a means to understand the intrinsic factors unique to HF stem cells that are necessary for SCC initiation, and thereby allows for the determination of potential targets downstream of Ras/Raf signaling for chemoprevention.
To screen for signaling pathways downstream of Ras that could be activated during the initiation of hyperplasia and/or during epithelial to mesenchymal transition (EMT) in K15-CrePR; KrasG12D and K15-CrePR; KrasG12D; p53ff mice, candidates were selected based on known downstream effectors (). We examined several signaling pathways downstream of Ras, including Map Kinases (Erk and p38) and Akt.
Figure 1. Ras signaling pathways examined in SCC prone KrasG12D induced hair follicle stem cells and in SCC resistant KrasG12D induced hair follicle transit amplifying cells. P-Erk, p-Akt, p-S6 and p-p38 stained hair follicle hyperplasia and cyst (more ...)
First, Erk1/2 activation was examined by IHC for phosphorylated Erk (p-Erk). This signaling effector of the Ras pathway was found at high levels in hyperplastic hair follicles and the basal cells of the epidermal cyst structures of skin with KrasG12D
expression originating from HF stem cells. This indicates that administration of an inhibitor of MEK, an upstream regulator of Erk1/2 activity, might provide a preventative response to KrasG12D
induced tumorigenesis. AZD6244 is one such inhibitor that may prove useful.21
This potential target is further supported by previous transgenic animal studies that manipulated MEK activity.22,23
We also examined the p38 arm of the Ras signaling by p-p38 staining. Though this marker was detected during hyperplasia and in epidermal cysts, it was also found throughout the hair follicle in control skin. This indicates that attempting to inhibit this pathway may not be useful, as it may affect normal skin homeostasis.
Second, we examined the Akt arm of Ras signaling. Using IHC for p-Akt, it was determined that Akt signaling was indeed found in some hyperplastic hair follicles and in epidermal cyst structures. Further downstream of Akt, we examined both phosphorylated mTor and phosphorylated NFκB. Phospho-mTor was evident in hyperplastic hair follicles and cyst structures at low levels compared with the robust activity of p-Akt. Rapamycin, a potent inhibitor of mTor signaling, has been suggested as a potential chemopreventative agent by studies in head and neck squamous cell carcinomas and murine chemical carcinogenesis.24-26
Rapamycin, or a similar analog, may have some preventative effect in the initial stages in tumorigenesis in our model and in patients with Kras-inducing SCC. To examine another output of Akt signaling, we examined p-NFκB. NFκB signaling has been implicated in a wide range of tumorigenesis processes, including EMT and inflammation.27
p-NFκB was also detected during tumorigenesis initiation in this model system. Bortezomib and Bay-117082, inhibitors of NFκB signaling, have recently been shown to be effective in inhibiting tumorigenesis in a model of lung cancer.28
Notably, this lung cancer model utilizes the same genetic insults we used in our mouse model system. Additionally, bortezomib has been shown to have some limited effect on human cases of head and neck squamous cell carcinomas.29
This suggests that these inhibitors may also be useful in our model of cutaneous SCC.
This examination of Ras signaling indicates that the inhibition downstream of Kras at the level of MEK, mTor or NFκB individually, or in combination, may represent a chemopreventative therapeutic regimen that can be administered simultaneous with tumor initiation in our mouse model of SCC. If successful, this may represent a potentially useful method to inhibit SCC formation in patients taking B-Raf inhibitors or in patients with compromised immune systems. However, activation of ERK, AKT and mTor (p-Erk1/2, p-Akt and p-mTor) was not detected in cells of the bona fide SCC, suggesting that these downstream pathways are not required to sustain the dedifferentiated state. This striking observation indicates that inhibition of these pathways may not be therapeutically useful following the onset of SCC in our model or in patients presenting with high grade Kras-derived SCCs. How the cancer cells evolve to shed the necessity for activity of pathways downstream of Ras or utilize alternate Ras signaling pathways warrants further investigation.
Identifying the molecular basis of sensitivity to Ras activity
In an alternate approach, since the direct descendants of the hair follicle stem cells are completely refractory to KrasG12D and KrasG12D; p53KO induced tumorigenesis, a molecular comparison between transit amplifying cells and the parental stem cells could point toward new targets for tumorigenesis prevention. Though very closely related in hierarchy, the intrinsic properties that facilitate tumorigenesis have been lost during the transition to the transit amplifying cell type. To reveal the nature of these intrinsic properties, cell populations purified just following induction of KrasG12D expression alone and/or with p53KO could be compared in detail on the genetic, epigenetic, transcriptome and proteome levels. Novel or known mediators of Kras signaling not found in the transit amplifying population could provide targeting candidates for further exploration.
Targeting EMT to SCC progression
mouse models are excellent systems to study KrasG12D
induced epithelial to mesenchymal transition (EMT). EMT is thought to be a necessary precursor to invasiveness and metastasis, and this process results in the spindle shaped cells of the SCC produced in K15-CrePR
In both K15-CrePR
skin, hair follicle stem cells undergo EMT following a brief phase of hyperplasia. This has been concluded by antibody staining of ectopic Tenascin-C, high levels of Vimentin, ectopic Keratin 8 and more recently, by ectopic N-cam staining (unpublished data). By purifying these cells from initiation of hyperplasia through induction of EMT, a detailed transcriptome and proteome profile can be generated. Since our model system can also incorporate a LSL-Yfp
allele that generates YFP expression exclusively in KrasG12D
expressing cells, we can be confident that cells expressing these markers were once epithelial cells. This is important in order to distinguish them from nearby cancer associated fibroblasts, which express many of the same markers. These data could yield a wealth of information from an in vivo cancer that is undergoing crosstalk with its naturally occurring microenvironment, which contrasts to traditional xenograft studies, which creates an unnatural environment with crosstalk cues that may or may not be truly representative. Theoretically, if EMT can be pharmacologically blocked, the tumor cells may revert to a more keratinocyte-like nature, which could thus be redirected from the path toward squamous cell carcinoma and instead become terminally differentiated skin cells.
Finally, though EMT is found in K15-CrePR; KrasG12D skin, these cells do not proliferate into bona fide SCC. Only in K15-CrePR; KrasG12D; p53ff skin do transformed cells undergo a switch to high proliferation and then to SCC development. A comparison between these two mouse models may inform on how this switch occurs. The nature of the pathways induced by KrasG12D in the context of p53KO in hair follicle stem cells may further provide novel targets for reversion back to a non-proliferating cell.