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. 2019 Oct 7;218(10):3212-3222.
doi: 10.1083/jcb.201907178. Epub 2019 Sep 5.

Hair follicle regeneration suppresses Ras-driven oncogenic growth

Cristiana M Pineda  1 , David G Gonzalez  1 , Catherine Matte-Martone  1 , Jonathan Boucher  1 , Elizabeth Lathrop  1 , Sara Gallini  1 , Nathan R Fons  2 , Tianchi Xin  1 , Karen Tai  1 , Edward Marsh  1 , Don X Nguyen  3 , Kathleen C Suozzi  1 , Slobodan Beronja  4 , Valentina Greco  5   6
Affiliations

Hair follicle regeneration suppresses Ras-driven oncogenic growth

Cristiana M Pineda et al. J Cell Biol. .

Abstract

Mutations associated with tumor development in certain tissues can be nontumorigenic in others, yet the mechanisms underlying these different outcomes remains poorly understood. To address this, we targeted an activating Hras mutation to hair follicle stem cells and discovered that Hras mutant cells outcompete wild-type neighbors yet are integrated into clinically normal skin hair follicles. In contrast, targeting the Hras mutation to the upper noncycling region of the skin epithelium leads to benign outgrowths. Follicular Hras mutant cells autonomously and nonautonomously enhance regeneration, which directs mutant cells into continuous tissue cycling to promote integration rather than aberrancy. This follicular tolerance is maintained under additional challenges that promote tumorigenesis in the epidermis, including aging, injury, and a secondary mutation. Thus, the hair follicle possesses a unique, enhanced capacity to integrate and contain Hras mutant cells within both homeostatic and perturbed tissue, demonstrating that in the skin, multiple, distinct mechanisms exist to suppress oncogenic growth.

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Figures

Figure 1.
Figure 1.
Hras mutant cells outcompete wild-type neighbors yet are integrated into normal follicular tissue. All epithelial nuclei are in green (K14H2BGFP), and recombined cells are in red (tdTomato). (A) Left: Cartoon schematic depicting targeted activation to the HFSC compartment using the K19CreER system. Right: Two-photon images of Hras+/+ and HrasG12V/+ follicles during the growth phase. (B) Western blot analysis reveals that Hras signaling is active in the HrasG12V/+ K19CreER skin during the growth phase of follicle cycling as assessed by phospho-MAPK (n = 7 Hras+/+, 8 HrasG12V/+) and phospho-PI3K expression (n = 3 Hras+/+, 4 HrasG12V/+ mice). (C) Two-photon representative images of the same Hras+/+ and HrasG12V/+ follicles from first to second rest phase (∼1 mo) show a significant expansion of tdTomato in the HrasG12V/+ follicles. (D) Top: Imaris tdTomato volumetric analysis schematic showing (from left to right) a two-photon image of the HFSC during rest phase, a volumetric mask of the entire follicle, a volumetric mask of the tdTomato population within the entire follicle, and an isolated volumetric mask of only the tdTomato population. Bottom: The tdTomato expansion in HrasG12V/+ follicles is statistically significantly higher than in Hras+/+ follicles (n = 20 follicles per genotype across four mice per genotype). (E) Analysis of the average number of mitotic (left) and apoptotic (right) events per hair follicle from two-photon images reveals enhanced behaviors in HrasG12V/+ follicles yet proper temporal maintenance of these behaviors within the appropriate cycle phases (mitotic events: n = 64 follicles across 4 Hras+/+ mice, average follicle length 149.6 µm and n = 45 follicles across 5 HrasG12V/+ mice, average follicle length 166.85 µm; apoptotic events: n = 25 follicles across 5 Hras+/+ mice, average follicle length 243.27 µm and n = 40 follicles across 6 HrasG12V/+ mice, average follicle length 251.09 µm). (F) Targeted dermal papilla (DP) ablation during the rest phase locks follicles in permanent quiescence. Follicle 1 is ablated, while follicle 2 is not, and the asterisk denotes the autofluorescent halo produced after ablation (n = 50 follicles across 4 HrasG12V/+ mice, n = 30 follicles across 3 Hras+/+ mice; no ablated follicles ever regrew in either group). Insets are at higher exposure of the red channel to highlight the dermal papilla before and after ablation. (G) Cartoon summary depicting follicular HrasG12V/+ integration and obedience of normal tissue programs. For significance values, *, P < 0.05 and ****, P < 0.0001, as determined by an unpaired, two-tailed t test. Error bars indicate standard deviation and n.d. indicates not detected. All scale bars represent 50 µm.
Figure 2.
Figure 2.
The Hras mutant epithelium nonautonomously impacts the mesenchyme and follicular regeneration. All epithelial nuclei are in green (K14H2BGFP), and recombined cells are in red (tdTomato). (A) Top: Two-photon revisits reveal that K19CreER;HrasG12V/+ follicles reenter the growth phase more rapidly than K19CreER;Hras+/+ controls. Follicles are labeled A–C for ease of recognition, and cycling follicles are labeled with an asterisk. Bottom: Quantification of follicle staging over an 8-wk (W1–W8) period after tamoxifen induction (n = 1,704 follicles across 4 Hras+/+ mice and n = 1,413 follicles across 3 HrasG12V/+ mice). (B) Top: Cartoon schematic depicting the cycling and noncycling regions of the ear skin and imaged region. Bottom: Two-photon images of the hair follicles in the permanently quiescent region of skin at the tip of the ear in Hras+/+ and HrasG12V/+ mice show that while wild-type follicles do not cycle, HrasG12V/+ follicles do (observed in five K19CreER;HrasG12V/+ mice). TAM, tamoxifen. (C) In utero injection cartoon that depicts targeted lentiviral-Cre delivery to the amniotic sack at embryonic day 9.5. Injection with lentiviral-Cre into the HrasG12V/+;K14H2BGFP;tdTomato system directly tags and activates HrasG12V/+ cells in a mosaic fashion. (D) Two-photon images of Hras+/+ (left) and HrasG12V/+ (right) hair follicles during the “second rest phase” (P55). Mosaic activation of Hras using lentiviral-Cre delivery demonstrates that wild-type follicles (green) within the HrasG12V/+ skin reenter the growth phase precociously along with their Hras mutant (red) neighbors while follicles in the Hras+/+ mouse remain appropriately in rest phase (observed in n = 2 LV-Cre HrasG12V/+ mice compared with n = 3 LV-Cre Hras+/+ mice). (E) Cartoon summary depicting the enhanced regenerative power of HrasG12V/+ follicles in both cycling and noncycling skin. All scale bars represent 50 µm.
Figure 3.
Figure 3.
Follicular tolerance of Hras is maintained long-term in uninjured tissue. All epithelial nuclei are in green (K14H2BGFP), and recombined cells are in red (tdTomato). (A) Two-photon images of the epidermis (left) and hair follicles (right) of a K19CreER;HrasG12V/+;K14H2BGFP;tdTomato mouse 1 yr after activation reveal that the skin appears grossly normal and the mutant cells (in red) are still present yet contained to the HFSC niche. (B) Two-photon revisit images of the same K19CreER;HrasG12V/+ follicles immediately after dermal papilla ablation (day 0, labeled with an asterisk) and at the indicated time points up to 13 mo. Mutant cells (in red) remain present yet contained to the HFSC niche and the ablated follicles never grow unlike their unablated neighbor. (C) Tumor emergence graph for K19CreER;Hras+/+ (n = 0/9), K19CreER;HrasG12V/+ (n = 2/10, well differentiated cSCCs), and K19CreER;HrasG12V/+;TGFβfl/fl (n = 5/7, poorly differentiated cSCCs) mice monitored over the course of 1 yr after activation. (D) Hematoxylin and eosin images of K19CreER;Hras+/+ (top left) and K19CreER;HrasG12V/+ (bottom left) clinically normal back skin from 1-yr-old mice. On the right are representative images of a tumor developed in a K19CreER;HrasG12V/+ mouse 1 yr after activation. The staining reveals verrucous squamous proliferation with acanthosis, hyperkeratosis and mild atypia consistent with well-differentiated squamous cell carcinoma. (E) Photograph of a cSCC tumor from a K19CreER;HrasG12V/+;TGFβfl/fl mouse 4 mo after tamoxifen activation. (F) Hematoxylin and eosin images revealing islands of atypical keratinocytes within the dermis extending into subcutaneous fat with marked atypia and associated mixed inflammatory infiltrate consistent with moderately differentiated, necrotic invasive squamous cell carcinoma. (G) Two-photon images of the ear skin from the same mouse from E and F at day 0 and 4 mo after tamoxifen activation reveal normal follicular architecture despite the persistent presence of mutant cells (tdTomato+). Follicles are labeled A–H as landmarks. Scale bars in two-photon images represent 50 µm. Scale bars in histology images represent 200 µm.
Figure 4.
Figure 4.
Follicles are resistant to oncogenic growth, even in the face of additional tumor promoting insults. All epithelial nuclei are in green (K14H2BGFP), and recombined cells are in red (tdTomato). (A) Cartoon schematic detailing the various approaches for testing the buffering effect. (B–D) Two-photon revisits show that targeted ablation of the bulb region in Hras+/+ (B), HrasG12V/+ (C), and HrasG12V/+;TGFβfl/fl (D) follicles during the growth phase results in follicular deformation and subsequent regeneration. (E) Mutant follicles rapidly regenerate after injury, and within 1 mo after ablation, ∼75% of mutant follicles are cycling again compared with only 35% of wild-type follicles (n = 23 Hras+/+ follicles across two mice, n = 34 HrasG12V/+ across four mice, n = 19 HrasG12V/+;TGFβfl/fl follicles across three mice). All error bars indicate standard deviation. (F) Cartoon summary depicting rapid, normal regeneration of HrasG12V/+ after injury. All scale bars represent 50 µm.
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