Wounding mobilizes hair follicle stem cells to form tumors

Abstract

A wide variety of human cancers are associated with injury. Although stem cells participate in tissue regeneration after wounding, it is unclear whether these cells also contribute to epithelial tumors. Human basal cell carcinomas (BCCs) are associated with misactivation of Hedgehog (Hh) signaling, commonly through acquisition of mutations in Smoothened (Smo). We have found that expression of an activated form of Smo by stem cells of the hair-follicle bulge and secondary hair germ does not induce robust Hh signaling or produce BCCs. However, wounding recruits these cells from the follicle to the wound site, where downstream Hh signal transduction is derepressed, giving rise to superficial BCC-like tumors. These findings demonstrate that BCC-like tumors can originate from follicular stem cells and provide an explanation for the association between wounding and tumorigenesis.

Fig. 1.

SmoM2-expressing bulge or SHG-derived cells form superficial BCC-like tumors at sites of injury. (A) Intact K15:SmoM2 dorsal skin, 11 wk after oncogene induction, displays largely normal IFE and telogen hair follicles, similar to wild-type skin. (B) Magnified view of the boxed area in A, showing a follicle with a focally hyperplastic bulge (arrow). (C) RU486-induced K15:SmoM2 skin, 10 wk after wounding, possesses superficial BCC-like tumors (arrows) at the site of the healed wound (dashed line). Control skin does not form tumors after wounding. (D) Magnified view of BCC-like tumors in C. (E) β-Gal staining indicating that K14-Cre^ERT and K15-Cre^PR1 exhibit largely reciprocal domains of activity, irrespective of SmoM2 expression, 3 d post induction. (F) Quantitation of β-Gal+ cell location 3 d after induction. (G) Intact K14:SmoM2:LacZ dorsal skin, 11 wk after oncogene induction, contains extensive IFE hyperplasia and BCC-like downgrowths (arrows), without obvious involvement of the bulge (Inset). (H) K14:SmoM2:LacZ skin, 10 wk after wounding, exhibits BCC-like downgrowths equally abundant at the healed wound site (dashed line) and in intact skin. (I) Magnified view of tumors from wounded K14:SmoM2:LacZ skin. (J) Quantitation of BCC-like neoplastic downgrowths, either at the healed wound site or in intact skin (*P = 0.017). G–I were stained for LacZ expression. In non–LacZ-expressing control animals, no consistent β-Gal staining was detected (data not shown).

Fig. 2.

Wounding induces the migration of bulge and SHG-derived cells into sites of injury. (A) β-Gal+ bulge and SHG-derived cells from K15:LacZ and K15:SmoM2:LacZ skin migrate into the epithelial tongue (asterisk) at the leading edge of the contracting wound, 3 d after injury. (Right) Magnified views of the boxed areas. (B) β-Gal staining of healed dorsal skin, 10 wk after wounding, indicates that labeled cells infrequently persist in K15-Cre^PR1 skin. (Right) Infrequently retained bulge or SHG-derived β-Gal+ cells contribute normally to the healed epithelium in the absence of oncogene expression but form tumors in the presence of SmoM2. (C) Immunofluorescence staining of normal telogen follicles (Left), IFE-derived tumors of K14:SmoM2 skin (Center), and wound-induced tumors of K15:SmoM2 skin (Right).

Fig. 3.

SmoM2-expressing bulge and SHG cells fail to activate robust Hh signaling. (A) In situ staining for Gli1 in wound-induced tumors from K15:SmoM2 skin. (B) β-Gal staining reveals that cells that had undergone K15-Cre^PR1-mediated recombination are retained in the bulge and SHG of unwounded skin for at least 10 wk, irrespective of SmoM2 expression. (C) In situ staining for Gli1 in wild-type telogen skin (Left) and unwounded K15:SmoM2 skin (Right). Arrow indicates the site of normal Gli1 expression near the SHG and dermal papilla. (D) Gli1 is modestly up-regulated at a site of focal hyperplasia in the bulge of unwounded K15:SmoM2 skin (arrowhead). (E) Immunofluorescence staining for the YFP tag of SmoM2 and Gli2 in K15:SmoM2 wound-induced tumors (Top), in unwounded K15:SmoM2 follicles (Middle), and in non-SmoM2-expressing control follicles (Bottom). Arrows indicate the SmoM2-expressing cells from unwounded K15:SmoM2 follicles (blue, DAPI).

Fig. 4.

Bulge or SHG-derived BCC-like tumors are induced by incisional or excisional wounding but not by depilation. (A) β-Gal staining indicates that bulge and SHG-derived cells that had undergone K15-Cre^PR1-mediated recombination contribute to cells of the lower anagen bulb in hyperplastic follicles from depilated K15:SmoM2:LacZ skin. (B Upper) Follicles of K15:SmoM2 skin are grossly normal 10 wk after depilation. Note that anagen hair follicles had subsequently progressed to telogen without developing aberrations, aside from occasional focal hyperplasia (Right, arrows). (Lower) β-Gal staining demonstrates that bulge and SHG cells that had undergone K15-Cre^PR1-mediated recombination are retained in K15:SmoM2 skin 10 wk after depilation. (C) BCC-like tumors arise from healed excisional wound sites of K15:SmoM2 skin injured 5 wk after induction of SmoM2. (D) Tumors (arrows) arise at the healed incisional wound junction of K15:SmoM2 mice 10 wk after injury but do not form in healed control K15-Cre^PR1 skin.