Hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole-rat

Abstract

The naked mole-rat is the longest living rodent with a maximum lifespan exceeding 28 years. In addition to its longevity, naked mole-rats have an extraordinary resistance to cancer as tumors have never been observed in these rodents. Furthermore, we show that a combination of activated Ras and SV40 LT fails to induce robust anchorage-independent growth in naked mole-rat cells, while it readily transforms mouse fibroblasts. The mechanisms responsible for the cancer resistance of naked mole-rats were unknown. Here we show that naked mole-rat fibroblasts display hypersensitivity to contact inhibition, a phenomenon we termed "early contact inhibition." Contact inhibition is a key anticancer mechanism that arrests cell division when cells reach a high density. In cell culture, naked mole-rat fibroblasts arrest at a much lower density than those from a mouse. We demonstrate that early contact inhibition requires the activity of p53 and pRb tumor suppressor pathways. Inactivation of both p53 and pRb attenuates early contact inhibition. Contact inhibition in human and mouse is triggered by the induction of p27(Kip1). In contrast, early contact inhibition in naked mole-rat is associated with the induction of p16(Ink4a). Furthermore, we show that the roles of p16(Ink4a) and p27(Kip1) in the control of contact inhibition became temporally separated in this species: the early contact inhibition is controlled by p16(Ink4a), and regular contact inhibition is controlled by p27(Kip1). We propose that the additional layer of protection conferred by two-tiered contact inhibition contributes to the remarkable tumor resistance of the naked mole-rat.

Fig. 1.

Assay of anchorage-independent growth in mouse and naked mole-rat skin fibroblasts. Cells were transfected with the vectors encoding different forms of LT antigen (LT, LTK1, and LTΔ434–44) and oncogenic Ras, and plated in soft agar. Figure shows representative microphotographs of colonies generated after 3 weeks at magnification ×20. (A) Mouse skin fibroblasts. (B) Naked mole-rat skin fibroblasts. (C) NMRSF2, a mutated line of naked mole-rat skin fibroblasts that lost early contact inhibition.

Fig. 2.

Early contact inhibition in naked mole-rat fibroblasts. Growing and confluent naked mole-rat (A) and mouse (B) fibroblasts. Naked mole-rat fibroblasts arrest cell proliferation when few cell-cell contacts are formed, a phenomenon we termed early contact inhibition. Naked mole-rat cells do not form a dense monolayer like the mouse cells. NMR SF, naked mole-rat skin fibroblasts; NMR LF, naked mole-rat lung fibroblasts; MSF, mouse skin fibroblasts, MLF, mouse lung fibroblasts.

Fig. 3.

Naked mole-rat fibroblasts reach lower confluent cell density than mouse fibroblasts. (A) Comparison of cell densities attained by naked mole-rat and mouse skin fibroblasts. Cells were seeded on grided plates, and cell numbers were counted daily for each plate. MSF, mouse skin fibroblasts; NMRSF Mut, naked mole-rat skin fibroblast line that lost early contact inhibition; NMRSF, normal naked mole-rat skin fibroblasts. (B) Comparison of cell densities attained by naked mole-rat and mouse lung fibroblasts. MLF, mouse lung fibroblasts; NMRLF, naked mole-rat lung fibroblasts. Cells were seeded on grided plates and cell numbers were counted daily. The experiments were repeated at least four times, using fibroblast lines from different animals (except for NMRSF Mut). Error bars, standard deviations. (C) Analysis of DNA synthesis by thymidine incorporation in growing and confluent naked mole-rat and mouse cells. Early contact inhibited cells do not synthesize DNA. The experiments were repeated three times, and standard deviations are shown.

Fig. 4.

Inactivation of both p53 and pRb pathways is required to abolish early contact inhibition. Naked mole-rat skin (NMR SF), lung (NMR LF), mouse skin (MSF), and lung (MLF) fibroblasts were transfected with plasmids encoding SV40 Large T antigen (LT), or LT mutants LTK1 that targets only p53 family (K1), or LTΔ434 that targets only pRb-family (Δ434). Cells were seeded on grided plates, and cell numbers were counted daily. (A) SV40 Large T antigen but not K1 or Δ434 derivatives abrogates early contact inhibition in naked mole-rat skin fibroblasts (NMR SF) and causes the cells to grow to high density. (B) SV40 Large T antigen but not K1 or Δ434 derivatives abrogates early contact inhibition in naked mole-rat lung fibroblasts (NMR LF) and causes the cells to grow to high density. (C) SV40 Large T antigen and its mutants do not affect cell density of mouse lung fibroblasts (MLF). (D) SV40 Large T antigen and its mutants do not affect cell density of mouse skin fibroblasts (MSF). All of the experiments were repeated at least three times, and error bars show, standard deviations.

Fig. 5.

Analysis of apoptosis in naked mole-rat cells transfected with SV40 large T antigen (LT) or its mutant derivatives K1, which binds p53 family of proteins, and Δ434, which binds pRb family members. (A) Apoptosis in naked mole-rat skin fibroblasts (NMR SF) transfected with the plasmids encoding large T antigen constructs. Apoptosis was assayed on days 8 (black bars) and 14 (white bars) after transfection, using TUNEL. Day 14 is the time when cells start entering early contact inhibition. Striped bars correspond to cells grown in the presence of 5 μM apoptosis inhibitor Z-VAD-FMK for 14 days. (B) Same experiment as in (A) done with naked mole-rat lung fibroblasts (NMR LF). (C) Apoptosis in mouse skin fibroblasts (MSF) transfected with the plasmids encoding large T antigen constructs. Apoptosis was assayed on days 8 (black bars) and 14 (white bars) after transfection, using TUNEL. (D) Same experiment as in (C) with mouse lung fibroblasts (MLF). (E) Analysis of cell proliferation in naked mole-rat skin fibroblasts in the presence of 5 μM apoptosis inhibitor Z-VAD-FMK. Corresponding growth curves without Z-VAD-FMK are shown in Fig. 3A. Cells were transfected with the plasmids encoding large T antigen constructs and seeded on grided plates. Cell numbers were counted daily. The experiments were repeated at least three times and error bars show standard deviations. (F) Analysis of cell proliferation in naked mole-rat lung fibroblasts in the presence of ZVAD-FMK as described in (E). Corresponding growth curves without ZVAD-FMK are shown in Fig. 3B. All experiments were repeated three times, and error bars, standard deviations.

Fig. 6.

Early contact inhibition in naked mole-rat cells is associated with accumulation of p16 while regular contact inhibition is associated with accumulation of p27. Human SF, human skin fibroblasts; MLF, mouse lung fibroblasts, MSF, mouse skin fibroblasts, NMR LF, naked mole-rat lung fibroblasts; NMR SF, naked mole-rat skin fibroblasts; NMR SF2 Mut, mutated naked mole-rat skin fibroblast line that lost early contact inhibition. The number at the end of the cell line name corresponds to the animal it was derived from. g, growing cells; c, confluent cells. (A) Western blot analysis of p27 in growing and confluent human, naked mole-rat, and mouse fibroblasts. p27 is strongly induced in cells undergoing regular contact inhibition such as human, mouse, or mutated naked mole-rat cells. The identity of the naked mole-rat protein was verified with two different antibodies. (B) Western blot analysis of p16 in growing and confluent human, naked mole-rat, and mouse fibroblasts. p16 accumulates in early contact inhibited naked mole-rat fibroblasts but not in human, mouse, or mutated naked mole-rat cells. The identity of the naked mole-rat protein was verified with two different antibodies. (C) p16 accumulates in naked mole-rat cells seeded at high density (HD). The first two lanes show growing cells (day 7) and confluent cells (day 20). The next three lanes correspond to naked mole-rat cells that were seeded at high density. The number above the lane indicates days after plating. (D) pRb becomes hypophosphorylated in early contact inhibited naked mole-rat cells.

Fig. 7.

A model comparing contact inhibition in naked mole-rat to mouse and human. Naked mole-rat cells have two tiers of contact inhibition: early contact inhibition mediated by p16 and regular contact inhibition mediated by p27. In contrast, human and mouse only have regular contact inhibition. The presence of two-tiered contact inhibition may provide naked mole-rats an increased protection against tumor development.