Targeting p21 to stimulate irradiated muscle stem cell function and muscle regeneration

摘要 在过去的30年里，青少年癌症患者的生存率有了显着提高。今天，80%的 儿童和青少年将在初步诊断后存活五年。然而，作为青少年癌症幸存者， 活得更长，他们经历细胞毒性癌症治疗的后遗症，直到成年。为此，少年 癌症幸存者患有早发性肌肉减少症。肌肉减少症，通常困扰老年人， 骨骼肌的严重加速流失因为放射治疗可以针对活跃的祖细胞， 幼年肌肉的辐射可能会产生长期的后果。骨骼肌是天生的 肌肉干细胞（卫星细胞，SC）的群体。在成人骨骼肌中，SC存在于静止的 状态然而，在出生后的生长过程中，SC有助于肌细胞的发育和成熟， 幼年骨骼肌我们最近已经证明了SC衍生的肌纤维在 青春期前小鼠骨骼肌的发育（Bachman et al，Development 2018）。幼年SC丢失 导致肌肉纤维尺寸和力量产生能力的立即和显著缺陷。它没有 如果对幼年鼠骨骼肌的照射可以破坏循环SC群，则已经确定。 此外，辐照SC功能障碍的后果和机制尚未阐明。我们 初步数据表明，分次放射治疗（8.2戈伊MWF）可导致立即 幼年SC库减少。然而，抗辐射SC的群体确实持续存在。这些无线电- 抗性SC在体外增殖和经历肌原性定型的能力降低。一致 由于这些缺陷，受辐射的幼年骨骼肌的再生潜力受损。内照射的 SC功能障碍与细胞周期抑制剂和DNA应激传感器p21的表达上调一致 （Cdkn1a）。因此，我建议的目标是确定辐照对幼年SC池的影响， 鉴定p21表达升高是否是辐射SC功能障碍的原因。我们将解决这些问题 目的1）确定抗辐射SC库是否主要由以下组成： 目的2）探讨分次照射是否能诱导幼仔的标记保留细胞（LRC）增殖， 骨骼肌由于内在SC功能障碍导致严重延迟的再生 p21的敲低刺激辐射SC功能和肌肉再生。总之，这些目标将使 阐明青少年放疗介导的骨骼肌衰退的细胞和分子机制， 潜在的治疗

Abstract Over the last 30 years, the survival rate of juvenile cancer patients has improved dramatically. Today, 80% of children and adolescents will survive five years beyond initial diagnosis. However, as juvenile cancer survivors live longer, they experience the after-effects of cytotoxic cancer therapies into adulthood. To this end, juvenile cancer survivors suffer from early-onset sarcopenia. Sarcopenia, normally afflicting the elderly population, is the severe and accelerated loss of skeletal muscle. Because radiation therapy can target active progenitors, it is possible that irradiation of juvenile muscle can have long-term consequences. Skeletal muscle is endowed with a population of muscle stem cells (satellite cells, SCs). In adult skeletal muscle, SCs reside in a quiescent state. However during postnatal growth, SCs contribute myonuclei to the development and maturation of juvenile skeletal muscle. We have recently demonstrated a role for SC-derived myonuclear contribution to the development of prepubertal murine skeletal muscle (Bachman et al, Development 2018). Loss of juvenile SCs results in immediate and significant deficits in muscle fiber size and force generation capacity. It is has not been established if irradiation to juvenile murine skeletal muscle can disrupt the cycling SC population. Additionally, neither the consequences nor mechanism of irradiated SC dysfunction have been elucidated. Our preliminary data indicates that that a fractionated radiation treatment (8.2 Gy MWF) can result in an immediate reduction in the juvenile SC pool. However, a population of radio-resistant SCs does persist. These radio- resistant SCs have a reduced capacity to proliferate and undergo myogenic commitment in vitro. Consistent with these deficits, irradiated juvenile skeletal muscle has impaired regenerative potential. Intrinsic irradiated SC dysfunction coincides with upregulated expression of the cell cycle inhibitor and sensor of DNA stress p21 (Cdkn1a). Thus, the goal of my proposal is to determine the impact of irradiation on the juvenile SC pool and identify if elevated p21 expression is responsible for irradiated SC dysfunction. We will address these questions in the following aims: Aim 1) To determine if the radio-resistant SC pool primarily consists of infrequently dividing label-retaining cells (LRCs) Aim 2) To examine whether fractionated irradiation of juvenile skeletal muscle leads to severely delayed regeneration due to intrinsic SC dysfunction Aim 3) To determine if knockdown of p21 stimulates irradiated SC function and muscle regeneration. Together, these aims will shed light on the cellular and molecular mechanisms of juvenile radiotherapy-mediated skeletal muscle decline and potential therapies.