YAP1 dysfunction promotes molecular properties linked to breast cancer susceptibility

YAP1 is a co-transcription factor that promotes malignant and stem cell properties in cancer. We previously found that YAP1 dysregulation is associated with aging in human mammary epithelia. With increased age, YAP1 expression changes in luminal epithelial cells, the prospective breast cancer cell of origin. Because age is a significant risk factor for breast cancer, we tested if YAP1 dysregulation acted early in cancer progression by conferring cellular states associated with increased cancer susceptibility. Here we find, that with increased age and genetic risk for developing cancer, human breast tissues showed significantly increased YAP1 expression and cultured primary human mammary epithelial cells (HMEC) showed significantly increased expression of both YAP1 and its transcriptional targets. Increased YAP1 expression in cultured HMEC induced gene expression changes associated with increased cancer susceptibility such as genes associated with: a stem cell fate, cells with increased telomerase activity, breast cancer progression, and increased age and genetic breast cancer risk. Further, overexpression of YAP1 in post-stasis HMEC- finite lifespan cells which have bypassed a retinoblastoma-mediated senescence barrier- promoted properties related to an increased growth potential. We found that YAP1 dysregulation in finite post-stasis epithelial cells allows for access to gene programs and functions that are typically thought to be restricted to stem cells. We hypothesize that YAP1 acts early in breast cancer progression, before development of a tumor, to impose cancer susceptible molecular states. Overall design: General experimental design: Post-stasis human mammary epithelial cells (HMEC) were cultured in tissue culture plastic in M87A media. YAP1 or control constructs were transduced into cells to determine the effect of YAP1 on post-stasis HMEC. In some samples, MYC was further added to induce immortalization. In some samples, clonal immortalization events occurred. Cells were collected at different passages during post-stasis and immortal growth. Experiments were done with 2 different biological specimens: 184D and 240LB. Biological Replicate 1: Specimen 240LB. Cells were transduced with p16sh to make cells post-stasis. Control post-stasis cells were collected at passage 11. YAP1 was transduced into cells at passage 10 and cells were collected at passage 12. YAP1 transduced cultures underwent clonal immortalization around passage 18 when they were collected. Fully immortal YAP1 transduced cells were collected at passage 25. Biological Replicate 2: Specimen 240LB. Cells were transduced with p16sh to make cells post-stasis. YAP1 or control virus was transduced into cells at passage 10 and cells were collected at passage 12. YAP1 transduced cultures underwent clonal immortalization around passage 16 when they were collected. Fully immortal YAP1 transduced cells were collected at passage 22. Biological Replicate 3 (All samples collected in triplicate): Specimen 184D. Cells were transduced with p16sh to make cells post-stasis. YAP1 or control virus was transduced into cells at passage 10 and cells were collected at passage 12. Control cells approached replicative senescence at passage 18; control and YAP1 transduced cells were collected at passage 17. YAP1 transduced cultures underwent rare immortalization event(s) around passage 18 when they were collected. Fully immortal YAP1 transduced cells were collected at passages 20 and 24. Control and YAP1 post-stasis cells were also transduced with MYC at passage 12 and immortalized cells were collected at passages 15 and 21.

YAP1是一种共转录因子（co-transcription factor），可促进癌症的恶性表型与干细胞特性。既往研究发现，YAP1表达失调与人类乳腺上皮细胞的衰老相关。随着年龄增长，作为乳腺癌潜在起源细胞的腔上皮细胞中，YAP1的表达会发生改变。鉴于年龄是乳腺癌的重要风险因素，本研究探究了YAP1失调是否通过赋予细胞更高的癌症易感性状态，在癌症进程早期发挥作用。

本研究发现，随着年龄增长与癌症遗传风险升高，人类乳腺组织中YAP1的表达显著上调；而培养的原代人类乳腺上皮细胞（human mammary epithelial cells, HMEC）中，YAP1及其转录靶基因的表达均显著升高。在培养的HMEC中，YAP1表达上调可诱导与癌症易感性升高相关的基因表达改变，包括干细胞命运相关基因、端粒酶活性增强的细胞相关基因、乳腺癌进程相关基因，以及与年龄增长和乳腺癌遗传风险升高相关的基因。

此外，在停滞后期（post-stasis）的HMEC——即绕过了视网膜母细胞瘤介导的衰老屏障的有限增殖寿命细胞——中过表达YAP1，可促进与生长潜能增强相关的表型。本研究发现，有限增殖能力的停滞后期上皮细胞中YAP1失调，可使其获得通常仅干细胞才具备的基因程序与功能。我们提出假说：YAP1可在肿瘤发生前的乳腺癌进程早期，赋予细胞癌症易感性的分子状态。

## 实验设计
总体实验方案：将停滞后期的人类乳腺上皮细胞（HMEC）于组织培养塑料皿中，以M87A培养基进行培养。将YAP1或对照载体转导至细胞中，以探究YAP1对停滞后期HMEC的影响。部分样本中额外转入MYC以诱导细胞永生化；部分样本可发生克隆性永生化事件。在停滞后期及永生化生长阶段的不同传代时期收集细胞。本实验使用2种不同的生物标本：184D与240LB。

1. 生物学重复1：标本240LB。通过转导p16sh使细胞进入停滞后期。于第11代收集对照停滞后期细胞。于第10代将YAP1转导至细胞中，并于第12代收集细胞。YAP1转导的细胞培养物在第18代左右发生克隆性永生化，此时收集细胞；于第25代收集完全永生化的YAP1转导细胞。
2. 生物学重复2：标本240LB。通过转导p16sh使细胞进入停滞后期。于第10代将YAP1或对照病毒转导至细胞中，并于第12代收集细胞。YAP1转导的细胞培养物在第16代左右发生克隆性永生化，此时收集细胞；于第22代收集完全永生化的YAP1转导细胞。
3. 生物学重复3（所有样本均进行三次重复收集）：标本184D。通过转导p16sh使细胞进入停滞后期。于第10代将YAP1或对照病毒转导至细胞中，并于第12代收集细胞。对照细胞在第18代左右进入复制性衰老，故于第17代收集对照细胞与YAP1转导细胞。YAP1转导的细胞培养物在第18代左右发生罕见的永生化事件，此时收集细胞；于第20代与第24代收集完全永生化的YAP1转导细胞。于第12代将MYC转导至对照与YAP1转导的停滞后期细胞中，并于第15代与第21代收集永生化细胞。