In vitro cell-line model to evaluate oncogenic functions associated with the HOXB13 protein with X285K variant
A few recent reports uncovered a HOXB13 variant (X285K) predisposing to prostate cancer in men of West-African ancestry. The protein function associated with this inherited variant is unknown. To elucidate the oncogenic mechanisms of the X285K protein, we designed an in vitro cell-line model to evaluate oncogenic functions associated with the X285K protein. Replacement of the wild-type HOXB13 protein with the X285K protein resulted in a gain of the E2F/MYC signature, validated by a gain in the expression of Cyclin B1 and c-MYC, without affecting the androgen response signature.  In conclusion, Functional studies revealed a unique gain-of-function oncogenic mechanism of X285K protein in regulating E2F/MYC signatures. Overall design: To study the functions mediated by X285K, we devised a strategy to replace the wild-type (WT) HOXB13 with X285K in LNCaP95, a castration-resistant prostate cancer (CRPC) cell line. Treatment with doxycycline (Dox) will displace the endogenous WT HOXB13 with exogenous WT or X285K HOXB13. A total of 11 stable clones, including five LN95WT (exogenous WT would replace endogenous WT after DOX), three LN95X285K (exogenous X285K would replace endogenous  WT after Dox), and three LN95NT (non-targeting vector) were generated. Each of the 11 clones was subjected to 4 different treatment conditions, with (Dox+) or without (Dox-) Dox (sample named are annotated as DoxP or DoxN), and in the absence (R1881-) or presence (R1881+) of synthetic androgen R1881 (sample names with R if R1881 is added). We generated RNA-sequencing (RNA-Seq) data from these 44 cell line samples.