Fig. 4: Wild-type p53 and oncogenic Ras paracrine STAT activation stimulates the growth of human cancer cells.

(a–j) In vitro experiments showing that media conditioned with p53-overexpressing or irradiated cancer cells stimulate cell proliferation via STAT signaling. Western blot of protein lysates prepared from MCF-10A cells expressing oncogenic HRas (Ras^ONC) or wild-type p53 (p53^OE) alone or together and blotted with anti-p53 (a) or anti-pERK (b) or anti-GAPDH (as loading control). (c) Western blots showing STAT activity in MCF-10A cells cultured in media conditioned with MCF-10A (controls) or with MCF-10A cells expressing Ras^ONC or p53^OE alone or together. (d) p53 or GAPDH western blot images from MCF-10A cells expressing oncogenic Ras or not under normal or irradiated conditions. (e) Western blot image showing STAT and GAPDH or phospho-STAT and GAPDH in cells conditioned with media conditioned with irradiated MCF-10A cells or MCF-10A cells expressing Ras^ONC from (d). (f) Conditioned media stimulate cell growth, as determined by cell number under the indicated conditions: MCF-10A cells cultured in media conditioned with MCF-10A control cells or MCF-10A cells overexpressing p53^OE or Ras^ONC or coexpressing both. Error bars denote SD values. P values are derived from Student’s t test analyses. (g) The growth of MCF-10A cells in media conditioned with irradiated MCF-10A cells in the presence or absence of p53 overexpression is shown. Ganetespib (25 nm) treatment suppressed cell growth. Error bars denote SD values. P values are derived from Student’s t test analyses. (h) Western blots showing p53 expression in lung cancer cells transfected with p53 expression construct or not. GAPDH represents the loading control. (i, j) Western blot images showing STAT activity (pSTAT) in lung cancer cells cultured in media conditioned with unmodified cells or with cells overexpressing p53. Total STAT and GAPDH protein levels were used as loading controls. These experiments are shown in biological triplicates in (j). (k) Effect of media conditioned with p53 overexpressing lung cancer cells on cell number in the presence or absence of Ganetespib. Ganetespib (25 nm) treatment had no to minimal effect on the growth of control cells but it significantly suppressed the growth of cells growing in conditioned media. (l) Effect of media conditioned with irradiated lung cancer cells on cell number in the presence or absence of Ganetespib (25 nm). (m) Western blot images showing p53 expression in A549 cells under normal and irradiation conditions. GAPDH represents a loading control. (n) Western blot image showing total STAT and pSTAT levels in A549 cells grown in media conditioned with other A549 cells or with irradiated A549 cells. (o) Image of a nude mouse showing the size of tumor xenografts (green circles) 8 weeks after flank injection of 1 × 10⁶ A549 cells. Left flank inoculants consisted of normal A549 cells, while right flank received an equal mixture of normal and irradiated A549 cells. (p) Quantification of tumor size from (o). Sample size N = 06 animals per group. Tumor sizes (0.523 × length × width × height) were calculated with a digital caliper. To the exception of one animal (animal ID:2451) that developed a larger tumor on the left flank (homogenous inoculants), the remaining five animals developed noticeably larger tumors from the heterogenous inoculants. (q) Graphical representation of right to left flank tumor size ratio. At 8 weeks post inoculation, two of the five animals received Ruxolitinib (10 mg/kg) by oral gavage for 3 weeks. The remaining animals were treated with DMSO vehicle control for the same duration. Caliper measurements determined tumor size in treated versus vehicle control animals. Right to left tumor size ratios are shown at 1 and 2 weeks following treatment initiation. (r) Western blot from lysates derived from tumor xenografts harvested from animals treated with Ruxolitinib or DMSO vehicle controls were probed with phospho-STAT or STAT or GAPDH antibodies. All error bars denote SD. P values are derived from Student’s t test analyses. Effect size (d) values for f, g, k and l are greater than 0.8.