P53 mutations in stromal fibroblasts sensitize tumors against chemotherapy

Authors


Abstract

The efficacy of chemotherapy is usually viewed as the outcome of cancer-cell-autonomous processes while the contribution of stroma is being overseen. Here we show that p53 mutations in stromal fibroblasts, a genetic lesion that is detectable in primary breast, prostate and probably other cancers, while they accelerate tumorigenesis they also sensitize tumours against conventional chemotherapy by doxorubicin and cis-platinum. The mechanism by which p53 of stromal fibroblasts affects the response of a tumour against chemotherapy is likely to involve the induction of senescence in the fibroblasts which in turns results in the production of growth factors acting onto the cancer cells by paracrine mechanisms. Our findings identify stromal fibroblasts as important modulators of the efficacy of anticancer therapy. © 2008 Wiley-Liss, Inc.

Fibroblasts represent the major cellular component of cancer-associated stroma. Although their role in accelerating cancer growth, and in certain instances even to cause malignant conversion, has been demonstrated, the molecular factors regulating this process remain largely unknown.1–4 Mutations in p53 tumour suppressor gene, besides their presence and well studied cell-autonomous role in cancer cells, have also been described in stromal fibroblasts of breast and prostate cancers in humans and experimental animals.5–8 Recently, by performing a series of tumour reconstitution experiments, involving the co-inoculation of breast cancer cells with fibroblasts differing in their p53 status, we showed that p53 mutations in stromal fibroblasts, which represent a common lesion in primary breast and other cancers, exert a positive effect on cancer growth.9 This tumour-promoting role of stromal p53 deficiency, which was subsequently confirmed in a transgenic mouse model of prostate cancer involving the assessment of primary malignant lesions,6 suggests that at least in certain cases the transition of stromal fibroblasts into a cancer associated state may be associated with the acquisition of p53 deficiency.6, 7, 9 Although the precise mechanism for this p53-dependent paracrine stimulation of tumour growth by stromal fibroblasts remains poorly understood, it is likely to involve the differential regulation of secreted growth factors such as the stromal cell-derived factor 1.10 Consistently with this notion, recently published results of proteomic analyses have demonstrated the p53-controlled post-translational modifications on certain secreted proteins, providing mechanistic basis on the paracrine communication between cancer cells and stromal microenvironment.11

Besides its well-established role in the development of the disease, p53 is also considered as a major regulator of the efficacy of anticancer therapy.12, 13 This action of p53, in the regulation of the efficacy of anticancer therapy, apparently is not only limited to the cell-autonomous action of p53 within the cancer cells but is also extended to the tumour stroma and particularly the vascular endothelium at which repression of p53 sensitizes p53-deficient tumours to experimental radiotherapy and chemotherapy.14 These findings prompted us to address if p53 mutations in stromal fibroblasts can also modulate the response of a tumour against conventional chemotherapy.

Material and methods

Mice, xenograft development and treatment

SCID mice were originally obtained by Jackson laboratories (ME) and subsequently maintained in our laboratory. Care of animals was in accord to Institutional guidelines. For xenograft development 2.1 × 106 MCF7 breast or PC3 prostate cancer cells and 0.7 × 106 MEFs per mouse were re-suspended in 0.1 ml of serum-free DMEM and then injected s.c. in SCID mice. Subsequently, animals were observed daily for tumour development. Female animals were used for the inoculation of MCF7 breast cancer cells while male mice were used for the inoculation of the PC3 prostate cancer cells. About 6–8 week old animals were used for all experiments. As soon as tumours became palpable, mice were treated by a single i.p injection of doxorubicin (DOX) (4 mg/Kg) or cis-platinum (5 mg/kg). The period following treatment until tumours became undetectable by palpation was scored. All experiments have been repeated at least three times independently (n = 4–7 animals per group).

Cell culture and proliferation assays

Human mammary epithelial adenocarcinoma cells MCF7, and PC-3 prostate adenocarcinoma cells were originally obtained from American Type Culture Collection (VA) and maintained in DMEM containing 10% FBS and antibiotics/antimycotics. MEFs were isolated at E11.5 from females heterozygous for p53 mice that have been mated with heterozygous males, by using standard procedures and were subsequently genotyped to assess the p53 zygosity status as described previously.9 For both the tumour reconstitution and the in vitro assays, early passage MEFs, undergone less than 7 population doublings were used. Tissue culture reagents were obtained from Invitrogen. For cytotoxicity assays approximately 20 × 103 p53-null and wild-type fibroblasts were exposed to DOX (0,2 μM) or cis-platinum (120 μM) for 24 hr, media were collected 48 hr later and added onto PC3 and MCF7 cells. Cell number for PC3 and MCF7 cells when exposed to conditioned media ranged in different independent experiments performed between 10 × 103 − 20 × 103. Their final number was counted after 2 days. Each experiment was performed in triplicates and average values were calculated for 3 independent experiments by counting cells in 3 optic fields per experiment containing at least 100 cells per optic field.

Western blot analysis

Expression of p21 in MEFs was evaluated in cells lysed by using RIPA reagent (Pierce) and total protein was subjected to western blot analysis. Antibodies for p21 and actin were obtained from Santa Cruz and Chemicon (CA).

H2O2 treatment and beta-galactosidase staining

MEFs were exposed to 300 μM H2O2 for 2 hr and subjected to standard b-gal staining for identification of senescent cells as previously described.15

Statistical analysis

Statistical analysis of the results was performed by using the student's t-test.

Results and discussion

Initially we asked if p53 of stromal fibroblasts affects the rate of tumorigenesis and the efficacy of anticancer therapy in xenograft models of the disease in vivo. Therefore, we have reconstituted PC3 prostate and MCF7 breast human tumours in immuno-incompetent SCID mice, with MEFs differing in the p53 status. Following cell inoculation we monitored the latency for tumour onset prior and after application of conventional chemotherapy by DOX and cis-platinum. These two anticancer agents are used widely in the clinical practice and act in principle by triggering apoptosis in the cancer cells. It is noted that SCID mice have normal estrous cycle and thus, they can support the growth of hormone-dependent cells in vivo, such as those used in our study, without the supplementation of exogenously provided hormones that may interfere with the outcome of the experiment.9, 16 As shown in Figures 1a and 2a both MCF7 breast and PC3 prostate cancers containing p53-null fibroblasts developed tumours faster than their counterparts with wild-type p53, confirming and extending previously reported data showing that p53 mutations in stromal fibroblasts accelerate tumorigenesis.6, 9 Subsequently, as soon as tumours became palpable, animals were subjected to chemotherapy by DOX and cis-platinum. Our results showed that when the animals bearing tumours containing p53-null-fibroblasts and were treated with the anticancer agents DOX and cis-platinum they exhibited higher sensitivity than their counterparts reconstituted with wild type fibroblasts (Fig. 1b and Figs. 2b and 2c). Thus, the status of p53 in the stroma fibroblasts was sufficient to modulate the sensitivity of tumours, otherwise identical as regards their neoplastic component, against chemotherapy. It is noted that the size of the tumour, when therapy was applied, does not seem to affect the outcome of this experiment (our unpublished observations), thus differential sensitivity to chemotherapy due to the different time needed for the establishment of palpable tumours among the 2 experimental groups does not account for these results.

Figure 1.

Paracrine modulation of anticancer drug efficacy by p53 in stromal fibroblasts. Tumours reconstituted by PC3 prostate (left panel) or MCF7 breast (right panel) cancer cells develop faster (a) but are also more sensitive to chemotherapy (b) by doxorubicin (upper panel) or cis-platinum (lower panel) when admixed with p53-null MEFs, than their counterparts with wild-type MEFs. Results of a representative of at least 3 independent experiments are shown (n = 4–7 animals per group).

Figure 2.

p53 deficiency in stromal fibroblasts accelerates the onset of PC3 prostate and MCF7 breast tumours (a) but renders them more sensitive to chemotherapy by doxorubicin (b) and cis-platinum (c). Summary of the results presented in Figure 1 is shown. The period following treatment until tumours became undetectable by palpation was scored. The results were analyzed by two parameter two-tailed student's t-test and P values are presented in the graph. The identity of the cancer cells and the p53 status of stromal fibroblasts is shown.

Then we asked if fibroblasts secrete soluble growth factors at different amounts depending on their p53 status upon exposure to chemotherapeutic agents. Consistently with the results of the in vivo experiment described above, conditioned media from treated with these anticancer agents wild type fibroblasts, stimulated the growth of MCF7 and PC3 cells more efficiently (p < 0.05) than those from treated, p53-null fibroblasts (Fig. 3). Thus, p53 deficient fibroblasts, following treatment with anticancer agents, apparently release growth factors less efficiently than their wild type counterparts, which may act onto cancer cells and stimulate their growth by paracrine mechanisms. The mediation of growth inhibitory effects of media from p53-null fibroblasts could not be excluded from the present experiments; however, in both cases our results are consistent with the modulatory effects of fibroblasts' p53 during chemotherapy.

Figure 3.

Conditioned media from DOX (upper panel) or cis-platinum (lower panel) - treated wild type fibroblasts stimulated the growth of PC3 and MCF7 cells more efficiently than media from p53-null fibroblasts treated with the same anticancer drugs. Results of a representative of at least 3 independent experiments performed in triplicates are shown. The results were analyzed by 2 parameter two-tailed student's t-test.

Considering that p53 is involved in the regulation of cellular senescence17 and that at least replicative senescence is associated with an elevation in growth factor release,18 we wanted to test if our findings can be attributed—at least in part—in the differential susceptibility against senescence between the p53-null and the wild type fibroblasts. Therefore, fibroblasts differing in the p53 status were exposed to DOX and cis-platinum under the same conditions as those used before to obtain conditioned media, and senescent cells were quantified by staining for cytoplasmic b-galactosidase (bGal). As shown in Figures 4a and 4b the ratio of bGal-positive cells was considerably higher (p < 0.001) in the wild type than in the p53-null fibroblasts after exposure to DOX and cis-platinum. Exposure to H2O2 was used as a positive control for the induction of senescence. This is also supported by the more efficient induction of p21/waf1 tumour suppressor in wild type than in p53-null MEFs, following exposure to DOX or cis-platinum (Fig. 4c). p21/waf1 is an important regulator of cell cycle arrest and entrance into senescence.19

Figure 4.

(a) Representative b-gal staining of p53 -null and wild type proliferating MEFs (control), or treated with H2O2, DOX or cis-platinum. Following exposure to H2O2, DOX or cis-platinum, wild type MEFs are considerably more sensitive to senescence than their p53-null counterparts. Diagrammatic presentation of the average of 3 independent experiments is shown in (b). Each experiment was performed in triplicates and average values were calculated for 3 independent experiments by counting cells in 3 optic fields per experiment containing at least 100 cells per optic field. (c). western blot analysis for p21/waf1 in wt and p53-null MEFs following exposure to DOX and cis-platinum. Treatment with the anticancer agents was performed as described above. Actin was used as a loading control. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Thus, we concluded that therapy with drugs such as those used in our study results in the induction of senescence in wild type stromal fibroblasts, which in turn induces the production and secretion of growth factors by the latter, capable of acting upon the cancer cells. When however mutations in p53 are present in stromal fibroblasts, the aforementioned mechanism is not fully operational, the production of paracrine growth factors acting onto the cancer cells is compromised, and therefore tumours appear more sensitive against anticancer therapy. As already suggested elsewhere,14 this contradictory function of p53 suppressing tumorigenesis and facilitating anticancer therapy cell-autonomously, and conferring drug resistance non-autonomously, may be due to the various different functions of p53 acting at the same time in different cell types as a regulator of apoptosis or operating as a survival factor by triggering cell cycle arrest and eventually senescence. Thus, it is likely that failure of anticancer therapy in certain instances can be due at least in part to the content of tumours in stromal fibroblasts and on the efficacy by which therapy induces cell cycle arrest and senescence in the stromal fibroblasts.

Collectively, our findings identify stromal fibroblasts as important modulators of the efficacy of anticancer therapy. Furthermore, they imply that specific, transient inhibition of senescence, i.e., by suppression of p53 activity, during or shortly after application of therapy may increase the efficacy of anticancer therapy.

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