TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53β promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53β increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53β is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53β depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53β induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression.https://doi.org/10.7554/eLife.14734.001
Most cancers are caused by a build-up of mutations that are acquired throughout life. One gene in particular, called TP53, is the most commonly mutated gene in many types of human cancers. This suggests that TP53 mutations play an important role in cancer development.
It is widely considered that the TP53 gene normally stops tumors from forming, while mutant forms of the gene somehow promote cancer growth. Evidence from patients with cancer has shown, however, that the relationship between TP53 mutations and cancer is not that simple. Some very aggressive cancers that resist treatment and spread have a normal TP53 gene. Some cancers with a mutated gene do not spread and respond well to cancer treatments. Recent studies have shown that the normal TP53 gene produces many different versions of its protein, and that some of these naturally occurring forms are found more often in tumors that others. However, it was not clear if certain versions of TP53’s proteins contributed to the development of cancer.
Now, Gadea, Arsic, Fernandes et al. show that Δ133p53β, one version of the protein produced by the TP53 gene in human cells, helps tumor cells to spread to other organs. Tests of 273 tumors taken from patients with breast cancer revealed that tumors with the Δ133p53β protein were more likely to spread. Patients with these Δ133p53β-containing tumors were also more likely to develop secondary tumors at other sites in the body and to die within five years.
Next, a series of experiments showed that removing Δ133p53β from breast cancer cells grown in the laboratory made them less likely to invade, while adding it back had the opposite effect. The same thing happened in colon cancer cells grown in the laboratory. The experiments showed that Δ133p53β causes tumor cells with the normal TP53 gene or a mutated TP53 gene to spread to other organs.
Together the new findings help explain why some aggressive cancers develop even with a normal version of the tumor-suppressing TP53 gene. They also help explain why not all cancers with a mutant version of the TP53 gene go on to spread. Future studies will be needed to determine whether drugs that prevent the production of the Δ133p53β protein can help to treat aggressive cancers.https://doi.org/10.7554/eLife.14734.002
Cancer is driven by somatically acquired point mutations and chromosomal rearrangements, that are thought to accumulate gradually over time. Recent whole cancer genome sequencing studies have conclusively established that the tumor suppressor gene, TP53, is the most frequently mutated gene in a wide range of cancer types. In tumors expressing wild-type (WT) TP53 gene, numerous experimental and clinical data have shown that viruses or cellular oncogene proteins target the p53 pathway, promoting abnormal cell proliferation. Altogether these data strongly suggest that defects in TP53 tumor suppressor activity are a compulsory step to cancer formation. In addition, ample data have also demonstrated that TP53 gene, whether WT or mutant, has a paramount biological and clinical role in response to cancer treatment (Brosh and Rotter, 2009; Do et al., 2012; Jackson et al., 2012; Muller et al., 2009).
We previously reported that the human TP53 gene expresses at least twelve p53 isoforms through alternative splicing of intron-2 (Δ40) and intron-9 (α, β, γ), initiation of transcription in intron-4 (Δ133) and alternative initiation of translation at codon 40 (Δ40) and codon 160 (Δ160). This leads to the expression of p53 (α, β, γ), Δ40p53 (α, β, γ), Δ133p53 (α, β, γ) and Δ160p53 (α, β, γ) protein isoforms that contain different transactivation domain, oligomerisation domains and regulatory domains (for review Joruiz and Bourdon, 2016).
All animal models (zebrafish, drosophila and mouse) of p53 isoforms and experimental data in human cells of diverse tissue origins have consistently shown that p53 isoforms regulate cell cycle progression, programmed cell death, replicative senescence, viral replication, cell differentiation and angiogenesis. Several clinical studies reported that abnormal expressions of p53 isoforms are found in a wide range of human cancers including breast and colon cancers and that p53 isoforms are associated with cancer prognosis (Joruiz and Bourdon, 2016). However, it is not known whether they are just markers or play an active role in cancer formation and progression. Recently, we reported that Δ133p53β promotes cancer stem cell potential and metastasis formation in a xenograft mouse model (Arsic et al., 2015). However, its physiopathological role, its molecular mechanism, its association with cancer progression and the effect of TP53 mutations on its activities have never been investigated.
To date, it is currently thought that all the tumor suppressor activities associated with TP53 or the oncogenic activities associated with mutant TP53 gene expression are implemented by the canonical full-length p53 protein (also named TAp53α or p53α). It is well established that TAp53α , is a transcription factor rapidly activated to restore and maintain cell integrity in response to alteration of cell homeostasis, while most TAp53α missense mutants have compromised tumor suppressor activities and have acquired transforming activities.
Compelling evidence indicates that WT TAp53α protein, plays a pre-eminent role in preventing human cancer formation and progression to metastasis. In particular, WT TAp53α protein prevents cell migration and invasion which are the main traits of Epithelial to Mesenchymal Transition (EMT), which itself involves global genome reprogramming that drives cancer progression to metastasis (McDonald et al., 2011; Suva et al., 2013). Conversely, most TAp53α missense mutant proteins promote EMT, inducing cancer cells to leave their primary tumor site and disseminate, leading to metastasis (Bernard et al., 2013; Gadea et al., 2007; Muller et al., 2009; Roger et al., 2010). However, there remain many enigmas concerning the association of p53 expression with cancer progression and treatment largely due to the natural existence of multiple alternative transcripts from the TP53 gene. (De Roock et al., 2009; Brosh and Rotter, 2009; Petitjean et al., 2007; Soussi, 2007).
The main focus of this study is the role of Δ133p53β in cancer progression. We show that Δ133p53β is associated with poor prognosis in breast cancer, particularly in luminal A breast cancer patients who express WT TP53. This indicates that Δ133p53β, which is a physiological gene product of TP53, is a predictor of cancer cell invasion and increased risk of death. We corroborate the clinical analysis by demonstrating experimentally that Δ133p53β promotes invasion using a panel of breast cancer cell models expressing WT and mutant TP53 gene. Similar results were obtained in a panel of colon cancer cell lines. Depletion of endogenous mutant Δ133p53β prevents invasiveness despite unaltered strong expression of mutant TAp53α. Reciprocally, introduction of WT Δ133p53β promotes invasion of WT TP53 cells devoid of endogenous Δ133p53β expression. Altogether, our data indicate that cell invasiveness is regulated by Δ133p53β irrespective of whether they harbor a mutant or a WT p53 gene.
Challenging the paradigm that WT TP53 always acts as a tumor suppressor protein, our data show that the WT TP53 gene can also promote invasiveness by encoding Δ133p53β. Hence, our data established that the cell decision is not exclusively determined by canonical full- length p53 protein (TAp53α), either mutant or WT, but by the modular protein complex of p53 isoforms, which reprogram epithelial cells to pro-metastatic cells.
The association of Δ133p53 (α, β, γ) isoforms with prognosis has not previously been investigated in breast cancer. As there is no antibody specific of Δ133p53α, Δ133p53β or Δ133p53γ protein isoform, we developed a nested RT-PCR method that specifically detects and identifies Δ133p53α, Δ133p53β or Δ133p53γ mRNA variants in tumor samples. Briefly, the RT-PCR method is based on the previously described RT-PCR method (Khoury et al., 2013) but its sensitivity and specificity have been improved (A fully detailed protocol is provided in the 'Extended Experimental Procedure' section and the primer sequences are described in Table 1). It enables to specifically detect and identify expression of Δ133p53α, Δ133p53β or Δ133p53γ mRNA variants in tumor samples. This nested RT-PCR analysis allows thus to investigate the association of Δ133p53α, Δ133p53β or Δ133p53γ expression with the clinico-pathological markers and/or patient clinical outcome.
Total RNAs were extracted from 273 randomly selected primary breast tumors (Figures 1A,B and Table 1). Expression of Δ133p53α mRNA was detected in 97/273 (35.5%), Δ133p53β mRNA in 23/273 (8.4%) and Δ133p53γ mRNA in 56/221 (25.3%) of the breast tumors analyzed (Table 2). To verify that detection of Δ133p53β mRNA is associated with Δ133p53β protein expression, protein extracts from 8 large breast tumors were analyzed by SDS-PAGE and western-blot with KJC8, a beta p53 specific antibody (Figure 1—figure supplement 2). Samples identified as expressing Δ133p53β mRNA also express Δ133p53β at the protein level, confirming the RT-PCR analysis data and that Δ133p53β protein is expressed in tumor samples.
Δ133p53β or Δ133p53γ are tightly associated with Δ133p53α expression [21/23 (91.3%); 53/56 (94.6%), respectively]. Almost all tumors expressing Δ133p53β express Δ133p53α. However, only 25% of tumors expressing Δ133p53γ also express Δ133p53β (14/56, Table 3). The strong association of Δ133p53α with either the Δ133p53β or Δ133p53γ isoforms is consistent with the fact that the three mRNAs are initiated from the same promoter in intron-4 of the human TP53 gene (Bourdon et al., 2005).
We analyzed the association of Δ133p53α, Δ133p53β, and Δ133p53γ with the current clinico-pathological subtypes of breast cancer: luminal A (ER+, PR+, HER2-), luminal B (ER+, PR−, HER2-), luminal/HER2+ (ER+, PR+/-, HER2+), HER2 positive (ER-, PR-, HER2+), and triple negative (ER-, PR-, HER2-), according to the recommendations of St Gallen International expert consensus (Goldhirsch et al., 2013). None of the three isoforms were associated with histological cancer type, number of metastatic lymph nodes, tumor size or grade (Table 4) and neither Δ133p53α nor Δ133p53γ were associated with breast cancer subtypes. However, Δ133p53β was significantly less frequently expressed in HER2 positive and luminal/HER2+ tumors than in other breast cancer subtypes (2/69, Fisher’s exact test, p≤0.042, Figure 1C). TP53 mutations were identified in 64 tumors (64/260, 24.6%). Δ133p53β expression was significantly associated with TP53 mutation status (χ2 = 5.625, p≤0.018, Figure 1C), whereas Δ133p53α and Δ133p53γ expression levels were not (Table 2). In all tumors, Δ133p53α, Δ133p53β or Δ133p53γ carried the same TP53 gene mutation, indicating that all the corresponding mRNAs derive from cancer cells and not from normal stromal or inflammatory cells.
Altogether, these data indicate that almost all tumors expressing Δ133p53β and/or Δ133p53γ also express Δ133p53α. However, Δ133p53β is distinct from Δ133p53α and Δ133p53γ, in that it is less frequently detected in breast tumors overexpressing HER2 and more frequently expressed in tumors expressing mutant p53 than in tumors expressing WT p53. Therefore, Δ133p53α, Δ133p53β, and Δ133p53γ are not randomly expressed.
The association of Δ133p53α, Δ133p53β, and Δ133p53γ expression with cancer patient outcome (cancer progression/disease-free survival and overall survival/death) was investigated by univariate analysis of our cohort of breast tumors. Δ133p53β was associated with cancer progression (CP) and death (CP: χ2 = 5.953, p≤0.015, death: χ2 = 7.126, p≤0.008) (Figure 1C), while Δ133p53α and Δ133p53γ were not. This was further confirmed by Kaplan-Meier log-rank analyses (Kaplan-Meier log-rank test, CP: χ2 = 6.221, 1 df, p≤0.013; death: χ2 = 5.731, 1 df, p≤ 0.017, Figures 1D,E,F, Figure 1—figures supplement 1A,B and C). Importantly, Δ133p53β expression was also associated with cancer progression in WT TP53 breast cancer patients (Kaplan-Meier log-rank test, CP: χ2 = 5.232, p≤0.022) (Figure 1—figure supplement 1I), indicating that the association between ∆133p53β expression and cancer progression is not due to TP53 mutation. (Of note the Kaplan-Meier log-rank analyses were also performed for Δ133p53α or Δ133p53γ in WT TP53 breast cancer patients. Their respective expression was not found associated with clinical outcome of WT TP53 breast cancer patients.)
In addition to Δ133p53β expression, other well-established markers of cancer prognosis such as the breast cancer subtypes, TP53 mutation, tumor grade, lymph node metastasis (absence or presence) and tumor size (>20 mm) were, as expected, also associated with cancer patient outcome in our cohort (Figure 1—figures supplement 1D–H). To clarify the univariate analyses and adjust for possible confounding variables, the association of overall survival and disease-free survival with Δ133p53β, the breast cancer subtypes (luminal-A, luminal-B, HER2+, triple-negative and luminal/HER2+), TP53 mutation, tumor grade, lymph node metastasis and/or tumor size (>20 mm) were investigated using the multivariate Cox’s regression analysis. The cohort was composed of 112 luminal-A, 43 luminal-B, 49 luminal/HER2+, 20 HER2+ and 49 triple-negative tumors. We observed that the luminal-A subtype was, as expected, the most significant independent predictor of disease-free survival (HR = 3.09, 95% CI, 1.78 to 5.38, p<1.10–4) and overall survival (HR = 4.15, 95% CI, 2 to 8.62, p<1.6 10–4, Table 5A). Then, we examined by multivariate Cox’s regression analysis the level of inter-dependence between Δ133p53β, TP53 mutation, tumor grade, lymph node metastasis (absence or presence) and tumor size (>20 mm) in the luminal-A breast cancer population (n = 112). We determined by an Omnibus test of model coefficient that the Cox-regression model is fitted (χ2 = 17.589, 1 df, p<2.7 10–5), indicating that the number of luminal-A tumors in our cohort is sufficient to support the conclusions. Among all the parameters included in the analysis (Δ133p53β, TP53 mutation, tumor grade, lymph node metastasis and tumor size [>20 mm]), Δ133p53β stood out as the most significant independent predictor of cancer recurrence and death within the luminal-A subgroup regardless of TP53 mutation status or presence of lymph node metastasis (Hazard ratio [HR], 7.93; 95% CI, 2.52 to 25; p<4.31 10–4; [HR], 3.29; 95% CI, 1.125 to 9.63; p<0.03, Table 5B, respectively). It implies that at the time of surgery, detection of Δ133p53β in primary luminal-A breast tumors of patients devoid of lymph node would predict cancer progression. Of note, the multivariate Cox-regression analysis was also performed for Δ133p53α and Δ133p53γ but no significant association with the clinical outcome of luminal-A breast cancer patients was found.
Thus, Δ133p53β in luminal-A breast cancer, which predominantly expresses WT TP53, enhances on average by 8 times the risk of recurrence and by 3 times the risk of death in an otherwise excellent prognostic group. This indicates that WT TP53 gene can be associated with cancer recurrence and death if it expresses Δ133p53β.
The clinical study suggests that Δ133p53β may confer a more invasive phenotype to breast cancer cells. We then compared the ability of three different cancer cell lines to invade into type1- Collagen. We used: (1) luminal MCF7 cells (WT TP53 and ER+), which depend on estrogen and EGF (Epidermal Growth Factor) for their growth and are neither locally invasive nor metastatic in mouse models, (2) the triple-negative MDA-MB-231 (mutant TP53-R280K, ER-), which was derived from a pleural effusion metastasis, and (3) its ‘D3H2LN’ variant (mutant TP53-R280K, ER-), which was selected for their enhanced tumor growth and widespread metastasis in mice (Jenkins et al., 2005).
As expected, MCF7 cells were very weakly invasive and MDA-MB-231 D3H2LN cells showed a significantly higher invasiveness as compared to parental MDA-MB-231 cells (Figure 2A). The difference between MCF7 cells and the two MDA-MB-231 cell lines may result from their TP53 status, since mutant TAp53α protein can activate cell migration, invasion and metastasis (Gadea et al., 2007, 2002; Muller et al., 2009; Roger et al., 2010). However, the difference in invasion between the two MDA-MB-231 cell types expressing the same mutant TP53 gene suggests the presence of another mechanism activated in the D3H2LN variant cell line. We examined whether this could be due to differential expression of the p53 isoforms. By quantitative real-time-PCR (RT-qPCR) using primers and probe (TaqMan) specific of the different subclasses of TP53 mRNA variants TAp53, Δ133 and alpha, beta and gamma (intron-9 splice variants) (Khoury et al., 2013), we found that whereas the two cell lines expressed similar levels of TAp53 and alpha TP53 mRNA variants, the more invasive MDA-MB-231 D3H2LN expressed higher levels of Δ133p53, beta and gamma mRNA variants (Figure 2B). We confirmed, by western blotting, the highest expression level of p53 protein isoforms in MDA-MB-231 D3H2LN cells by using a sheep polyclonal anti-p53 antibody (Sapu), which recognizes each p53 protein isoform with high specificity (Marcel et al., 2013). By western blotting, eight bands at 28, 35, 38, 40, 42, 45, 47, and 51 kDa were revealed in mock-transfected cells. (Figure 2C, mock lanes a and b). All these bands correspond to genuine p53 protein isoforms as they disappear after transfection with siRNA siE7 which targets exon-7 that is present in all p53 mRNA variants (lanes 'siE7') (Aoubala et al., 2011; Camus et al., 2012; Terrier et al., 2012). In agreement with the RT-qPCR data, TAp53α protein expression is similar between MDA-MB-231 and MDA-MB-231 D3H2LN, while all the other p53 protein isoforms, notably ∆133p53β are expressed at higher levels in MDA-MB-231 D3H2LN. This suggests that the enhanced invasiveness of MDA-MB-231 D3H2LN is not due to variation of mutant TAp53α protein expression level (Figure 2C, all lanes compare b to a).
To address the role of mutant Δ133p53 isoforms in the enhanced invasiveness of MDA-MB-231 D3H2LN cells, we analyzed cells depleted either of all Δ133p53 isoforms (by using the siRNAs si133-1 or si133-2, which specifically target the 5’UTR of all Δ133 mRNAs [Aoubala et al., 2011]), or of all β isoforms (by using siβ, [Camus et al., 2012; Terrier et al., 2012]), or of all p53 isoforms except Δ133p53 (α, β, γ) by using siTAp53, a siRNA targeting p53 mRNA splice variant containing TP53 exon-2 (Figure 3A). We assessed knockdown efficacy by RT-qPCR and western blotting using the Sapu and KJC8 antibodies (Figures 3D and Figure 3—figure supplement 1A). Importantly, depletion of Δ133 or β p53 variants (by transfection of si133-1, si133-2 or siβ) significantly decreased the invasiveness of mutant TP53 MDA-MB-231 D3H2LN cells without altering the expression of mutant TAp53α protein (Figure 3D). However, depletion of all p53 isoforms except Δ133p53 (α, β, γ) by siTA did not change the invasive activity of MDA-MB-231 D3H2LN cells, indicating that the simultaneous depletion of all TAp53 (including mutant TAp53α) did not impair MDA-MB-231 D3H2LN cell invasion (Figures 3B and Figure 3—figures supplement 1A). To confirm the role of the Δ133p53β isoform in promoting invasion potential, we re-introduced a si-resistant Δ133p53β-R280K isoform in MDA-MB-231 D3H2LN cells in which all Δ133p53 (α, β, γ) isoforms had been knocked down with si133-1 or si133-2. As expected, expression of the si133-1- and si133-2- resistant Δ133p53β-R280K rescued invasion (Figure 3C and Figure 3—figure supplement 1B). Similarly, Δ133p53β-R280K expression rescued invasion when cells were depleted of all β isoforms by siβ (Figure 3C and Figure 3—figure supplement 1C). We then compared the effects of Δ133p53α-R280K, Δ133p53β-R280K and Δ133p53γ-R280K isoforms on cell invasion. As the siRNAs si133-1 and si133-2 specifically target the 5’UTR of all Δ133 mRNAs, we tested their respective contribution to the invasive phenotype by re-introducing each of them in MDA-MB-231 D3H2LN cells in which all Δ133p53 (α, β, γ) isoforms had been knocked down with si133-1 or si133-2. All three mutant Δ133p53 isoforms rescued invasion in cells depleted of ∆133p53 variants, with Δ133p53β-R280K conferring the highest invasive activity compared to Δ133p53α-R280K or Δ133p53γ-R280K (Figure 3C and Figure 3—figure supplement 1B). These results indicate that mutant Δ133p53 isoforms, notably the Δ133p53β variant, are sufficient to promote invasion in mutant TP53 MDA-MB-231 D3H2LN cells, consistently with an active role for Δ133p53β isoform in cancer progression.
EMT is a gatekeeper process involving global cell reprogramming leading to profound phenotypic changes that include enhanced invasiveness along with loss of epithelial cell-cell adhesion proteins such as E-Cadherin and concomitant gain of mesenchymal protein expression including Vimentin and N-Cadherin. EMT is a reversible process, thus mesenchymal cells can reverse their phenotype and re-express epithelial markers (Thiery et al., 2009). Interestingly, depletion of mutant Δ133 or β p53 isoforms reversed mesenchymal traits of MDA-MB-231 D3H2LN cells, by inducing a significant induction of E-Cadherin protein expression and a marked decrease in Vimentin expression, particularly in cells transfected with siβ. Knockdown efficacy was assessed by western blotting using the p53 pantropic polyclonal sapu and β-specific KJC8 antibodies (Figure 3D). Thus, inhibition of endogenous mutant Δ133 or β p53 isoforms induced a switch in the expression of these epithelial features which reflects global reversion of EMT cell reprogramming. Interestingly, re-epithelialization was impaired upon depletion of all p53 isoforms (Figure 3D, lane siE7), suggesting that other p53 splice variants control epithelial features. Differential induction of E-Cadherin expression was also observed at the mRNA level (Figure 3E).
We then addressed whether the introduction of WT Δ133p53β in MCF7 cells that endogenously express all p53 isoforms except Δ133p53β and Δ133p53γ (Figure 3—figure supplement 1D) promotes invasion. As shown in Figure 3F and Figure 3—figure supplement 1E, WT Δ133p53β triggered MCF7 cell invasion into type1-Collagen.
Altogether, the results in MDA-MB-231 D3H2LN and in MCF7 indicate that mutant and WT Δ133p53β promote invasion of WT and mutant TP53 breast cancer cells. In mutant TP53 cells expressing Δ133p53β, depletion of TAp53α does not inhibit cell invasion, while depletion of the Δ133p53 or β p53 isoforms decreases invasion of cells expressing mutant TAp53α. In WT TP53 cells expressing all p53 isoforms except Δ133p53β, introduction of WT Δ133p53β promotes invasion. It suggests an active role for WT and mutant Δ133p53β in cancer progression corroborating the clinical analysis.
To determine whether Δ133p53β can promote invasion in other cancer types regardless of TP53 mutation status, we studied a panel of WT and mutant TP53 cell lines derived from colon carcinoma, another relevant model for cancer cell invasion, by comparing their ability to invade into Matrigel. The cell lines used were derived from the primary colorectal carcinoma (CRC): HCT116 (WT TP53) and SW480 (mutant p53R273H), and from metastatic tumors: LoVo (WT TP53), SW620 (mutant p53R273H) and CoLo205 (mutant p53, Y103 del27bp). Of note, the SW620 cell line was derived from a metastasis of the primary tumor from which the SW480 cell line was derived one year earlier. Compared to cells derived from primary colon tumors, metastasis-derived cell lines expressing WT or mutant TP53 exhibited a higher ability to invade Matrigel (Figure 4A) and had no E-Cadherin at cell-cell junctions (Figure 4B). Of note, although the SW620 and SW480 cell lines have a mutated TP53 gene (R273H) and come from the same patient, only SW620 are highly invasive, indicating that the expression of mutant TAp53α-R273H protein is not sufficient to explain invasive activities. We thus measured Δ133p53 mRNA expression by RT-qPCR (Taqman) and found it significantly higher in the invasive cell lines whether they express WT or mutant TP53 gene, i.e. LoVo, SW620 and CoLo205, as compared with HCT116 and SW480 cell lines (Figure 4C), in agreement with the results obtained in the breast cancer cell lines. To confirm the role of Δ133p53 isoforms in cell invasion, we knocked-down these isoforms in mutant TP53 SW620, or WT TP53 LoVo CRC cells by using si133-1 and si133-2, which led to a significant decrease invasive capacity in both cell lines (Figure 4D and Figure 4—figures supplement 1A and B). As expected, re-introduction of si133-1-resistant WT Δ133p53β rescued invasiveness of LoVo cells depleted of Δ133p53 isoforms by si133-1 (Figure 4—figures supplement 1C and D). This indicates that Δ133p53 isoforms, notably Δ133p53β, regulate invasion, irrespectively of TP53 mutation status.
Among WT TP53 colon carcinoma cells, HCT116 are weakly invasive and endogenously express all p53 protein isoforms, including Δ133p53β (Figure 4E). We investigated whether we could modify HCT116 cell invasion by modulating the ratios of p53 isoforms. First, we depleted endogenous expression of different p53 isoforms using specific siRNAs and compared the invasiveness of HCT116 cells depleted of all p53 proteins other than Δ133p53 isoforms (i.e. transfected with the siTAp53 siRNA) or depleted of β isoforms but still expressing TAp53α (i.e. transfected with the siβ siRNA) (Aoubala et al., 2011; Marcel et al., 2014) (Figure 4F).
HCT116 cells transfected with siTAp53 are more invasive than cells transfected with siNS or siE7, while HCT116 cells transfected with siβ, which depleted p53β, ∆40p53β and ∆133p53β are less invasive than cells transfected with siNS (Figure 4E and F). This suggests that the ratio of TAp53 and β isoforms, including ∆133p53β, defines the invasive activity of HCT116 cells. Altogether our data indicate that the invasive capacity of colon cancer cell lines, as in the breast tumor cell lines, correlates with and depends on the expression of Δ133p53 isoforms, regardless of the TP53 mutation status.
To determine whether the Δ133p53β-enhanced invasion directly impacts on the mode of cell migration, we transfected HCT116 with GFP- WT Δ133p53β and tracked them by video-microscopy. We observed two populations of cells, cohesive epithelial cells adherent to the glass and rounded cells loosely attached on top of cohesive cells. GFP-tagged Δ133p53β expression elicited a significant increase of rounded cells with dynamic bleb-like structures on their surface (Figure 5A, Video 1 and Figure 5—figure supplement 1A as control). While recording, we noticed that some GFP-Δ133p53β -positive cohesive adherent cells detached progressively from the substratum, became rounded (white arrowhead, Figure 5A) and migrated (white arrow, Figure 5A). Propidium iodide staining and FACS analysis showed that blebbing cells were alive and were not undergoing apoptosis (Figure 5—figure supplement 1B). The number of blebbing cells was 25 times greater than the number of adherent cells in HCT116 cells transfected with Δ133p53β (Figure 5B). Similar rounded and detached cells were obtained using myc-tagged isoforms, ruling out any role of GFP in the observed phenotype (Figure 5B). Since loss of adhesive structures and concomitant acquisition of a rounded-blebbing movement are hallmarks of epithelial-amoeboid transition (EAT), a derivative of EMT which produce highly invasive cells, we further confirmed the EAT-like phenotype of Δ133p53β-expressing cells by measuring E-cadherin and β1-integrin levels, as the loss of both is a marker of EAT (Friedl and Wolf, 2003). As shown in Figure 5C,E-cadherin and β1-integrin, expressed at high levels in adherent cohesive cells, were barely detected in blebbing cells, implying they had experienced EAT. Δ133p53β also enhanced cell migration and invasion, since its expression in HCT116 cells elicited a two-fold increase in migration (Figure 5D) in uncoated transwell chambers and conferred a 5 times higher invasive capacity in Matrigel (Figure 5D) compared to GFP-only-transfected controls.
Our data indicate that expression of WT Δ133p53β promotes EAT in WT TP53 HCT116 cells which endogenously express low levels of Δ133p53β. We therefore next investigated whether we could modify EMT/EAT features in WT TP53 CRC cells that endogenously express high levels of Δ133p53β. The WT TP53 colon carcinoma LoVo cells are strongly invasive and endogenously highly express Δ133p53 isoforms at the mRNA and protein levels (Figure 4A,C). Knockdown of Δ133p53 isoforms in LoVo cells remarkably decreased 3-D cell scattering, a process which shares cellular features reminiscent of cells undergoing EMT/EAT (Figure 5E and Figure 5—figure supplement 1C). To investigate the impact of p53 isoforms-mediated cell scattering on the process of migration, we performed wound-healing assay. LoVo transduced with control shNS progressively detached and migrated as individual cells (arrows, Figure 5F). LoVo depleted of Δ133p53 isoforms still remain cohesive and migrate very slowly into the gap. In this case, some rare clusters of cells leave the cohesive epithelium to enter into the gap (arrowheads, Figure 5F). Interestingly, the cluster of cells remains compact and cells move collectively, but with a reduced velocity compared to individual migrating cells with unaffected ∆133p53 expression (Figure 5F and Videos 2 and 3). Thus endogenous Δ133p53 protein isoforms regulate both the mode of cell motility and the ability to implement adherens junctions, with a subsequent impact on cell velocity.
Altogether, our data indicate that Δ133p53β expression elicits hallmarks of EAT/EMT in CRC cells.
All the genetic evidence in the clinic and animal models indicate, unequivocally, the pivotal and fundamental role of the TP53 gene in cancer formation, progression and response to treatment. TP53 is ubiquitously expressed and it regulates different cell responses such as cell repair, proliferation, senescence, differentiation, cell migration and cell death in response to any change of tissue/cell homeostasis, thus maintaining tissue/cell integrity upon stress or damage. Consistently, TP53 mutation is the most frequently mutated gene in human cancers and its mutation status is associated with poor clinical outcome. Any cancer treatments change cell homeostasis and therefore trigger TP53-mediated cell responses. It is currently impossible to accurately predict response to cancer treatment in the clinic because of the large variety of TP53-mediated cell responses. In addition, as missense mutations of TP53 do not totally abrogate its tumor suppressor activity and can confer additional biological activities to TP53 gene, it is very difficult to choose the most efficient cancer treatment based on TP53 mutation status (Kruiswijk et al., 2015; Lang et al., 2004; Li et al., 2012; Meek, 2015). This indicates that major features of the TP53 gene and its pathway have still to be uncovered and understood and this represents a critical bottleneck preventing major breakthroughs in cancer treatment.
To date, it is thought that the diverse biological activities associated with TP53 gene expression are carried out by a single protein isoform, p53 (TAp53α). However, like most human genes, TP53 gene does not express one but at least 12 different p53 protein isoforms which have distinct domain and activities.
The WT canonical p53 protein (TAp53α) prevents cancer invasion by inhibiting EMT (Gadea et al., 2007; McDonald et al., 2011; Muller et al., 2011; Roger et al., 2010; Suva et al., 2013) while the mutant canonical p53 protein (mutant TAp53α) was shown to promote cell invasion (Adorno et al., 2009; Gadea et al., 2002, 2004; Muller et al., 2009; Roger et al., 2010; Gadea et al., 2007). However, despite this clear distinction between WT and mutant TP53, the situation in the clinic is not clear cut, as a mutant TP53 expression in primary tumors does not systematically lead to metastasis formation and WT TP53 is frequently expressed in metastasis (Dong et al., 2007; Kalo et al., 2007; Oren and Rotter, 2010).
Little is known about the biological roles and clinical relevance of p53 isoforms in cancer. In this study, we analyzed the expression of Δ133p53 isoforms (α, β, γ) in relation to clinical outcome in primary breast cancers. We determined that all tumors expressing Δ133p53β or Δ133p53γ also co-express Δ133p53α. This indicates that Δ133p53 (α, β, γ) isoforms are not randomly expressed in breast cancers, confirming that the internal promoter of TP53 and the alternative (β, γ) splicing of p53 mRNA are regulated in tumor cells (Aoubala et al., 2011; Marcel et al., 2014; 2010; Tang et al., 2013).
We also determined that Δ133p53β expression is significantly associated with cancer recurrence in breast cancer patients, even in tumor expressing WT TP53. In particular, Δ133p53β enhances on average by eight times the risk of recurrence and by three times the risk of death in luminal A breast cancer patients, an otherwise excellent prognostic group. Importantly, detection of Δ133p53β at time of surgery in primary luminal-A breast tumors of patients devoid of lymph node would predict cancer progression. Therefore, far from having its function inactivated in Luminal A breast cancer, the WT TP53 gene would promote breast cancer recurrence and increased risk of death when it expresses Δ133p53β.
To corroborate the clinical data, we experimentally investigated how Δ133p53β could confer cell invasion and motility to WT and mutant TP53 breast cancer cells. First, we observed that the invasive activity of MCF7, MDA-MB-231 and MDA-MB-231 D3H2LN cells is positively correlated to the Δ133p53 mRNA expression level. This was also observed in a panel of WT or mutant TP53 colon cancer cell lines. In mutant TP53 MDA-MB-231 D3H2LN breast cancer cells, depletion of endogenous mutant Δ133p53 isoforms reduces cell invasion, despite unaltered and strong expression of mutant full-length p53 (TAp53α) protein. Re-introduction of si-133-resistant mutant Δ133p53α-R280K, Δ133p53β-R280K or Δ133p53γ-R280K in MDA-MB-231D3H2LN cells depleted of Δ133p53 isoforms restores their invasive phenotype, with mutant Δ133p53β-R280K being significantly the most potent. Importantly, depletion of the p53 protein isoforms with siTA that targets only the p53 proteins containing the transactivation domains (i.e TAp53α, TAp53β, TAp53γ, Δ40p53α, Δ40p53β, Δ40p53γ) without altering expression of Δ133p53 isoform did not change the invasive activity of MDA-MB-231 D3H2LN. This indicates that the simultaneous depletion of all TAp53 (including mutant TAp53α) did not impair MDA-MB-231 D3H2LN cell invasion.
It has previously been reported that overexpression of mutant TAp53α promotes integrin and epidermal growth factor receptor (EGFR) recycling, thus driving invasion (Muller et al., 2009). However, expression of the mutant TAp53α is not sufficient to explain why the triple negative breast cancer MDA-MB-231 and its highly metastatic MDA-MB-231 D3H2LN derived clone have different invasive activity while they both express the same mutant TP53-R280K gene. MDA-MB-231 D3H2LN cells showed a significantly higher invasiveness as compared to parental MDA-MB-231 cells. Interestingly, MDA-MB-231 D3H2LN expressed higher levels of Δ133p53 mRNA variants and proteins (Figure 2B and C). Similar results were observed in mutant TP53 colon cancer cell lines. The SW480 and SW620 colon carcinoma cell lines derive respectively from the primary and secondary tumors resected from the same patient (Hewitt et al., 2000). Although SW480 and SW620 cells express the same mutant TP53-R273H gene, SW480 cells are far less invasive than the SW620 cells. Interestingly, SW480 cells express much less Δ133p53 mRNAs than the SW620 cells. Importantly, depletion of endogenous Δ133p53 isoforms from SW620 cells reduces cell invasion, despite unaltered and strong expression of mutant TAp53α protein. This indicates that the role of endogenous mutant Δ133p53 isoforms in promoting invasiveness may be extended to other tissue types and that endogenous Δ133p53 isoform expression regulates cancer cell invasion in TP53 mutant colon and breast cancer cells, irrespective of the full-length p53 (TAp53α) expression.
Importantly, the invasive activity of Δ133p53 isoforms is not associated with TP53 mutation status since depletion of ∆133p53 isoforms in WT TP53 colon carcinoma LoVo cells abolishes their scattering and invasive activities. ∆133p53 isoform expression thus explains the inconsistent clinical association between TP53 mutation and metastasis.
Interestingly, the introduction of WT Δ133p53β in the poorly invasive WT TP53 MCF7 breast cancer cells that express all WT p53 isoforms but Δ133p53β, enhances MCF7 invasive activity. In WT TP53 HCT116 colon cancer cells, that express all WT p53 protein isoform including low level of Δ133p53β protein, we demonstrated that HCT116 invasive activity can be controlled (enhanced or inhibited) by siRNAs specific of different p53 isoforms to modulate expression of different subsets of p53 protein isoforms. Hence HCT116 invasive activity is inhibited after depletion of Δ133p53 or β isoforms by siRNA si133 or siβ respectively while HCT116 invasive activity is enhanced after depletion of WT full-length p53 proteins (TAp53α, TAp53β, TAp53γ, Δ40p53α, Δ40p53β and Δ40p53γ) by siRNA siTA. Furthermore, ectopic expression of WT Δ133p53β in HCT116 cells enhances their invasive activity.
Altogether, Δ133p53β expression provides a rationale for invasive cancers expressing WT TP53 and non-invasive cancers expressing missense mutant TP53. These data suggest that epigenetic mechanisms that stem Δ133p53β expression, i.e. alternative splicing and induction of the TP53 internal promoter favour cancer invasion and dissemination of metastatic cells, regardless of TP53 mutation status. Our data indicate that all three ∆133p53 isoforms (∆133p53α, Δ133p53β and Δ133p53β) promote invasion. This is in accordance with recent reports showing that ∆133p53α stimulate angiogenesis and cell invasion (Bernard et al., 2013; Roth et al., 2016). Here we identified the ∆133p53β isoform as being the most efficient promoter of invasion, corroborating its role as a predictive indicator of cancer relapse and death in the clinic.
We investigated then how Δ133p53β regulate cell invasion in cancer cell lines. Since EMT leads to profound phenotypic changes due to genome-scale epigenetic reprogramming and post-transcriptional regulation, we studied the regulation of EMT by Δ133p53β to illustrate a molecular mechanism of Δ133p53β. Our data indicate that expression of Δ133p53β promotes acquisition of a rounded-blebbing movement, which is associated with decrease of E-cadherin and β1-integrin in colon cancer HCT116 cells. These phenotypic changes are hallmarks of epithelial-amoeboid transition (EAT), a derivative of EMT which produces highly invasive cells. Depletion of ∆133p53 isoforms also induces E-Cadherin expression and concomitantly inhibits Vimentin expression in invasive breast cancer MDA-MB-231 D3H2LN cells. Accordingly, knockdown of Δ133p53 isoforms in colon cancer LoVo cells decreased 3-D cell scattering, a process associated with EMT/EAT. Our study supports a critical role for Δ133p53β (whether WT or mutant) in the induction of cell invasion and EMT/EAT.
In summary, our data lead us to conclude that the biological activities associated with WT or mutant TP53 gene expression are not carried out by the single full-length p53 protein (TAp53α). p53 isoforms underlie the dual role of WT TP53 gene in preventing or promoting cancer cell invasion. The invasive activities of cancer cells expressing WT or mutant TP53 gene can be enhanced or inhibited by respectively increasing or reducing expression of Δ133p53β (Figure 5G). Testing the prognostic discriminatory potential of Δ133p53β could influence clinical practice and may offer appropriate and yet unexplored therapeutic options for mutant or WT TP53 tumors expressing Δ133p53β.
Primary, previously untreated and operable breast cancers from 273 Caucasian women, with sufficient tumor tissue surplus to diagnostic requirements and with complete clinical and pathological data, were analyzed. To minimize variations such as different surgical techniques and skills, which would have an effect on the rates of cancer recurrence and death, all tumors were provided by only one surgeon, Prof Alastair Thompson, breast cancer surgeon at Ninewells hospital in Dundee. Moreover, all patients were treated at Ninewells Hospital according to established clinical protocols. Furthermore, to minimize variation in tumor handling, all tumors were collected, stored, processed, extracted and stained by the Tayside Tissue Bank according to validated and standardized protocols at Ninewells Hospital. RNA quality was assessed using the BioAnalyzer 2100 prior to RT-PCR analysis and all samples with a ratio of 28S/18S < 1.2 were discarded. In addition, the Tayside Tissue Bank collected and verified the clinico-pathological data established by two anatomo-pathologists. Furthermore Tayside Tissue Bank collected and anonymised all the clinical data (treatment, patient follow-up).
The median age at diagnosis of the breast cancer patient cohort was 61.5 years (range 28.7 to 89.1 years) and median follow-up period was 6.85 years (range 0.29 to 13.7 years). Tumor tissues were macro-dissected by a specialist breast pathologist and snap frozen in liquid nitrogen prior to storage at −80ºC. The samples were examined following Local Research Ethics Committee approval under delegated authority by the Tayside Tissue Bank (www.taysidetissuebank.org).
Immunohistochemical staining was carried out on 4 μm sections of formalin-fixed paraffin-embedded tumors, as previously described (Purdie et al., 2010).
To generate cDNA from tumor total RNA extracts, two independent reverse transcription of 300 ng of total RNA were performed and pooled (Khoury et al., 2013). The quality of the reverse Transcription (cDNA) was assessed by quantification of the Actin and p53 cDNAs using quantitative Real-Time PCR (Sybr Green) previously described (Khoury et al., 2013). All cDNA samples for which Actin or p53 cDNA could not be detected in less than 30 cycles were discarded as it indicates that the reverse-transcription did not work properly. This step reduces the number of false-negative samples that would bias the statistical analysis.
The sensitivity and specificity of the RT-PCR analysis were improved by performing 2 successive PCR of 35 cycles each and by using 2 sets of nested primers for each Δ133p53 mRNA variants. Therefore the RT-PCR analysis specifically amplifies Δ133p53α, Δ133p53β or Δ133p53γ mRNA. The primer sequences are provided in Table 1 and a detailed protocol is described in the 'extended experimental procedures'.
In the first PCR of 35 cycles, all Δ133p53 mRNA variants were amplified using a forward primer corresponding to the 5’UTR of the Δ133p53 mRNA (TP53 intron-4) and a reverse primer corresponding to the exon-10. Then, the product of the first PCR (1ul) is re-amplified in the second PCR of 35 cycles using a nested forward primer located in the 5’UTR of the Δ133p53 mRNA (intron4) and a nested reverse primer spanning the exon junction between exon-9 and exon-10 to specifically amplify the DΔ133p53α cDNA. To specifically amplify the Δ133p53β cDNA, the product of the first PCR (1ul) is re-amplified in the second PCR of 35 cycles using the nested forward primer located in the 5’UTR of the Δ133p53 mRNA (intron4) and a nested reverse primer corresponding to the exon9b encoding the β C-terminal amino-acid sequence. To specifically amplify the Δ133p53γ cDNA, the product of the first PCR (1ul) is re-amplified in the second PCR of 35 cycles using the nested forward primer located in the 5’UTR of the Δ133p53 mRNA (intron4) and a nested reverse primer spanning the exon junction between exon-9 and exon-9g that encodes the γ-C-terminal amino-acid sequence. The RT-PCR analysis was tested in p53-null cells (H1299) and on a panel of cell lines expressing TP53 gene (MCF7, MDA-MB-231, HCT116). The products of the second PCR were then validated by sequencing and after electrophoresis on 1% agarose gel.
For the RT-PCR analysis of the tumor samples, the products of the second PCRs were validated for all tumors by electrophoresis on 1% agarose gel. The PCR products of at least 2 tumor samples were sequenced to validate identity of the amplified Δ133p53 mRNA variants. The samples giving rise to a PCR product of the expected size were deemed positive for the expression of the corresponding Δ133p53 mRNA variant otherwise they were deemed negative. The two successive PCR of 35 cycles using two nested sets of primer maximize sensitivity and specificity of amplification. It enables to detect and identify the expression of each Δ133p53 variants even if expressed in a small population of tumor cells, taking account thus of tumor cell heterogeneity. It allows thus to compare their expression and respective association with the clinico-pathological markers and/or patient clinical outcome. The expression of p53 isoforms and analysis of p53 mutation were performed as described in the extended experimental procedures.
The statistical clinical analysis was performed as described previously (Bourdon et al., 2011). All statistical comparisons were made using Wilcoxon test and a p-value < 0.05 was considered to be statistically significant.
Human p53 isoform constructs (Bourdon et al., 2005) were sub-cloned into pEGFPC1 (Clontech) or pLPCmyc. Antibodies were purchased from BD-transduction laboratories, Santa Cruz and GE-Healthcare. Dilutions are indicated in extended experimental procedures.
Cells were cultured at 37°C in the presence of 5% CO2 in McCOY’5A or DMEM media (Sigma) for colon cancer and breast cancer cells, respectively. HCT116 (RRID:CVCL-0291), MDA-MB231 (RRID:CVCL-0062), MCF7 (RRID:CVCL-0031), LoVo (RRID:CVCL-0399), SW480 (RRID:CVCL-0546), SW620 (RRID:CVCL-0547), Colo205 (RRID:CVCL-0218) were purchased from ATCC. MDA-MB231 D3H2LN (RRID:CVCL-D257) were purchased from PerkinElmer. All cell lines were tested for mycoplasma contamination after thawing using Lonza mycoalert assay (Reference number: LT27-236). Transfections of p53 isoforms and siRNA were carried out using JetPEI kit (Qbiogen) and Interferin (Polyplus), respectively, according to the manufacturers’ instructions. For adhesion assays, non-adherent and adherent cells were collected 24 hr after transfection and counted using the Countess cell counting system (Invitrogen).
Time-lapse DIC microscopy was performed on a Leica DMIRE2 inverted microscope with an automatic shutter and GFP filter sets, as described in the extended experimental procedures.
Non-adherent and adherent cell extracts were obtained and analyzed by western blotting. Detailed protocols are described in the extended experimental procedures. Cells were lysed and protein complexes were co-immunoprecipitated using GFP-Trap beads (Chromotek) as previously described (Arsic et al., 2012). Sources of antibodies are provided in the extended experimental procedures.
Total RNA was extracted with the RNeasy Mini Kit (Qiagen) and treated with DNase (Qiagen) prior to reverse transcription, which was carried out using oligo(dT) (Invitrogen) and M-MLV reverse transcriptase (Invitrogen). Sub-classes of p53 mRNA isoforms were quantified by Real-Time PCR (TaqMan) as previously described (Aoubala et al., 2011; Moore et al., 2010). All measurements were normalized to the expression of the TATA box-binding protein (TBP) gene.
Immunohistochemical staining was carried out on 4 μm sections of formalin-fixed paraffin-embedded tumors with the mouse monoclonal anti-estrogen receptor alpha (ER) antibody 6F11 (Novocastra Laboratories Ltd), progesterone receptor (PR) antibody clone 16, (Novocastra Laboratories Ltd) and mouse monoclonal anti-HER2 antibody CB11 (Novocastra Laboratories Ltd). Additional analyses were performed according to histological tumor grade (Bloom and Richardson, 1957) and graded by a specialist consultant breast pathologist. ER status (as ER negative 0–3 versus ER positive 4–18) was determined by the Quickscore method (Detre et al., 1995). HER2 scoring was performed as previously described (Purdie et al.,2010).
Approximately 10 mg of tumor tissue (>40% of tumor cells) was homogenized in 750 μl QIAzol lysis reagent (Qiagen Ltd, Crawley, West Sussex, UK) and total RNA was extracted (Qiagen). RNA quality was assessed using the BioAnalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA), prior to RT-PCR analysis and all samples with a ratio of 28S/18S < 1.2 were discarded. To generate cDNA from tumor total RNA extracts, two independent reverse transcription of 300ng of total RNA were performed using the Cloned AMV Reverse Transcription kit (Invitrogen) following manufacturer recommendation (Khoury et al., 2013). The quality of the reverse Transcription (cDNA) was assessed by quantification of the Actin and p53 cDNAs using quantitative Real-Time PCR (Sybr Green) previously described. Primers are provided in Table 1 (Khoury et al., 2013). All cDNA samples for which Actin or p53 cDNA could not be detected in less than 30 cycles were discarded. In the first PCR of 35 cycles, all Δ133p53 mRNA variants were amplified using a forward primer corresponding to the 5’UTR of the Δ133p53 mRNA (TP53 intron-4) and a reverse primer corresponding to the exon-10. Per reaction mix (1 sample): 5x Green buffer Go Tag (Promega): 10 μl; dNTP (10 mM): 1 μl; forward Primer (10 uM): 1 ul; reverse Primer (10 μM) 1 μl; Go Taq DNA (Promega) 0.25 μl; 2 μl of cDNA complete at 50 μl final volume with milliQ water.Thermal cycling conditions for PCR amplification (1st PCR reactions) for p53 isoforms are as follows: [94°C for 3 min (1 cycle)], [94°C for 30 s, 63°C for 30 s, 72°C for 1 min 30 s (35 cycles)] and finally [72°C for 8 min (1 cycle)].
Then, the product of the first PCR (1ul) is re-amplified in the second PCR of 35 cycles using a nested forward primer located in the 5’UTR of the Δ133p53 mRNA (intron4) and a nested reverse primer spanning the exon junction between exon-9 and exon-10 to specifically amplify the D133p53α cDNA. To specifically amplify the Δ133p53β cDNA, the product of the first PCR (1 ul) is re-amplified in the second PCR of 35 cycles using the nested forward primer located in the 5’UTR of the Δ133p53 mRNA (intron4) and a nested reverse primer corresponding to the exon-9β encoding the β C-terminal amino-acid sequence. To specifically amplify the Δ133p53γ cDNA, the product of the first PCR (1ul) is re-amplified in the second PCR of 35 cycles using the nested forward primer located in the 5’UTR of the Δ133p53 mRNA (intron4) and a nested reverse primer spanning the exon junction between exon-9 and exon-9γ that encodes the γ-C-terminal amino-acid sequence. Per reaction mix (1 sample) : 10 μl; dNTP (10 mM): 1 μl; forward Primer (10 uM): 1ul; reverse Primer (10 μM) 1 μl; Go Taq DNA (Promega) 0.25 μl; Add 1 μl of c-DNA from1st PCR; complete at 50 μl final volume with milliQ water. Thermal cycling conditions for PCR amplification (2nd PCR reactions): [94°C for 3 min (1 cycle)], [94°C for 30 s, 60°C for 45 s, 72°C for 1 min 30 s (35 cycles)] and finally [72°C for 8 min (1 cycle)].
The PCR products are then analyzed by electrophoresis on 1% agarose gel. The RT-PCR analysis was tested in p53-null cells (H1299) and on a panel of cell lines expressing TP53 gene (MCF7, MDA-MB-231, HCT116). The products of the second PCRs were validated for all tumors by electrophoresis on 1% agarose gel. The PCR products of at least 2 tumor samples were sequenced to validate identity of the amplified Δ133p53 mRNA variants.
TP53 mutation status was determined from p53 PCR fragment as previously described (Bourdon et al., 2005).
The primary outcomes in this study were breast cancer-specific overall survival (abbreviated to overall survival) and breast cancer-specific disease-free survival (abbreviated to disease-free survival or cancer recurrence throughout the text), and accordingly, non-breast cancer deaths were censored at the time of death (i.e. at the time of their death, the women were considered to have survived breast cancer but died of other causes).
The human p53 isoform constructs used in this report were previously described (Bourdon et al., 2005). They were sub-cloned into the EcoRI and BamHI sites of pEGFPC1 (Clonetech) to obtain GFP-tagged proteins or in the BamHI and EcoRI sites of pLPCmyc to obtain myc-tagged proteins. Constructs were amplified using the Nucleobond PC 500 kit (Macherey-Nagel) according to the manufacturer’s instructions. Human Δ133p53 isoforms (α, β and γ) were cloned into pMSCVhyg (Clontech Laboratories) plasmid for retrovirus production. pSIREN-Luc (luciferase)-shRNA was purchased from Clontech. The mouse anti-E-Cadherin (clone 36) and the mouse anti-Beta1-Integrin, were purchased from BD-transduction laboratories and were diluted at 1/400° and 1/250°, respectively. The mouse N-Cadherin (clone 32/BD Transduction laboratories), and the rabbit polyclonal anti-GFP (Invitrogen, Life Technologies, A-6455) were used at 1/400°, 1/250°, 1/300° and 1/2000°, respectively.
The sheep pantropic p53 antibody Sapu was diluted at 1/5000°. The rabbit polyclonal KJC8 antibody specific of the β p53 isoforms was diluted at 1/4000° (Bourdon et al., 2005). The Horse-Radish Peroxidase (HRP)-conjugated anti-IgG antibodies were purchased from GE-Healthcare and were diluted at 1/5000°. The Western Lightning Chemiluminescence (ECL) reagents were purchased from PerkinElmer.
Time-lapse DIC microscopy was performed with a 63x oil-immersion objective (HC x PL APO 1.32–0.6 oil CS), sample heater (37°C) and homemade CO2 incubation chamber. Images were captured with micromax CCD camera (1300Y/HS) imaging software, converted into TIFF files and edited and compiled with Metamorph software. The exposure time was 500 ms for GFP and 300 ms for light. Images were captured every 3 sand during 5 min or every 4 min during 12 hr.
Media containing blebbing cells was spun down at 1200 rpm for 5 min and the pellet constituted of blebbing invasive cells was lysed. The remaining adherent cells were gently scraped in lysis buffer. The two populations of cells were analyzed separately for each condition (see Figure legends). Total protein concentration was determined using the BCA kit (Promega). Protein samples were electrophoretically separated on 8% SDS-PAGE gels for E-Cadherin and Beta1-Integrin. An equal amount of total protein (30 µg) was loaded into each lane. Proteins were then transferred onto nitrocellulose membranes. Membranes were blocked with TBS/0,1% Tween 20 containing 3% milk for one hour and then incubated overnight with the primary antibodies diluted in TBS/0,1% Tween 20 containing 3% milk. After several washes in TBS/Tween, membranes were incubated with anti-rabbit or anti-mouse Ig antibodies linked to HRP. Membranes were developed with ECL according to the manufacturer’s instructions. All scanned autoradiographs were quantified using AIDA/2D densitometry software.
Tissues were dissected and snap frozen in liquid nitrogen. Tissue chunks were placed in an Eppendorf tube containing RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and PBS, pH: 7.4, supplemented with protease inhibitor Complete (Roche) and iron beads. Tubes were then placed in Qiagen Retsch MM 300 tissue lyser and shook 4 times for 45 s (frequence 20). Samples were then centrifuged at 12000RPM for 10 min at 4C. Pellets were discarded and supernatants were sonicated (20 s, 30% amplitude). Protein concentration was determined using BCA Protein Assay Kit (Thermo Scientific). Protein extracts were then analyzed by SDS-PAGE as described (20 ug protein extract were loaded per well). Actin was used as a loading control. The rabbit polyclonal antibody KJC8 specific of the beta p53 isoforms was diluted at 1 μg/ml in 4.5% milk in PBS/Tween and incubated overnight at 4C.
The quantification of cell invasion was carried out in Transwell cell culture chambers containing fluorescence-blocking polycarbonate porous membrane inserts (Fluoroblock; #351152; BD Biosciences; pore size 8 μm). 100 μl of 1 mg/mL Matrigel with reduced growth factors (a commercially prepared reconstituted BM from Englebreth-Holm-Swarm tumors, # 354230; BD Biosciences) were prepared in a Transwell. Cells were transfected as monolayers before trypsinization and plating in 2% FCS-containing media on top of a thick layer (around 500 μm) of Matrigel contained within the upper chamber of a Transwell. Controls were left untreated. The upper and lower chambers were then filled with, respectively, 2% FCS- containing media and media with 10% FCS, thus establishing a soluble gradient of chemo-attractant that permits cell invasion throughout the Matrigel. Cells were allowed to invade at 37°C, 5% CO2 through the gel before fixing for 15 min in 3.7% formaldehyde. Cells that had invaded through the Matrigel were detected on the lower side of the filter by GFP fluorescence and counted. Six fields of the filter were counted and each assay was performed at least three times in triplicate for each condition.
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Joaquín M EspinosaReviewing Editor; University of Colorado School of Medicine, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "TP53 drives invasion through expression of its Δ133p53β variant" for consideration by eLife. Your article has been favorably evaluated by Charles Sawyers as the Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
The manuscript by Gadea et al. reports a number of interesting observations about the role of a specific mRNA isoform arising from the TP53 locus, Δ133p53β, in cancer cell invasion and metastasis.
The paper starts with a strong set of observations establishing a correlation between Δ133p53β mRNA expression and poor prognosis in breast cancer. The authors performed a first-in-kind expression analysis for the various Δ133 isoforms and observed that only the β isoform correlates with more aggressive disease and lower survival rates.
Next, the authors embarked on a series of cell-based assays using breast and colorectal carcinoma cell lines and both loss- and gain-of function experiments to demonstrate that Δ133p53β promotes cell motility, invasion and migration, by conferring a sort of 'epithelial to amoeboid' transformation.
Finally, the authors attempt to dissect the mechanism by which Δ133p53β exerts this function, and present some data hinting at a role for the p53 family member p63 as a mediator of Δ133 pro-metastatic functions.
Overall, the reviewers agreed that the work presented is very interesting and the scholarship is very good, but also noted that the last portion of the manuscript (i.e. molecular mechanisms involving p63 isoforms) is poorly developed and the data is not as strong as in the first part of the manuscript.
A main issue is that throughout the first part of the paper the authors demonstrate that Δ133 can exert its functions independently of mutations in DNA binding domain within the TP53 locus that are commonly observed in cancer, suggesting that its effects are independent of DNA binding. However, during their mechanistic investigations in the later part of the paper, the authors sometimes perform experiments with only the wild type or mutant Δ133 protein, involving different p63 isoforms depending on the cell line employed (ΔNp63 in some scenarios, TAp63 in others). The final product is a rather confusing scenario where it is unclear if mutations in the DBD affect Δ133 function, and also unclear which p63 isoform is involved.
Accordingly, the reviewers agreed to encourage resubmission of a significantly revised manuscript addressing the following Major Points:
1) Removal of the p63 chapter. The reviewers agreed that the paper would be better served by removing the last chapter on mechanism - basically Figures 6 and 7 - and strengthening the main observations throughout Figures 1–5 by addressing the various technical aspects listed below. Though the association with p63 is of interest, the experiments presented lack major controls and depth to draw solid conclusions. Reviewers concluded that developing the p63 aspect of the paper would require an amount of work not feasible within the review process and that it could become the subject of a follow up study.
2) Improved expression analysis in tumor samples:
2.1) The data on the expression of the different p53 isoforms in tumors only rely on a nested RT-PCR analysis. This could lead to misleading interpretation especially when used to compare expression of different isoforms. For example, are the tumors 5 and 8 in Figure 1B negative or positive for the γ isoform? It is important to verify these data with an orthogonal approach.
2.2) Since an antibody specific for the β isoform is available, it will be important also to verify expression at protein level.
2.3) It is not clear if the analysis in Figure 1—figure supplement 1E-I was limited to Luminal A tumors. Conducting the analysis without considering breast cancer subtypes could in fact be problematic given the difference p53 isoforms and mutations have different distribution across breast cancer sub-types and given lineage specificities in the behavior of tumors.
3) Another major concern is related to the specificity of the siRNA and their association with the observed phenotypes. A rescue experiment with the appropriate controls will be required to definitely prove that the described differences are due to Δ133 p53 β isoform. Also to have a better sense of the specific functional role of the different p53 isoforms, it will be important that all experiments be performed with all the described siRNAs and upon over expression of the different p53 isoforms.https://doi.org/10.7554/eLife.14734.021
- Gilles Gadea
- Nikola Arsic
- Pierre Roux
- Gilles Gadea
- Nikola Arsic
- Pierre Roux
- Kenneth Fernandes
- Judit Remenyi
- Marie P Khoury
- Philip R Quinlan
- Alastair M Thompson
- Jean-Christophe Bourdon
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We are grateful to MRI (Montpellier Rio Imaging) for constructive microscopy, P. Fort for critical comments on the manuscript. The Tayside Tissue Bank is supported by Breast Cancer Campaign, Cancer Research UK (CRUK) and by NHS Tayside through the Chief Scientist Office and Health Sciences Scotland (formerly the Scottish Academic Health Science Collaboration, AHSC). JC Bourdon is a fellow of Breast Cancer Now (2012MaySF127). K Fernandes, J Remenyi, P Quinlan and AM Thompson are supported by Breast Cancer Campaign (BCC: 2010NovPR50, BCC, 2010NovPR16 and BCC, TB2009DUN, respectively). M Khoury was supported by the ‘Association pour la Recherche contre le Cancer’ (contract AO/3/5099). G Gadea, N Arsic and P Roux are supported by CNRS and INSERM.
Human subjects: Samples were examined following Local Research Ethics Committee approval under delegated authority by the Tayside Tissue Bank (www.taysidetissuebank.org).
- Joaquín M Espinosa, Reviewing Editor, University of Colorado School of Medicine, United States
© 2016, Gadea et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.