An Essential Role of Maspin in Embryogenesis and Tumor Suppression—Response (2017)

We are writing to respond to Drs. James C. Whisstock (JCW) and Philip I. Bird (PIB) who submitted a letter (1) to Cancer Research (CR) regarding a recent publication by Dzinic and colleagues in CR entitled “An Essential Role of Maspin in Embryogenesis and Tumor Suppression” (2).Maspin, also known as serpinB5, is an epithelial-specific protein in the serine protease inhibitor (serpin) superfamily (3). Earlier, the group Drs. JCW and PIB published an article entitled “Maspin Is Not Required for Embryonic Development or Tumour Suppression” (4). In their letter to CR, Drs. JCW and PIB state that the results of Dzinic and colleagues contradict their published results (4). This is NOT ENTIRELY FACTUAL. In their study, Drs. JCW and PIB's group generated viable maspin knockout mice (Maspin KO) through the intercrossing of “floxed mice heterozygous for the Serpinb5tm1.1pib mutation” (4). Based on the description, their “floxed mice heterozygous for the Serpinb5tm1.1pib mutation” were derived from cytomegalovirus-driven Cre-mediated recombination that targeted the maspin exon 4, which was flanked by two lox P sequences (4). In comparison, breeding Scheme 2 in the study of Dzinic and colleagues employed a design similar to that of Drs. JCW and PIB, with the exception that the oocyte-specific Zp3Cre was used to delete the maspin exon 4. As shown in Fig. 2A (2), viable animals with all three genotypes according to Mendelian distribution were produced. Thus, the results of Dzinic and colleagues (2) in part reproduced the results of Drs. JCW and PIB (4).It is true, however, that the overall findings of Dzinic and colleagues differ significantly from the results of Drs. JCW and PIB (4). The letter of Drs. JCW and PIB to CR includes a series of comments on these differences, leading to their conclusion that “The title of a new article on maspin by Dzinic et al. is inconsistent with the data contained in it” (1). They specifically point out that the results of Dzinic and colleagues contradict their conclusion that “Maspin is not required for embryonic development.” However, they did not perform and/or report all of the required experiments to support this contention. Nor was the broad conclusion by Drs. JCW and PIB based on evidence that directly contradicts the results of an earlier study by Gao and colleagues (5). In brief, Gao and colleagues used a direct gene-targeting strategy to delete “a 2 kb region of the maspin gene that contains the entire exon 7, part of intron 6 and the entire 3′-UTR region” in mouse embryonic stem cells and showed that homozygous loss of maspin led to embryonic lethality at the peri-implantation stage (5). To examine whether the lack of embryonic lethality in the Maspin KO mice generated by the group of Drs. JCW and PIB (4) as well as through Scheme 2 by Dzinic and colleagues (2) was due to a remaining lox P site in the DNA (4), breeding Scheme 1 was designed by Dzinic and colleagues to generate Maspin KO mice in the absence of any lox P insertion. In principle, this breeding scheme is similar to the strategy of Gao and colleagues (5). As shown in Table 1 and Supplementary Table S2 (2), of 6 litters of the F3 mice, no Maspin KO mice were born alive, thus confirming the conclusion of Gao and colleagues that maspin is essential for embryogenesis.In their letter to CR (1), Drs. JCW and PIB criticized the study of Dzinic and colleagues for not having enough mice and speculated that there was a problem in the genotyping. It is not clear whether Drs. JCW and PIB were concerned about the results of our Scheme 1, Scheme 2, or both. It is also not clear by what criteria Drs. JCW and PIB deemed our number of animals insufficient. As summarized in Supplementary Table S2 (2), Schemes 1 and 2 generated 6 and 10 litters of F3 offspring, respectively. It is obvious that the total number of live births in Schemes 1 to 27 was skewed by the number of unborn mice. If the Maspin KO mice were born alive, we would have approximately 36 mice generated. The numbers of animals in each breeding scheme are sufficiently powered to reach statistically significant conclusions.In their letter to CR, Drs. JCW and PIB stated that “Notwithstanding the unsubstantiated conclusion that the knockout mice from Scheme 2 arise from an experimental artefact affecting genome structure that compensate for maspin loss…” (1). The words “experimental artefact” quoted in the letter by Drs. JCW and PIB were NOT used by Dzinic and colleagues. Instead, the concept of compensatory adaptation was used to compare the results of Scheme 2 with those of our Scheme 1 by Dzinic and colleagues (2). We believe that under certain circumstances, animals may use compensatory mechanisms to bypass the impact of maspin knockout in order to survive. In support to this notion, the expression of the cellular prion protein PrPC was found upregulated in maspinflox/Δ and maspinΔ/Δ mice (Fig. 3B and Supplementary Fig. S3; ref. 2). It is likely that future studies on how maspin knockout may be compensated for are of common interest for the field and may uncover the mechanisms underlying the survival of the Maspin KO mice generated by Drs. JCW and PIB as well.Further, we believe that the qualitative differences between the results of Dzinic and colleagues and the results of Drs. JCW and PIB' group are self-evident. The speculation by Drs. JCW and PIB regarding our genotyping problem is not supported. To rigorously screen for the floxed maspin DNA, Dzinic and colleagues used both Southern blotting and PCR methods. The resulting electrophoresis gels are shown in full molecular weight range (Fig. 1F and G; ref. 2). Dzinic and colleagues also used highly specific PCR-based mouse tail DNA genotype screening (Fig. 2B; ref. 2). To confirm the loss of maspin expression at the mRNA, Dzinic and colleagues designed specific PCR primers and showed highly specific detection of maspin in wild-type and maspinflox/Δ mice but not in maspinΔ/Δ mice (Fig. 3B; ref. 2). In parallel, Dzinic and colleagues showed the detection of maspin by Western blotting using the highly specific antibody against the unique maspin-reactive center loop sequence (Fig. 3A; ref. 2). This same antibody detected maspin, by IHC, in the mammary and prostate epithelial cells in wild-type and maspinflox/Δ mice, but not in corresponding tissues of maspinΔ/Δ mice (Fig. 4A and Supplementary Fig. S4; ref. 2).In comparison, the study by the group of Drs. JCW and PIB is underscored by a significant lack of quality controls. The only genotyping data presented were by Southern blotting. The authors showed no evidence of whether Southern blotting was also used for the routine genotype screening of the animals from the inter-cross of “floxed mice heterozygous for the Serpinb5tm1.1pib mutation,” or how else the genotypes of the offspring of the aforementioned intercross were confirmed. The PCR-based detection of maspin mRNA by primers “Maspin-PB1687/1688” generated amplicons of different sizes in different tissues, which were shown in cropped partial images of the gel electrophoresis (Fig. 2a; ref. 4). When these primers were tested in the study of Dzinic and colleagues, they detected multiple nonspecific bands in mouse tissues and in a mouse cell line that does not express maspin (Supplementary Fig. S2; ref. 2). The maspin antibody used by Drs. JCW and PIB was generated against recombinant maspin produced in bacteria and cross-reacted with multiple bands in mouse tissue extracts. In fact, the major band detected by this antibody does not match the molecular weight of maspin (Fig. 2b; ref. 4). The result of maspin IHC in the skin tissues of 8-week-old mice with this antibody is of poor quality, showing no significant distinction between the signals generated by wild-type and knockout mice (Fig. 2c; ref. 4). It is important to note that by Western blotting, this antibody detected a band of the same molecular weight as maspin in both the wild-type and the Maspin KO mice (Fig. 2b; ref. 4), raising a serious concern about the effectiveness of their maspin gene targeting strategy.In their letter to CR, Drs. JCW and PIB claim that the field has not resolved the issue of whether maspin acts as a tumor suppressor due to repeated failures to interpret the data of maspin differential expression. This claim contradicts the consensus in the field. In the period of January 20, 1994, to April 24, 2017, 610 publications on maspin in cancer by investigators from 34 countries on 5 continents were registered on Pubmed.com. As summarized in Fig. 1 in this response, 290 articles addressed the differential expression of maspin in clinical samples, and 320 preclinical studies focused on the biological functions and underlying mechanisms of maspin. Among the 320 preclinical studies, all 22 publications involving MDA-231 cell line, with the exception of a report by Drs. JCW and PIB's group (4), demonstrate a tumor-suppressive role of maspin. Overall, the number of articles presenting evidence for maspin as a tumor suppressor, or a molecular marker for better diagnosis and prognosis, is 540 out of 610 (88.5%).Drs. JCW and PIB suggested that “a simple explanation for the repeated failure of the field to consistently and convincingly link maspin to tumors cell biology is that maspin down-regulation via chromosomal deletion or epigenetic alternations is an occasional bystander effect of selection of PHLPP1 under-expression during cancer progression.” However, their summary of the Oncomine data on the maspin differential expression patterns (Fig. 4a; ref. 4) does not include critical clinical–pathological characteristics of the tumor tissues, such as tumor grades and tumor stages. Although the Kaplan–Meier plots showed similar association of maspin, PHLPP1, or p53 with patients' survival (Fig. 4b; ref. 4), the claim about the specific association between maspin and PHLPP1 was without further supporting evidence. As shown in Fig. 1 in this response, among the 124 publications on the mechanistic control of maspin expression, 87 described transcription factors involved in maspin expression, including p53, hormones, and cytokines. The other 37 studies demonstrate the reversible control of maspin transcription at the level of DNA methylation. Although polymorphism of maspin has been noted, there is no evidence that maspin is mutated or deleted. There is no independent report for the loss of the maspin gene due to loss of PHLPP1.In addition, the experimental evidence of Drs. JCW and PIB was insufficient to support their claim that maspin is not required for tumor suppression. First, Drs. JCW and PIB's group used viral transduction to re-express maspin in MDA-231 and MDA-435 cell lines. In vitro and in vivo characterization of the resulting mass cell populations showed no effect of maspin on tumor development and progression. In this study, there was no evidence that the tumor cell populations that had undergone viral transduction sustained the expression of maspin and the control DNA vector, respectively, in the durations dictated by the biological assays. To this end, given the aforementioned concern over the specificity of the authors' maspin antibody (Fig. 3c and d; ref. 4), the IHC staining with this antibody, as described, would not be reliable evidence to confirm the maspin expression in the xenograft tumors. Overall, the link between the biological phenotypes and the specific maspin-expressing population in the tumor mass cell cultures was not ascertained. Second, Drs. JCW and PIB's group analyzed skin tissues of 8-week-old Maspin KO mice and found no phenotypic differences (Fig. 2c; ref. 4). Such evidence cannot be used to exclude the possibility that maspin may suppress spontaneous tumorigenicity in epithelial organs such as the mammary gland. In comparison, in the study of Dzinic and colleagues, the Maspin KO mice exhibited multiple tumorigenic activities, including pulmonary adenocarcinoma, myoepithelial hyperplasia of the mammary gland, and hyperplasia of luminal cells of dorsolateral and anterior prostate (Figs. 4–6; Supplementary Figs. S4 and S7; ref. 2).In their letter to CR, Drs. JCW and PIB argue that the conclusion of Dzinic and colleagues on the tumor-suppressor role of maspin “cannot be sustained if the operation of an undefined compensatory mechanism in these animals is invoked” (1). Although the speculation by Drs. JCW and PIB that “another factor contributes to the reported tissue abnormalities in these animals” (1) is not without merit for scholarly debate, concerning the data of Dzinic and colleagues, this comment is an example of a paradox, i.e., setting up the expectation of biological observations by our understanding of the observation. It is not uncommon that tumor formation and progression start with singular molecular events. However, the progressive tumor outcome is always dependent on additional subsequent changes. For example, the loss of the APC tumor-suppressor gene leads to genetic instability (6). The current lack of critical insights into the molecular mechanisms of maspin is precisely why we should guard against premature expectations about what it can or cannot do.In the meantime, a logical approach to evaluate a genetic animal model is to compare the functions of maspin in a mouse model with that in human tissues or cells. In the study of Dzinic and colleagues, eight different tissues/organs were analyzed at three different time points for up to 1 year (Figs. 4–6; Supplementary Figs. S4 and S7; ref. 2). As a result, the phenotypes of the Maspin KO mice established, for the first time, the context-dependent regulation by maspin of epithelial terminal differentiation. For example, as in the Maspin KO mice, two types of epithelial cells in human breast tissue also exhibit distinct patterns of maspin expression, with more maspin in the myoepithelial cells and a lower level of maspin in the luminal epithelial cells. Similar to the observation that maspin differential expression is highly prevalent in human breast and prostate tumors, the Maspin KO mice displayed hyperplasia in the mammary or prostate glands, respectively, with 100% penetrance. Similar to the observation that maspin is differentially expressed and inhibits the invasive phenotype of lung adenocarcinoma, the Maspin KO mice developed grade 2–3 adenocarcinoma at 1 year of age. The phenotypic analysis of Dzinic and colleagues also included the corresponding organs in the maspinflox/Δ mice. For example, the dorsolateral prostate glands of the maspinflox/Δ mice exhibited hyperplasia. This result confirmed the haploinsufficiency of the maspin gene, which was first reported by Shao and colleagues (7). Overall, the concordance between the context-specific phenotypic profiles of the Maspin KO mice of Dzinic and colleagues and those observed in human tissues is strong.In their letter to CR, Drs. JCW and PIB further commented that “Rescue of the phenotype by introduction of a maspin transgene would resolve this, but was not carried out in the study. An accepted tumorigenesis protocol should also be used to examine if tumor formation or progression is affected in these mice, before concluding that maspin is a tumor suppressor” (1). It is worth noting that by a strategy analogous to that suggested by Drs. JCW and PIB, Luo and colleagues described evidence that IkkαAA/AA expression in the background of the TRAMP model for prostate cancer promotes tumor metastasis by downregulating the expression of maspin (8). Conversely, re-expression of maspin in the IkkαAA/AA primary prostate tumor cells abolished the effects of active IkkαAA/AA on tumor metastasis. Collectively, these independent findings support the tumor-suppressive action of maspin.In summary, we hope that this reply addresses for the field the comments raised by Drs. JCW and PIB, and serves as a catalyst for the authors to mutually engage in a productive dialogue about maspin, so that the field can advance based on scientific facts.See the original Letter to the Editor, p. 5207P.A. Pemberton has ownership interest (including patents) in Serplus Technology LLC. No potential conflicts of interest were disclosed by the other authors.