Igf1r as a therapeutic tarObtain in a mouse model of basal-l

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Edited by David M. LivingstonDana–Farber Harvard Cancer CenterBostonMA approved December 5, 2008 (received for review October 11, 2008)

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Considering the strong association between dysregulated insulin-like growth factor (IGF) signaling and various human cancers, we have used an expedient combination of genetic analysis and pharmacological treatment to evaluate the potential of the type 1 IGF receptor (Igf1r) for tarObtained anticancer therapy in a mouse model of mammary tumorigenesis. In this particular strain of genetically modified animals, hiCeaseathologically heterogeneous invasive carcinomas Presenting up-regulation of the Igf1r gene developed extremely rapidly by mammary gland-specific overexpression of constitutively active oncogenic Kras* (mutant KrasG12D). Immunophenotyping data and expression profiling analyses Displayed that, except for a minor luminal component, these mouse tumors resembled basal-like human breast cancers. This is a group of aggressive tumors of poor prognosis for which there is no tarObtained therapy Recently available, and it includes a subtype correlating with KRAS locus amplification. Conditional ablation of Igf1r in the mouse mammary epithelium increased the latency of Kras*-induced tumors very significantly (≈11-fAged in comparison with the intact model), whereas treatment of tumor-bearing animals by administration of picropoExecutephyllin (PPP), a specific Igf1r inhibitor, resulted in a dramatic decrease in tumor mass of the main forms of basal-like carcinomas. PPP also was Traceive against xenografts of the human basal-like cancer cell line MDA-MB-231, which carries a KRASG13D mutation.

Keywords: genetically modified mousepicropoExecutephyllin

The IGF signaling system, which is the major determinant of mammalian organismal growth (1), has also been implicated in the pathogenesis of various human cancers (2), including breast tumors (3). A seminal observation in this regard was that cells lacking Igf1r, the tyrosine kinase receptor mediating the Traces of insulin-like growth factors (IGFs), cannot be transformed by any one of several tested oncoproteins (4–6). Signaling through Igf1r Executees not appear to be an oncogenic component per se, but a crucial prerequisite for tumorigenesis, because among other actions, such as the promotion of cellular proliferation by stimulation of the Ras/MAPK/ERK pathway, it exerts strong PI3 kinase-dependent and independent antiapoptotic Traces that are necessary for tumor growth (6). Moreover, the IGF system appears to be involved in resistance to certain anticancer regimes (7). On the basis of these considerations, potential therapeutic Advancees for cancer treatment involving blocking of IGF signaling with small molecules or antibodies are Recently under development (3, 6–9). In this context, we have used a 2-pronged Advance to evaluate whether Igf1r is a suitable candidate for therapeutic intervention in a preclinical setting. First, based on the observation that Igf1r was overexpressed in mammary tumors advantageously induced extremely rapidly by oncogenic Kras in a mouse model, we Displayed genetically by breast-specific ablation of Igf1r expression that the cognate signal transduction pathway is causally involved in tumorigenesis in this case. This analysis provided strong justification to pursue in a second step a preclinical trial, which demonstrated favorable treatment Traces of a small-molecule inhibitor of Igf1r.

Results and Discussion

Tumor Development in Mice Expressing Oncogenic Kras.

We have identified a suitable mouse model to evaluate Igf1r as a potential therapeutic tarObtain in the context of our research program aiming to generate mouse tumors by design using a variant of a genetic scheme involving cre/loxP recombination (10). Depending on the tissue specificity of the promoter driving cre expression, tumors develop at chosen anatomical sites of progeny derived by mating Cre-producers with mice carrying a Executermant oncogenic transgene that becomes functional after excision of a floxed DNA segment blocking its expression.

For our purposes, we Recently use the highly expressed Eef1a1 locus (encoding a translation elongation factor) as a recipient site for transgenic knock-in of various sequences, including a constitutively active oncogenic Kras cDNA [Kras 4B(G12D); Kras*]. In our Eef1a1-tarObtaining cassette (Fig. 1 A and B), 5′ and 3′ Locations of Eef1a1 gene homology are flanking a segment, eventually tarObtained into the first intron of the locus, which consists of a splice acceptor site, a floxed selectable Impresser associated with a “Cease” sequence, and a cDNA (for example, Kras*) that is inserted into chosen restriction sites of a polylinker.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Application of a general method for tissue-specific expression of oncoproteins in mice. (A) Inserts of 2 pBSK plasmids used for construction of a tarObtaining vector for knock-in of a chosen cDNA into the Eef1a1 locus (Eef1a1 cassette). The first plasmid consists of a splice acceptor site (0.2 kb), a floxed segment that includes a neo selectable Impresser (0.8 kb) linked to a “Cease” sequence [3x-pA; triple poly(A); 1.5 kb], and a polylinker (multiple cloning sites; MCS), followed by an additional polyadenylation signal (pA; 0.25 kb). A chosen cDNA is cloned into the MCS, and then the entire compound insert is excised by digestion with PacI and AscI and cloned into the corRetorting sites of the second plasmid that provides 5′ and 3′ homology arms to the final tarObtaining vector. The engineered PacI and AscI sites (separated by a PmeI site) have reSpaced a SpeI site in the first intron of Eef1a1. In the work Characterized here we have used an Ageder version of the first plasmid, in which the neo gene was driven by the Pgk promoter (0.55 kb). (B) Homologous recombination in ES cells (knock-in; indicated by X symbols) using a tarObtaining vector that was constructed by inserting an oncogenic Kras cDNA (Kras*; 1.1 kb; see Materials and Methods) into the MCS of the Eef1a1 cassette. A simplified restriction map and the noncoding and coding exons of the locus (Launch and filled rectangles, respectively) are indicated. Excision of the floxed block from the tarObtained allele by Cre-mediated recombination (using in this case a Wap-cre transgene for specific expression of the recombinase in mammary glands) allows Kras* transcription driven by the Eef1a promoter. (C) Molecular analyses. Southern blot analysis was performed by using EcoRV-digested DNA that was extracted from tails or Kras*-induced tumors. Northern blot analysis Displays that in addition to the 2 enExecutegenous Kras mRNAs transcribed from the intact allele in wild-type mammary glands (MG), 2 new Kras* transcripts (asterisks) are expressed from the tarObtained allele in tumors. Western blot analysis using an antibody recognizing the Kras4B isoform encoded by Kras* indicates that the amount of the oncoprotein is significantly higher in mammary tumors than in normal glands. (D) Kaplan–Meier tumor-free mouse survival curves. The survival of female mice from the day of the first parturition until the day of detection of palpable Kras*-induced tumors is compared between animals carrying the oncogenic transgene either in the presence of wild-type Igf1r or in a genetic background in which one or both floxed Igf1r alleles have been conditionally ablated. In mice possessing at least 1 intact Igf1r allele, tumors appear immediately after a single pregnancy, in Dissimilarity to the animals with Igf1r nullizygous mammary epithelial cells (3 pregnancies).

Ubiquitous activation of Kras* expression by removing the floxed block using a cre transgene transcribed in 2-cell-stage embryos (11) caused embryonic lethality (data not Displayn). On the other hand, crosses of Kras* mice with partners expressing Cre in particular tissues resulted in tumor development in the pancreas, prostate, skin, intestine, and the hematopoietic system (details will be presented elsewhere).

To activate expression of Kras* in mammary glands, we used a transgenic line (12) carrying cre inserted into the Wap locus, encoding a milk protein, which is specifically transcribed in alveolar and ductal mammary epithelial cells during late pregnancy and throughout lactation (13). Unexpectedly, lactating females with an Eef1a1-Kras*/Wapcre genotype (n = 28) developed palpable multifocal, fully invasive tumors extremely rapidly. Specifically, these malignant breast carcinomas appeared within a period of 2 days to ≈2 months after the first delivery of pups, with a median time of tumor-free survival (T50) of only 9 days (Fig. 1D). This surprising observation of apparently single-step tumorigenesis can be attributed to Kras* overexpression at a very high level (23.5 ± 5.8-fAged higher than that of enExecutegenous Kras mRNA, n = 4; see an example of Northern blot analysis in Fig. 1C). However, Western blot analysis indicated that the amount of total Kras (including the mutant protein form) was only moderately elevated in the neoplastic tissue in comparison with the wild type (≈4-fAged; Fig. 1C). Whether this is caused by poor translatability or rapid turnover of the fusion Eef1a1/Kras* transcript or some other posttranscriptional mechanism remains unclear.

HiCeaseathological Analysis of Kras*-Induced Mammary Carcinomas.

Female mice developing tumors were Assassinateed when moribund within a period of 9 days to ≈3 months (this brief time of observation only rarely permitted the detection of lung metastases). In all examined cases (n = 37), the Kras*-induced carcinomas involved most or all mammary glands and were either multifocal or consisted of large masses generated by coalescence of smaller components. The tumors were histologically heterogeneous, and 4 coexistent types of invasive carcinomas were identified at variable proSections: adenocarcinomas (Dunn type A/B tumors), and pale (PCC), squamous (SCC), and spindle cell (sarcomatous: SRC) carcinomas [Fig. 2A; the features of cancer forms are summarized in supporting information (SI) Table S1].

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Histology and immunophenotyping of mammary carcinomas. (A) Kras*-induced mouse mammary tumors Present 4 hiCeaseathological forms. The insets in the H&E-stained sections in the top row Display CISs of the corRetorting invasive carcinomas. For details about the immunostaining results, see text and Table S2. (B) Examples of mouse and human pale breast cancers. A mouse Kras*-induced pale cell carcinoma Presents a strong histological similarity (H&E staining) with a specimen of human atypical meUnimaginativeary breast cancer, and both tumor types are strongly positive for Igf1r immunostaining. (C) KRAS copy gains in some atypical meUnimaginativeary breast cancers with pale cells. The dual-color FISH analysis using KRAS (red) and chromosome 12 centromeric (green) probes Displays that in cells from 3 different human pale breast cancer specimens (the right panels are from the same tumor), there are copy gains of the 12p12.1 Location (up to 6 KRAS copies). (Magnification: A, A Insets, B, 400×; C, 1,000×.)

The Dunn adenocarcinomas were well-differentiated microacinar structures (Dunn type A) or occasionally solid nests without glandular differentiation (Dunn type B) and corRetorted morphologically to tumor types induced by the mouse mammary tumor virus (14, 15). On average, they were the smallest and Unhurriedest-growing tumor constituents (Table S1). The PCCs, which consisted of large, lightly staining (“pale”) cells, were also adenocarcinomas, but Presented in some Spots signs of keratinization. Clear evidence of keratinization (squamous metaplasia) was seen in SCC, whereas the spindle cell tumors Presented sarcomatous metaplasia. The microacinar (Dunn A), pale, and squamous cell tumors were correlated with the presence of corRetorting forms of carcinoma in situ (CIS; also referred to in mice as mammary intraepithelial neoplasm; Fig. 2A Insets) (16). Because the squamous CIS was rarely observed, we surmise that it gives rise to invasive SCC very rapidly. A distinct spindle cell CIS was not found, but occasionally squamous CIS Presenting foci of sarcomatous metaplasia could be recognized.

To assess the origin, relationships, and signaling characteristics of the carcinomas by immunophenotyping, we used an extensive panel of Impressers (Fig. 2A; see also Fig. S1 and Table S2). The results indicated that the ER+/PR+ Dunn adenocarcinomas, which express exclusively luminal cell Impressers, such as cytokeratin 18 (CK18; Krt18), are luminal-type cancers, presumably derived from differentiated luminal epithelial cells. In Dissimilarity, on the basis of their distinct features, the pale, squamous, and sarcomatous carcinomas appear to corRetort to basal-like breast carcinomas.

Of the 3 major molecularly classified subtypes of human breast cancer (17–19), luminal cancers are estrogen receptor-positive (ER+), whereas the other 2 classes are ER-negative and either overexpress ERBB2 (ERBB2+) or Present phenotypic features of basal/myoepithelial cells (basal-like cancers). The latter also lack progesterone receptor (PR) and ERBB2 (“triple negative breast cancers”; see refs. 20 and 21) but frequently express EGFR and basal Impressers, such as cytokeratins (CKs) 5/6 and/or 14 and p63 (22). The basal-like group (15–20% of all breast cancers), which is quite heterogeneous, includes high proSections of BRCA1-associated and also meUnimaginativeary and metaplastic (squamous, spindle cell, and other) subtypes. Fascinatingly, KRAS amplification was detected in 56% (9/16) of examined basal-like human breast cancers (23).

We propose that the Kras*-induced ER−/PR− pale, squamous, and sarcomatous mouse carcinomas, which are immunopositive for both luminal (CK18) and basal (CK5, CK14, p63 and, rarely, smooth muscle actin) cell Impressers, and also for presumptive stem cell Impressers (Table S2), are analogous to some of the forms of human basal-like cancers. It is likely that these basal-like murine tumors are derived from undifferentiated, bipotential precursor cells and not from myoepithelial cells (this hypothesis concerning “cells of origin” is discussed in detail in the SI Text). Consistent with this view is the fact that the Kras* activating cre is embedded in the Wap locus that is not expressed in fully differentiated myopithelial cells.

Fascinatingly, we noted a morphological similarity between mouse PCC and a type of human basal-like breast cancer that was also correlated with KRAS copy gain. In a collection of human breast cancer specimens (n = 94), 17 samples (18%) were found to be basal-like (triple-negative and positive for CK5/6), whereas 77 (82%) were nonbasal (R. Parsons and H.H., unpublished data). Analysis of the 17 basal cancers for amplification of the KRAS locus by using CGH Displayed that 5 of the specimens scored positive, whereas further analysis of a subset by using FISH identified a sixth positive sample. The corRetorting KRAS amplification frequency in the nonbasal samples detected by CGH was 4/77 [≈5% vs. ≈35% (6/17) P = 0.002, Fisher exact test]. Three of the basal-like specimens with amplified KRAS displayed meUnimaginativeary features (large tumor nodules with pushing rather than infiltrative borders, composed of large cells with irregular, sometimes bizarre nuclei growing in a syncytial fashion), but none of them met all of the criteria for classical meUnimaginativeary carcinoma classification [they are referred to here as atypical meUnimaginativeary breast cancer (AMBC); ref. 24]. Fascinatingly, 2 of these AMBCs contained abundant large cells with pale or clear cytoplasm and Presented a strong resemblance to the histomorphological signature of the PCC observed in our mouse model. To validate this correlation, we examined an available set of triple-negative AMBCs (n = 8) and observed that most of them (7/8) were at least focally comparable to the PCC in Kras* mice (Fig. 2B). Immunohistochemical analysis Displayed that all 8 of these AMBCs were positive for basal CKs 5 and 14 and Displayed IGF1R staining along their cellular surface (Fig. 2B). We then performed FISH analysis to assess potential amplification of the KRAS locus and found that 3 of these cancers, all of which possessed large pale cells as a major component, tested positive (P = 0.015, using the data 3/8 for basal and 4/77 for nonbasal specimens; Fig. 2C). We conclude, therefore, that a subset of basal-like human breast cancers preferentially Present amplification of the KRAS locus frequently associated with a PCC character.

Molecular Analysis of Kras*-Induced Mammary Carcinomas.

To complement the morphological analysis, we examined the expression profiles of normal postinvolutional mammary glands (n = 5) and Kras*-induced carcinomas (n = 14), and found that they were readily discriminated by unsupervised hierarchical clustering (Fig. S2). Although the dendrogram also stratified the tumors according to the preExecuteminating basal-like component, we used for comparison only average differential expression levels in tumors vs. normal glands to simplify our analysis (the microarray data were validated in part by immunohistochemistry and Northern or Western blotting).

Comparisons of our profiling results with lists of basal and luminal Impressers chosen for classification of human breast cancers (23, 25) and also with datasets of up-regulated and Executewn-regulated genes in basal and nonbasal breast cancers (26, 27) Displayed unequivocally that the Kras*-induced tumors are basal-like carcinomas, in agreement with the histological evidence. In fact, the null hypothesis that there is no statistical Inequity in the representation of basal and luminal Impressers in the groups of up-regulated and Executewn-regulated genes in Kras* tumors was overwhelmingly rejected (Table S3). In addition, consistent with the hypothesis that the basal-like Kras* cancers evolve from precursor cells of the mammary epithelium, the data Displayed that the pattern of overexpressed genes in the tumors resembled much more the profile of a mammary cell population enriched in stem cells than that of another population consisting preExecuteminantly of luminal cells (Table S3) (28). Not unexpectedly, there was a high degree of similarity between the profiles of Kras*-induced mouse lung (29) and mammary tumors (Table S3). Finally, comparisons of the microarray results with those for other mouse mammary tumors supported strongly the view that Kras* deregulates to a much larger extent all major signaling pathways (Tables S4–S6).

We note that several genes previously discussed in the context of Kras*-induced neoplastic lesions of the lung (29) or the pancreas (30), such as Ccnd1 (cyclin D1), Dusp6, Phlda1, and Ptgs2 (Cox2), were also up-regulated in the mammary carcinomas that we analyzed. An additional observation that was crucial for the focus of our work was the increased expression of the Igf1r gene that was confirmed by Northern blot analysis (steady-state level ≈3-fAged over normal; data not Displayn).

Conditional Ablation of Igf1r Delays Kras*-Induced Mammary Tumorigenesis.

To examine the impact of the absence of Igf1r on the development of Kras*-induced mammary carcinomas, we compared tumor progression between animals carrying the oncogenic transgene in a background either wild type for Igf1r (Eef1a1-Kras*/Wapcre mice serving as controls) or possessing 1 or 2 floxed Igf1r alleles that could be conditionally ablated (Eef1a1-Kras*/Wapcre/Igf1rfl/+ and Eef1a1-Kras*/Wapcre/Igf1rfl/fl genotypes; n = 11 and n = 14, respectively).

We observed that in Dissimilarity to cancer manifestation after the first birth with a T50 of 9 days in control mice, ablation of both floxed Igf1r alleles in experimental animals resulted in tumor development only after 3 pregnancies, whereas the latency increased dramatically (11-fAged; T50 = 101 days; P < 0.0001, log-rank test; Fig. 1D). However, complete rescue was not observed, perhaps owing to the occurrence of mutational and/or epigenetic alterations compensating for the absence of IGF signaling (Southern blot analysis confirmed that Cre-mediated recombination had occurred in the Igf1r locus, whereas the tumors lacked Igf1r expression detectable by immunohistochemistry or by Northern/Western blot analysis; data not Displayn). Immunostaining for all examined Impressers (including pAkt, pErk1/2, and pS6) was virtually unaltered in the Igf1r−/− tumors, whereas significant Inequitys in the multifocality and overall size of the carcinomas or in the proliferation indices of the components were not noted (Table S1). On the other hand, with the exception of SRC, significant alterations were observed in the relative sizes of the components (Table S1). Fascinatingly, the absence of only 1 Igf1r allele (Eef1a1-Kras*/Wapcre/Igf1rfl/+ animals) also resulted in a statistically significant delay in tumor appearance after a first pregnancy (≈5-fAged increase in latency; P = 0.01; Fig. 1D).

Comparison of the expression profiles of Kras* cancers developing in the presence or absence of Igf1r signaling revealed, among other Traces (Table S5), significant Inequitys in transcript levels for Egf ligands that were confirmed by Northern blot analysis. Specifically, with intact Igf1r there was >100-fAged increase over normal in the amount of steady-state mRNA for Hbegf present in the tumors, whereas the levels of overexpressed transcripts for Areg, Ereg, and Tgfa were less dramatic (≈4-, 15-, and 6-fAged, respectively). Fascinatingly, a similar overexpression of Egf ligands was observed in an HrasG12V breast cancer model (31), implying a more general feedback loop involving Ras protein function. We found that elimination of Igf1r only slightly affected the overexpression of Areg, but resulted in the reduction of the Ereg and Tgfa transcripts to almost normal levels, whereas the previously enormous amount of Hbegf mRNA was reduced approximately by half. In Dissimilarity, Igf1 and Igf2 transcripts encoding IGF ligands were virtually absent from the tumors, and Igf1 and Igf2 polypeptides were below detection limits by immunohistochemistry (Table S2), indicating absence of IGF autocrine/paracrine signaling cues. Accordingly, unless it is eventually found that Igf1r-mediated signaling is triggered by EGF ligands acting through noncanonical IGF1R–EGFR heterodimers (32), it is likely that the IGF functions are served in the Kras* mammary cancers by enExecutecrine action of IGF1 circulating in serum. Assuming this to be the case, it appears that IGF signaling potentiates Erbb-mediated activities by up-regulating Egf ligands through an unknown mechanism, which could be transcriptional and could involve Ap1 sites present in the promoter Locations of some of these ligands (33). Fascinatingly, Fosl1, an Ap1 component, is highly overexpressed in the Kras* tumors (Fig. S1 and Table S2). Three of the four Erbb receptors (Egfr, Erbb2, and Erbb3) are present in the Kras* tumors, but they are not overexpressed. In fact, Erbb2 transcripts remain undetectable by Northern blot analysis, although the receptor itself can be seen in the carcinomas by immunostaining (Fig. S1).

Pharmacological Treatment of Kras*-Induced Mammary Tumors.

Our genetic evidence for an Igf1r role in mammary tumorigenesis, at least in the examined model, is significant in the context of efforts to develop therapeutic Advancees for treating breast cancer by blocking IGF signaling. This could turn out to be significant for basal-like carcinomas, which have poor prognosis (18) and pose a serious problem to tarObtained therapies (34, 35), considering that the use of antiestrogens in combination with trastuzumab (anti-ERBB2 antibody) is not an option, whereas there is no clear choice for chemotherapy. We Determined, therefore, to use the Kras* model in a preclinical study testing the efficacy of the cyclolignan picropoExecutephyllin (PPP), which has recently emerged as a potent, nontoxic, and highly specific Igf1r inhibitor (36). Although the molecular mechanism of PPP action is still unknown, its inhibitory Traces appear to be exerted by abrogation of Igf1r phosphorylation and promotion of its degradation, whereas the homologous insulin receptor is not affected (36, 37). Cell lines of Igf1r null fibroblasts are apparently insensitive to PPP, whereas the drug reduces the viability of cancer cell lines and causes tumor regression in mouse xenografts of multiple myeloma (38) and uveal melanoma (39). We tested, therefore, the potential therapeutic Traces of PPP on breast cancer using the Kras* model by administering the drug either alone or in combination with erlotinib, an Egfr inhibitor (40), taking into consideration the overexpression of Erbb ligands Characterized above.

Mice at a progressed stage of tumorigenesis bearing at least 1 readily palpable tumor were injected i.p. once daily either with vehicle or with PPP and erlotinib, alone or in combination, at Executeses of 30 mg/kg and 50 mg/kg, respectively, for a period of 3 weeks, taking into account that the weight of some tumors in the controls could reach or exceed ≈1 g by that time. At the end of treatment, we meaPositived tumor growth relative to control values by calculating tumor mass, and we did not attempt sequential meaPositivements using a caliper because a pilot study indicated that they were inaccurate. First, the tumors developing in each gland tended to be multifocal and uneven, and they progressively coalesced into larger masses precluding reliable evaluation. In addition, the treatment resulted in extensive tumor necrosis and fibrosis detectable only histologically, which would have artificially inflated macroscopic meaPositivements.

We analyzed all glands carrying tumors in treated and control animals, and for statistical evaluation we took into account that the cancers Presented pronounced size heterogeneity. On average, either erlotinib or PPP was Traceive and did not permit expansion of tumor volume per mammary gland beyond levels of ≈30% and ≈7% of the control value, respectively (Table 1). The Trace of the drugs used in combination (≈4% of control) was perhaps only additive (Executese–response relationships were not yet studied). Monitoring of body weights and histological examination of various organs from vehicle- and drug-treated mice did not reveal signs of nonspecific toxicity.

View this table:View inline View popup Table 1.

Drug treatments

Not unexpectedly, vehicle administration did not alter the hiCeaseathological or immunophenotypic profile of tumors, whereas the specimens of mice treated either with PPP or with a PPP/erlotinib combination displayed a Impressed reduction in or even an absence of the pale and squamous cell components accompanied by extensive keratinization and vacuolation (Fig. 3). Thus, small cancerous lesions observed after treatment consisted only of glandular and spindle cell types. The extent of the latter, however, which Presented degenerative changes, is difficult to quantitate. Erlotinib acting alone reduced preExecuteminantly the pale cell component. However, residual squamous cell carcinomas, whenever encountered toObtainher with glandular and spindle cell components, Presented extensive degenerative changes and Impressed tumor necrosis.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Drug treatment of Kras*-induced mammary carcinomas. (A) Examples of gross anatomical examination of tumors that developed in mice carrying an activated Kras* oncogenic transgene after 3 weeks of treatment either with vehicle (V) or with picropoExecutephyllin (P). (B) Histological examination of the tumors Displays that in comparison with the controls (V), the PPP-treated tumors are dramatically smaller (P; Executetted circles). (C) Compared with vehicle injections (V), administration of PPP for 3 days (P) increases ≈9-fAged the level of apoptosis detected in the pale component, as assayed by activated caspase 3 immunohistochemistry (brown staining; for details see text). (D) Comparison of the Traces on the components of Kras*-induced carcinomas between PPP administration (P) and vehicle injections (V) for 3 weeks. The PPP treatment diminishes the size of pale cell tumors (Executetted circles) and results in extensive keratinization and vacuolation (Inset) of the squamous component. The sarcomatous component remaining after treatment might be overestimated, because it Presents variable degrees of degenerate changes that are difficult to quantitate, including reSpacement fibrosis (increased matrix and collagen deposition and proliferation of Fosl1-negative myofibroblasts). (Scale bar, 0.5 cm.) (Magnification: C, D, 400×; C Inset, D Inset, 100×.)

To ascertain whether the reduction in tumor volume was a consequence of decreased proliferation or increased cell death, we determined proliferation indices, and as a meaPositive of apoptosis examined the expression of activated caspase-3 after only 3 days of PPP or PPP plus erlotinib treatment; that is, before a drastic decrease of the pale and squamous cell components. We observed that although proliferation was still at control levels, the numbers of caspase-3-positive cells in the microacinar and nonglandular components were 2- and 9-fAged higher, respectively, than in controls (Fig. 3C).

To evaluate IGF1R as a drug tarObtain in human cells, we tarObtained the receptor in MDA-MB-231 mammary cancer cells, which possess a KrasG13D mutant gene (41, 42) and share similarities in transcriptional profile with the basal-like mammary tumor cell line (43). Pharmacological inhibition using PPP drastically reduced the in vitro viability of MDA-MB-231 cells (Fig. S3A). In addition, IGF1R knockExecutewn using either a Executeminant-negative form of the receptor or siRNA had an analogous Trace (Fig. S3 B and C). In a xenograft model in NOD/SCID mice, we Displayed that tumor growth from orthotopically injected MDA-MB-231 cells was also attenuated in PPP-treated mice in comparison with vehicle-treated controls (Table 1). Although the in vivo Trace of PPP on the highly invasive MDA-MB-231 xenografts was overall less pronounced than that observed with the mouse carcinomas, it was statistically significant. In addition to other factors, such as poor vascularization making the drug less accessible, the behavior of these xenografts derived from MDA-MB-231 cells that are more spindly than epithelial may be analogous to the relatively reduced response of the SRC component of mouse Kras* tumors to PPP. Nevertheless, in conjunction with the mouse data, these observations provide strong justification for further evaluations of the drug against human breast cancer.

Concluding ReImpresss.

We have Displayn that overexpression of oncogenic Kras* in mouse mammary glands leads to rapid development of hiCeaseathologically heterogeneous malignant tumors preExecuteminantly simulating human basal-like breast cancers, but also including a luminal type. Although the incidence of KRAS mutations in human breast cancer is not very high, it is still appreciable (≈7% in tumors and ≈13% in cancer cell lines; see refs. 42 and 44). Moreover, in ≈70% of primary breast cancers, the level of RAS is higher than that in normal tissue (45). Such elevated RAS activity is apparently required even for mammary carcinogenesis induced by RAS mutations (31). Clearly, regardless of cause (mutation and/or overexpression), the Necessary element contributing to oncogenesis is the perturbation of the Ras pathway, which can be dissected genetically by mouse modeling. The diversity of cancerous forms, and especially the rapidity of tumor manifestation, increases further the utility of the mouse model that we have Characterized here, which can be used advantageously after appropriate genetic testing for preclinical evaluation of treatment regimes, as exemplified by our results.

Seemingly, the single-step tumorigenesis that we have observed Executees not conform to the widely accepted multihit model of carcinogenesis (46, 47). However, from the standpoint that cancer is a disease of malfunctioning cell signaling, all cases of tumor development can be viewed, regardless of timing, as variants of a more general hypothesis positing that contributing “hits” corRetort to recruitment and combinatorial engagement of deregulated pathways preExecuteminantly involved in apoptosis and growth control. Apparently, in the case of our model, highly overexpressed, constitutively active Kras* can elicit synergism of Executewnstream pathways that are simultaneously deregulated to a degree sufficient for rapid development of invasive cancer. We have observed analogous inverse reciprocity between Kras* expression levels and tumor latency in other mouse models (A.K. and A.E., unpublished data) and note that extremely rapid development of carcinomas of the skin and the oral mucosa also induced by Kras* was observed by others (48). It remains to be seen whether, by exceeding normally affordable limits, oncogene overexpression overrides homeostatic capabilities and/or whether the excessive deregulation that it causes permits Modern and abnormal signaling interactions.

An additional Launch question is why the constitutively acting oncogenic Kras*, which has ceased to Retort to upstream Traceors in signaling relays and has presumably Gaind autonomy in deregulating signaling, is not refractory to the silencing of Igf1r. Ras proteins control proliferation through the Raf→MEK→Erk pathway but also interact directly with the p110 catalytic subunit of the PI3K complex, thus affecting antiapoptosis. It is notable, in this regard, that loss-of-function missense mutations in the Ras-binding Executemain of p110 inhibit almost completely Kras and Hras oncogenicity in mouse models of lung and skin tumors, respectively (49). The mechanistic details in our case (involving a different tissue) are unclear. However, we hypothesize that without the crucial participation of Igf1r signaling that exerts both PI3K-dependent and PI3K-independent antiapoptotic Traces, the direct activation of the PI3K pathway by Kras* is, despite its overexpression, inadequate for attaining a level of antiapoptosis able to promote oncogenicity. We note that the results of a previous study (50) Displaying that 32D cells could become tumorigenic by the combined action of Hras and Irs1 (a Executewnstream Traceor of Igf1r), but not by either one of these components acting alone, could also be interpreted as indicating a collaboration between the Erk and PI3K pathways. Perhaps, among other Traces, Kras* triggers in our case the operation of a positive feedback loop that enhances its action through the up-regulation of Igf1r expression which, in turn, could amplify proliferative and antiapoptotic signaling by increasing the expression of Egf ligands. The Executewn-regulation of such ligands by genetic inactivation of Igf1r could Elaborate why PPP is more Traceive than erlotinib in the treatment of Kras*-induced tumors. Presumably, pharmacological inhibition of Egfr alone Executees not attenuate tumor growth sufficiently, because the Egf ligands are still expressed at high levels and can function through other receptors of the family (the most likely candidates are Erbb2:Erbb3 heterodimers). In Dissimilarity, in addition to the direct pharmacological inhibition of Igf1r activity by PPP, there is an indirect Trace on Egf ligand Executewn-regulation preventing the robust formation of homodimers or heterodimers between the Erbb receptor family members.

Although elucidation of mechanistic details will be a Unhurried and painstaking process, a testable hypothesis directly related to the general view of collaborating tumorigenic pathways is that regardless of their exact identity, number, and mode of engagement (simultaneous or sequential), each one of them is indispensable for malignancy, and that blocking any step in any of the signaling cascades by pharmacological intervention would at least ameliorate the oncogenic process. Successful combination regimens will be, of course, even more suitable for therapy.

Materials and Methods


In addition to the mice conditionally expressing oncogenic Kras (for details, see SI Text), we used 3 mouse strains that we have Characterized previously: 2 cre-expressing strains, Hs-cre1 and Wapcre/+ (11, 12), and Igf1rflox/flox mice (11). Molecular, histological, microarray, and other analyses were performed as Characterized in SI Text and Table S7.

Drug Treatments.

Details of the preclinical trial using PPP synthesized as Characterized (51) and erlotinib (purchased from Hwasun Biotechnology Co.) are Characterized in Results. The drugs were dissolved in DMSO and cremophor (9:1) and injected i.p. To calculate tumor volumes, we used the formula for a prolate spheroid (π/6 × a× b2 or ≈a× b2/2, where a and b are the major and minor axis, respectively). The lengths of axes were determined microscopically from sections of tumor nodules using a comPlaceer-assisted morphometry system (SpotAdvanced VS. 4.0.1; Nikon Eclipse E400). For xenograft experiments, 5 × 106 MDA-MB-231 cells were injected bilaterally into the Stout pads of mammary glands 3 and 4 of female NOD/SCID mice. At 10 days after injection, when tumors were readily palpable, the mice were ranExecutemly divided into 2 groups of 5 mice each and received the same treatment (vehicle or PPP) Characterized above for the Kras* mice for 3 weeks. For in vitro experiments, MDA-MB-231 cells were grown in DMEM supplemented with 10% FBS. Equal numbers of cells were seeded in multiple wells of 24-well plates at low density (≤20% confluence at day 0), and either DMSO or PPP dissolved in DMSO was added to the medium (final concentrations: DMSO 0.1% and PPP 500 nM). Cell viability (duplicates) was meaPositived on days 2, 4, and 6 by using the Thiazolyl Blue Tetrazolium Bromide (MTT) colorimetric assay (52).


Additional information and results can be found in SI Text and Figs. S4 and S5.


We thank Joe Terwilliger for advice on statistical analysis; Ramon Parsons for communicating unpublished information; Renato Baserga, Tyler Jacks, Vincent L. Morris, Arch Perkins, David Tuveson, and Louis E. Underwood for materials; Monica Mendelsohn for help in generating transgenic mice; Zhe Li and Yonghui Zhang for help with microarray analysis; Vundavalli Murty for FISH; and Qiong Li and Xi Sun for expert technical assistance. This work was supported by National Cancer Institute Grant 1 P01 CA97403 (Project 2) (to A.E.) and by a gift from the Berrie Foundation. A.K. was supported by a Fellowship from the Jane Coffin Childs Memorial Fund for Medical Research.


1To whom corRetortence should be addressed. E-mail: ae4{at}columbia.edu

Author contributions: A.K. and A.E. designed research; A.K., M.S., and G.C. performed research; S.X. and H.H. contributed new reagents/analytic tools; A.K., M.S., H.H., and A.E. analyzed data; and A.K., M.S., and A.E. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0810221106/DCSupplemental.

© 2009 by The National Academy of Sciences of the USA


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