A Truncated Phosphorylated P130cas Substrate Domain Is Sufficient to Drive Breast Cancer Growth and Metastasis Formation in Vivo

Tumor Biol. DOI 10.1007/s13277-016-4902-8

ORIGINAL ARTICLE

A truncated phosphorylated p130Cas substrate domain is sufficient to drive breast cancer growth and metastasis formation in vivo

Joerg Kumbrink1,2,3 & Ana de la Cueva 1 & Shefali Soni1,4 & Nadja Sailer1,5 & Kathrin H. Kirsch1

Received: 25 September 2015 /Accepted: 20 January 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016

Abstract Elevated p130Cas (Crk-associated substrate) levels proteins were tested in vivo in an orthotopic mouse model. are found in aggressive breast tumors and are associated with Breast cancer cells expressing the full-length SD and the func- poor prognosis and resistance to standard therapeutics in pa- tional smaller SD fragment (spanning SD motifs 6–10) were tients. p130Cas signals majorly through its phosphorylated injected into the mammary fat pads of mice. The tumor pro- substrate domain (SD) that contains 15 tyrosine motifs gression was monitored by bioluminescence imaging and cal- (YxxP) which recruit effector molecules. Tyrosine phosphor- iper measurements. Compared with control animals, the com- ylation of p130Cas is important for mediating migration, in- plete SD promoted primary tumor growth and an earlier onset vasion, tumor promotion, and metastasis. We previously de- of metastases. Importantly, both the complete and truncated veloped a Src*/SD fusion molecule approach, where the SD is SD significantly increased the occurrence of metastases to constitutively phosphorylated. In a polyoma middle T-antigen multiple organs. These studies provide strong evidence that (PyMT)/Src*/SD double-transgenic mouse model, Src*/SD the phosphorylated p130Cas SD motifs 6–10 (Y236, Y249, accelerates PyMT-induced tumor growth and promotes a more Y267, Y287, and Y306) are important for driving mammary aggressive phenotype. To test whether Src*/SD also drives carcinoma progression. metastasis and which of the YxxP motifs are involved in this process, full-length and truncated SD molecules fused to Src* Keywords p130Cas .Breastcancer .Substratedomain .Src*/ were expressed in breast cancer cells. The functionality of the SD . Metastasis . Bioluminescence imaging . Mouse tumor Src*/SD fragments was analyzed in vitro, and the active model

Electronic supplementary material The online version of this article (doi:10.1007/s13277-016-4902-8) contains supplementary material, Introduction which is available to authorized users. p130 Crk-associated substrate (p130Cas)1 is a scaffold protein * Kathrin H. Kirsch that integrates large multi-protein complexes in response to [email protected] growth factors, hormones, and integrin signaling [1, 2]. p130Cas contains a central substrate domain (SD) where 15 1 Department of Biochemistry, Boston University School of Medicine, YxxP repeats serve as phosphorylation sites for tyrosine ki- 72 E. Concord Street, Boston, MA 02118, USA nases. Through its interaction with Src family members, focal 2 Department of Medicine III, University Hospital Grosshadern, adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K), University of Munich, Munich, Germany 3 Present address: Institute of Pathology, University of Munich, Munich, Germany 1 The abbreviations used are the following: aa, amino acid; abs, antibod- 4 Present address: The Leona M. and Harry B. Helmsley Charitable ies; BLI, bioluminescence imaging; DOX, doxycycline; FAK, focal ad- Trust, New York, NY 10169, USA hesion kinase; IB, immunoblotting; KM, kinase inactive; MFP,mammary 5 Department of Biochemistry, University of Munich, fat pad; p130Cas, p130 Crk-associated substrate; Src*, attenuated kinase Munich, Germany activity; SD, substrate domain; WCE, whole cell extract. Tumor Biol. it recruits the adaptor proteins Crk and Nck to its phosphory- Cla I/Not I into the doxycycline-inducible retroviral expres- KM lated SD. These complexes modulate signal transduction path- sion vector pCXbsr containing HA-tagged Src* or Src . ways that control cell survival, proliferation, adhesion, migra- Primer sequences are provided in Table S1. All constructs tion, and invasion [1, 2]. All of these cellular programs are were verified by sequencing. The rat SD construct has been frequently deregulated during tumorigenesis [3]. Recently, described [10]. QIAGEN Plasmid Maxi Kit was used for functionally distinct amino-terminal variants of p130Cas were DNA preparation. identified that exert different binding activities to FAK, there- Cell culture and retroviral transductions Cell lines were by suggesting how p130Cas may be involved in the regulation obtained as follows: TAM-R, estrogen receptor (ER) positive, of multiple pathways [4]. Elevated expression and activity of tamoxifen resistant MCF-7-derivative (Robert Nicholson, p130Cas promote tumor progression in several cancer types Cardiff, UK); LM2, clone 4175, triple-negative, MDA-MB- including lung, pancreas, prostate, and mammary cancers [1, 231-derivative, constitutively expressing luciferase (Joan 2, 5, 6] and resistance to the anti-tumor drugs adriamycin Massague [13]). Culture conditions: TAM-R (phenol red- (Doxorubicin) and tamoxifen in breast cancers [7, 8]. free RPMI-1640, 5 % charcoal-stripped FBS, and 4 mM glu- We previously established a decoy approach (Src*/SD) to tamine); LM2 (DMEM, 5 % FBS). Media were supplemented modulate p130Cas SD downstream signaling [1, 9, 10]. Src*/ with antibiotics/sodium pyruvate and the cells were main- SD is a chimeric molecule composed of the c-Src kinase do- tained as described [14]. Retroviral transductions were carried main with reduced activity on cellular substrates [(Src*, tyro- out as described [14, 15]. sine 416 to phenylalanine (Y416F)] fused to the p130Cas SD. Protein analysis and cell fractionation Whole cell protein Src* constitutively phosphorylates the SD within Src*/SD extraction, fractionation, and immunoblotting were performed independent of upstream signals. Thereby, the phosphorylated as described [11]. Antibodies: mAbs: β-Actin (Sigma); SD in Src*/SD competes with endogenous p130Cas for PARP1 (clone Y17, recognizes 116 kDa full length, interacting proteins. As a result, the activity of several Millipore). Polyclonal: HA (12CA5, Roche Applied p130Cas-modulated signal transduction pathways and cellular Science); CasB (directed against the SD of p130Cas, Amy programs is altered [1, 9, 10]. H. Bouton, Charlottesville); phospho-tyrosine (p-Tyr; PY-99, Earlier, we investigated the effects of the Src*/SD molecule sc-7020, Santa Cruz Biotechnology). on breast tumor development in vivo by crossing mouse mammary tumor virus (MMTV)-Src*/SD mice and MMTV- aa 109 419 polyoma middle T-antigen (PyMT) mice [11]. PyMT mice A Src*/SD HA Src* SD (YxxP)15 are a commonly used breast cancer model, because they de- P PPP P P velop mammary gland tumors with short latency and the tu- P mor progression reflects that of human disease [12]. The aa 109 419 KM double-transgenic PyMT/Src*/SD mice displayed significant- Src /SD HA SrcKM SD (YxxP)15 ly accelerated tumor formation and developed more aggres- B sive lesions compared with single-transgenic PyMT animals Phospho 18 >500 0-5 >250 190 200>40 439 130 [11]. However, during the study period, no metastases were SitePlus observed with this model system. aa 109 200 300 400 419 Therefore, we aimed to answer the question whether the human1 1213 34951514 678 1011 12 134 15 Src*/SD molecule also drives metastasis formation in vivo rat SD and which part of the phosphorylated SD may be important 157 516 2 206 for this process by applying an orthotopic mouse model. Here, 3 109 207 315 4 260 315 we present that the full-length SD and a fragment containing 316

human 5 419 the SD motifs 6–10 [amino acids (aa) 207–315] significantly 6 207 259 enhanced tumor progression and metastasis formation. Our YLVP YQVP YDIP YDVP YAVP phosphorylation by Src, v-Crk binding studies indirectly show that the phosphorylated SD motifs Crk-, Nck- and Src-SH2 binding Crk-SH2 binding Crk- and Src-SH2 binding 6–10 (Y236, Y249, Y267, Y287, and Y306) of endogenous Fig. 1 Expression constructs used in this study. a Schematic of the HA- KM p130Cas may be important for tumor progression. tagged Src/SD fusion proteins. Src* attenuated Src kinase domain, Src inactive Src kinase domain, SD p130Cas substrate domain, P phosphorylation, aa amino acids. b Sketch of the relative positions and phosphorylation of the YxxP motifs in the p130Cas SD. Motifs 1–15 and Material and methods interaction partners are indicated. The symbols for the motif types and interactors are explained at the bottom of the figure. The number of Expression constructs The human p130Cas SD fragments studies detecting phosphorylation of each motif by proteomics is indicated in the gray box (PhosphoSitePlus, e.g., motif 1 was found (Fig. 1b) were generated by PCR with Pfu polymerase phosphorylated in 18 studies). The SD regions present in the generated (Stratagene) using p130Cas cDNA as template and subcloned constructs are displayed and boundaries are marked by aa numbers Tumor Biol.

Densitometric analysis of gel bands was performed using our laboratory and by others has demonstrated that not all of Image Studio Lite Version 5.2 (LI-COR Biosciences). Equal the YxxP SD motifs are phosphorylated by Src or required for adjustments of contrast and brightness of gel pictures and Crk binding [16, 17](Fig.1b). A PhosphoSitePlus analysis scanned films were applied to all parts of the image using [18] indicates that the central and carboxy-terminal sites 6–15, Adobe Photoshop CS2 version 9.0. Densitometric values of the primarily of the YDVP sequence, are more frequently phos- Src*/SD cytoplasmic (S) and membrane/pellet (P) bands were phorylated than the amino-terminal motifs 1 (YLVP), 3, 4, and related to the corresponding actin band values. The resulting 5 (YQVP sequences) (Fig. 1b). Motif 2 (YQVP) phosphory- values were related to the matching S values (set to one). lation was reported in >500 studies. This suggests that certain motifs are more important than others for p130Cas function. Mammary fat pad injections and survival surgery Animal To identify the SD region that mediates important p130Cas experiments were performed and the mice euthanized accord- functions during tumor progression, the Src*/SD approach ing to guidelines of and approved by the Institutional Animal was used as a tool. For this study, the human full-length SD Care and Use Committee (IACUC) at Boston University. At (construct 1) and the truncated SD fragments 2 to 6 (Fig. 1b) the day of injection, LM2 cells (passaged 1 day earlier) were were fused in frame to the attenuated Src kinase domain trypsinized and washed with regular growth medium and then (Y416F; Src*) or the inactivated Src kinase domain with cold PBS. 1 × 106 cells were diluted in 25 μlcoldPBS, (K295M; SrcKM) as controls. Stable DOX-inducible transduc- mixedwith25μl growth factor-reduced Matrigel (Fisher tants were established for the Src*/1 to Src*/6 as well as the Scientific), and kept on ice. The mixture (50 μl) was injected corresponding SrcKM controls and control (c; empty vector) with a 27.5-gauge needle into the fourth inguinal fat pad of constructs in metastatic LM2 breast cancer cells. LM2 cells anesthetized (3 % isoflurane) 8-week-old NOD/MrkBomTac- are derived from triple-negative MDA-MB-231 cells [13]. All Prkdcscid mice (Taconic). Proper injection of the cells into of the ectopic proteins were expressed, and as expected, the the mammary fat pad (MFP) was confirmed by biolumines- Src*/SD constructs were tyrosine phosphorylated, whereas cence imaging (BLI). Src*/SD induction in the injected cells the corresponding SrcKM/SD were not (Figs. 2a and S1). was achieved by continuous DOX administration (2 mg/ml) in Because signaling changes by expression of Src*/SD are drinking water. Primary tumor growth and metastases were strongly associated with induction of significant morphologic monitored by bioluminescence imaging (BLI) and caliper changes in other breast cancer cell lines [10], the morphology measurements as described [11]. of the LM2 transductants was analyzed. Bioluminescence imaging and analysis The Xenogen IVIS No alterations in cell morphology were observed in the Spectrum Instrument and Living Image Software Version 3.2 SrcKM controls (Fig. S1A and not shown) and Src*/4, 5, and (Caliper LifeSciences) were used for imaging, analysis, and 6 transductants (Fig. S1B). In contrast, induced Src*/1 or quantification of BLI signals. Animals were injected intraperito- Src*/3 expression induced cell rounding (Fig. 2b). After neally (IP) with 150 mg/kg D-luciferin (Gold Biotechnology) 48 h of DOX treatment, 72 % of Src*/1 and 51 % of Src*/3 diluted in Dulbecco’s phosphate-buffered saline (DPBS without cells rounded up whereas significantly less (P ≤ 0.01) magnesium and calcium, Fisher Scientific) 20 min prior to in rounded-up cells were observed in control cells (24 %). The vivo imaging. Up to five mice at a time were anesthetized same constructs were also active in a different breast cancer (1–3 % isoflurane), placed in a warmed imaging chamber with cell line (data not shown). This suggests that YxxP motifs 6– continuous isoflurane exposure (1–2%),andimagedforupto 10 within aa 207–315 and present in Src*/3 are important for 1 min. For ex vivo imaging, 150 mg/kg D-luciferin was injected p130Cas function. IP immediately prior to necropsy. Tissues of interest were ex- In our PyMT/Src*/SD mouse model, partial localiza- cised, placed individually into 12-well plates with 300 μg/ml tion of Src*/SD to cellular membranes by PyMT acceler- D-luciferin in DPBS, and imaged for up to 2 min. ated tumor development [11]. Therefore, the cellular localization of the SD proteins was examined. Whole cell Results extracts of Src*/1 and Src*/3 LM2 transductants were analyzed by cell fractionation and immunoblotting Identification of regions in the SD of p130Cas (Fig. 2c). TAM-R cells (rat SD) were included as control with potential function during tumor progression because we previously presented that Src*/SD expression is almost entirely restricted to the cytoplasm in these cells The rat Src*/SD (Src*/rat SD) decoy molecule (Fig. 1a)has [11]. TAM-R cells expressed the phosphorylated SD con- been previously shown by us to promote primary tumor struct preferentially in the cytoplasm (26 % of cytoplas- growth and a more severe phenotype in the PyMT breast mic levels were found at membranes) (Fig. 2d). LM2 cells cancer mouse model in vivo [11]. Based on these studies, expressed slightly higher Src*/1 and Src*/3 levels in the we were interested in which tyrosine motifs are especially cytoplasmic than in the membrane fraction. Importantly, a important for driving tumor development. Previous work from substantial portion of phosphorylated Src*/1 (60 %) and Tumor Biol.

p-Tyr A CasB LM2 Fig. 2 Expression of the full-length Src*/1 and the truncated Src*/3, c*/3*/1 containing an SD fragment (amino acids 207–315 of p130Cas), induces c */3 */1 morphological changes in LM2 cells. a–d LM2 cells stably transduced 100 kDa with the indicated inducible expression constructs were treated for 48 h with DOX or left untreated. * Src*, KM SrcKM, C control (empty vector). a Expression (CasB) and phosphorylation (p-Tyr) in WCE (30 μg) was 55 kDa confirmed by IB (actin, control). b Morphology of LM2 transductants (100,000 cells/well in 6-well plates) was analyzed by microscopy. Scale, 100 μm. Lower left panel, rounded up and flat/spread cells were counted actin 40 kDa after 48 h of DOX treatment in three fields from three independent 40 kDa actin xperiments per transductant (each field contained ∼70–180 cells) and the results were averaged. Percent rounded up cells of all cells and ’ B control +DOX */1 -DOX */1 +DOX standard deviation are shown. P values were calculated using Student s t test (*P ≤ 0.05). c WCE from the indicated LM2 and TAM-R transductants were fractionated into cytoplasmic (S) and crude membrane/pellet (P) fractions as described previously and analyzed by IB with CasB, p-Tyr, and actin abs. c control (empty vector). d Densitometric analysis of the experiment presented in c.Average protein expression (CasB) and phosphorylation (p-Tyr) of the indicated transduced constructs in the P fraction relative to the S fraction (set to one) and standard deviation from three (CasB) and two (p-Tyr) representative experiments are shown. P values were calculated using Student’s t test 100 */3 -DOX */3 +DOX (*P ≤ 0.05) 80 ** 60 ** 40 The full-length and a truncated phosphorylated p130Cas 20 SD promote metastasis formation % round up cells 0 */1 */3 In the PyMT/Src*/SD mouse model, we observed accelerated control primary tumor growth [11]. However, we could not detect +DOX metastases within the study period. To investigate whether LM2 TAM-R C the full-length and the truncated Src*/SD protein can promote */1 */3 c rat SD metastasis formation when substantial amounts of these phos- S P S P S P S P p130Cas 135 kDa phorylated SD molecules are located at membranes (as shown rat SD 100 kDa for the LM2 transductants), an orthotopic mouse model was 1 applied. LM2 cells have a high potential to form metastases 55 kDa when injected into the mammary fat pad of mice [13]. LM2 3 cells expressing the inducible Src*/1, Src*/3, or control con- actin 40 kDa structs were injected into the mammary fat pad of NOD-Scid p130Cas 135 kDa mice. Src*/1 and Src*/3 were utilized for these in vivo studies rat SD 100 kDa because out of all constructs tested in vitro, they were the only 1 ones that induced changes in cell morphology. The rationale

CasB p-Tyr CasB p-Tyr 55 kDa for this selection was that previously, we have shown that 3 transductants that displayed these morphological changes also actin 40 kDa had altered signaling and cell behavior [10]. Primary tumor growth and metastasis formation were monitored by BLI and 140 D 120 * 120 caliper measurements. 100 100 Representative expression and phosphorylation of the SD 80 80 chimeras in the primary tumors were confirmed by immuno- 60 60 40 40 blotting (Fig. 3a). Of note, SD phosphorylation is highly var- 20 20 iable but present in all tumors. In all groups, the injected cells 0 0 established primary tumors and in part formed spleen metas- rel. expression (CasB) (% of S fraction) SPSPSP SPSPSP */1 */3 SD rel. phosphorylation (pTyr) (% of S fraction) */1 */3 SD tases at later time points (Fig. 3b, BLI quantification in LM2 TAM-R LM2 TAM-R Fig. 3c, d). Compared with control cells, the primary tumor growth in mice injected with Src*/1 cells was significantly Src*/3 (53 %) was detected at membranes reflecting in accelerated (Fig. 3c). Primary tumors reached a volume of part the localization pattern found in PyMT/Src*/SD cells 500 mm3 on average within 29 days (±5.5, P ≤ 0.001) and indicated above. 37 days (±3.4, P ≤ 0.05) in Src*/1 and Src*/3 animals, Tumor Biol. respectively, compared with 41 days (±4.3) in control animals of 28 ± 8.8 days, P ≤ 0.05) and Src*/3 (42 ± 16.4 days, not (Table 1). While only 36 % of control mice developed metas- statistically significant) than in control animals (47 tases by 67 days, metastases were evident in 54 % of Src*/1 ± 16.9 days). At the end of the study period or when animals and 82 % Src*/3 mice (Fig. 4a and Table 1). Src*/1 animals had to be euthanized due to tumor burden, ex vivo imaging of could not be observed for longer than 50 days because of selected organs was performed (Fig. 4b and Table 2)toassay tumor burden or other pathologies that required euthanasia. for metastasis formation. In Src*/1 and Src*/3 animals, the Moreover, metastases were formed earlier in Src*/1 (average tumor cells formed multiple metastases at several organs

Fig. 3 The p130Cas SD drives */1 */3 primary breast cancer growth and A 1534 67- 67 8 metastasis. Mice were injected LM2-*/1 LM2-*/3 p-Tyr 100 kDa p-Tyr 55 kDa with LM2 transductants [*/1, */3, or control (empty vector)] into the CasB 100 kDa CasB 55 kDa mammary fat pad and analyzed actin 40 kDa actin by bioluminescence imaging 40 kDa (BLI) (b). a Expression (CasB) control control and phosphorylation (p-Tyr) of 1235 7 LM2-*/1LM2-*/3 1235 7 LM2-*/1LM2-*/3 the transgene in primary tumor 100 kDa lysates (40 μg) were analyzed by

IB (actin, control). Lysates of CasB 55 kDa p-Tyr LM2-*/1 and */3 cells were used actin 40 kDa as positive control, and in the upper panels, only 20 μgwas B control (animal 2-5) */1 (animal 2-5) loaded onto the gel due to the high day 7 day 22 day 29 day 36 day 7 day 14 day 22 expression in this positive control set. Numbers above the IBs are internal animal numbers. The dash indicates empty lane. b In vivo BLI at indicated days post cell injection (exposure time 1 s). Representative images for each cohort are shown. Color bars with different signal intensities are displayed. Black circles (solid line, primary tumor; dashed line, metastases) mark the areas */3 (animal 1-8) quantified in c and d. c Time */3 1-8 day 7 day 17 day 26 C 4.00E+09 course of primary tumor growth */1 2-5 as analyzed by BLI signals of the 3.50E+09 control 2-5 indicated areas in animals in b. 3.00E+09 The measurements ended by 2.50E+09 animal protocol after the first detection of metastases. d 2.00E+09 Quantification of BLI intensities 1.50E+09 of metastases at first detection of 1.00E+09 total Flux (photons/s) animals in b. e Comparison of the 5.00E+08 primary tumor growth in mice 0 injected with the indicated LM2 day 0 7 14 21 28 35 42 cells. n number of mice at the beginning of the study. Primary tumor volumes were determined 3.50E+08 1800 control (n=11) * D E 3 by caliper measurements as 3.00E+08 1600 */1 (n=11) described. P values were 2.50E+08 1400 */3 (n=11) calculated using Student’s t test 1200 ** (*P ≤ 0.05; **P ≤ 0.01) 2.00E+08 1.50E+08 1000 1.00E+08 800 * 5.00E+07 600 total Flux (photons/s) 0 400 200

primary tumor volume (mm ) (mm volume primary tumor 0 */1 2-5*/3 1-8 day 22 29 36 43 49 control 2-5 Tumor Biol.

Table 1 Tumor growth and 3 metastasis detection after Cohort n Tumor volume (500 mm ) Metastasis Average metastasis mammary fat pad injections of (within 67 days) detection LM2 transductants in mice Control 11 41 days (±4.3) 36 % 47 days (±16.9) Src*/1 13 29 days (±5.5, P ≤ 0.001) 54 % 28 days (±8.8, P ≤ 0.05) Src*/3 11 37 days (±3.4, P ≤ 0.05) 82 % 42 days (±16.4, n.s.)

n.s. not significant

(spleen, lungs, liver, kidneys) in a single animal, whereas in complexes. This is further supported by our previous studies control animals, majorly smaller single metastases to the describing that at membranes, the decoy activates extracellular spleen or lungs were observed. signal-regulated kinase (ERK) survival signaling [11]whereas These results show that the Src*/SD approach, including a in the cytoplasm, it inhibits it [10]. The fact that only a portion shorter fragment of the SD, promotes breast cancer progres- of the Src*/SD molecules is located at membranes in the LM2 sion in this orthotopic in vivo model. Expression of the trun- transductants indicates that the tumor-promoting activity is cated SD fragment led to a considerably higher number of dominant in this model system. detected metastases (more than twice of the animals devel- In the PyMT/Src*/SD mouse model, no metastasis forma- oped metastases compared with control mice). The full- tion was detected during the study period [11] although length SD enhanced primary tumor growth and led to a sig- p130Cas is known to drive metastases [1, 2]. Therefore, in nificantly earlier metastasis onset and increased metastasis an orthotopic model, we interrogated whether the decoy mol- formation. This suggests that the full-length SD mediates a ecule, when partially located at membranes, also promotes stronger tumor-promoting activity, including earlier dissemi- metastases and which part of the phosphorylated SD is impor- nation. Nevertheless, these studies also indirectly indicate that tant for this process. LM2 transductants, with a substantial the phosphorylated SD motifs 6–10 (aa 207–315 in wild-type portion of the Src*/SD molecules at membranes, were injected p130Cas) of endogenous p130Cas may be important for tu- into the mammary fat pad of NOD-Scid mice. Similar to the mor progression when a portion of the protein is found at PyMT model, primary tumor growth was enhanced. In addi- cellular membranes. tion, we could clearly demonstrate that the Src*/SD molecules, containing the full-length or a truncated SD (comprising aa 207–315 of wild-type p130Cas), drive metastasis forma- Discussion tion. Both Src*/SD proteins induced a considerably more severe phenotype as indicated by the detection of multiple metastases in several organs in single animals. Importantly, the Our studies demonstrate that the phosphorylated p130Cas SD control animals developed majorly smaller single metastases is important for p130Cas function during tumor progression only to the spleen or lungs. Furthermore, the full-length chi- in vivo. In an orthotopic mouse model, Src*/SD proteins (full- mera greatly accelerated primary tumor growth and metastasis length SD as well as a SD fragment containing motifs 6–10 of formation, whereas the effects of the truncated form were not the p130Cas SD) promoted tumor progression. The full- as prominent. However, the shorter fragment induced metas- length SD accelerated primary tumor growth and mediated a tases in more animals (82 % of the mice) than the full-length significantly earlier metastasis onset. Both the full-length and form (54 %). This may in part be explained by the fact that the truncated forms significantly increased metastasis animals expressing the full-length SD developed pathologies formation. within 50 days after tumor cell injection that required eutha- We recently uncovered that partial localization of Src*/SD nasia whereas the animals expressing the truncated protein to cellular membranes by the polyoma middle T-antigen could be observed for up to 67 days. Taken together, these (PyMT) accelerates PyMT-induced tumor growth and pro- results suggest that additional sequences in the full-length motes a more aggressive phenotype in vivo [11]. In the PyMT murine breast cancer model, this was attributed to the binding of the attenuated Src kinase domain to the PyMT transgene, which is a membrane-anchored viral protein. The Fig. 4 The p130Cas SD drives metastasis. Mice were injected with the findings that the Src*/SD fusion protein is partially located to indicated LM2 cells [*/1, */3, or control (empty vector)] into the membranes in LM2 cells and drives tumor progression in an mammary fat pad and analyzed by bioluminescence imaging (BLI). a orthotopic breast cancer model suggest that other mechanisms Percent metastases-free animals were calculated based on detections in of membrane targeting exist for this system. Importantly, via live animals as well as from ex vivo studies at the end of the study period. b Ex vivo BLI of the indicated organs. Different exposure times were the constitutively phosphorylated SD fragment, the chimera used as stated. Color bars with diverse signal intensities are displayed. might integrate membrane-associated active signaling DPBS + luc (luciferin), negative control Tumor Biol.

A Table 2 Ex vivo detection of metastasis in indicated organs after 100 mammary fat pad injections of LM2 transductants in mice (endpoint 90 maximum 67 days)

80 Organ Control (n =5) Src*/1(n = 3) Src*/3 (n =4) 70 60 Heart 0/5 0/3 0/4 50 Lungs 3/5 3/3 4/4 40 control (n=11) Kidneys 0/5 2/3 4/4 30 Liver 1/5 3/3 3/4 */1 (n=13) 20 Spleen 1/5 3/3 3/4 10 */3 (n=11) % metastases-free metastases-free animals % 0 day 02226323643455067 SD not present in the truncated molecule might be responsible B */1 (30 s exposed, day 36) for a stronger tumor-promoting activity. In addition to motifs 6–10, the motifs 11–15 are phosphorylated by c-Src and bound by Crk [17]. However, Crk binds with a lower affinity to the c-terminal SD motifs 11–15 than to the central tyrosine motifs 6–10 [17]. Nevertheless, testing of truncated SDs containing motifs 1–5or11–15 with the Src*/SD approach did not significantly affect the cellular behavior of two breast can- heart lungs kidneys cer cell lines (Fig. S1 and not shown). The finding that the truncated Src*/SD decoy drives in vivo tumor metastases provides new information that this region of endogenous p130Cas may be important for mammary cancer progression. This region contains the tyrosine motifs 6–10 of the human SD (Y236, Y249, Y267, Y287, and Y306). liver spleen DPBS + luc Phosphorylation of the SD is essential for the dynamic assem- (10 s exposed, day 50) */3 bly and disassembly of focal adhesions [19]. Each of these motifs was found to be phosphorylated in more than 190 pro- teomic studies as curated at PhosphoSitePlus [18], whereas most of the other SD motifs were significantly less often phosphorylated. Other studies demonstrated that the motifs 6–10 are central for Crk, c-Src, and Nck binding and migration heart lungs kidneys in vitro [16, 17], which are important processes for cellular transformation and tumor progression [2, 5]. The importance of these results is further indicated because p130Cas SD phosphorylation is a central step in the regulation of cell adhesion, migration, and invasion as well as proliferation and survival [6, 19–21]. In the past few years, p130Cas liverspleen DPBS + luc was also identified as a mechanosensor in response to extra- control (1 m exposed, day 50) cellular matrix-integrin engagement [22, 23]. Upon mechani- cal stretching, the SD tyrosines are exposed and accessible to phosphorylation by kinases leading to the activation of mechanotransduction signaling pathways [23]. Thereby, cells can respond and adapt to their environment by activating various cellular programs that may also affect the cells’ surround- heart lungs kidneys ings. These facts demonstrate the importance of p130Cas SD phosphorylation in tissue and cell homeostasis, and it is not surprising that deregulation of p130Cas expression and phosphorylation is involved in various pathogeneses. Elevated p130Cas levels in primary breast tumors correlate with increased rate of relapse and with poor response to tamoxifen liverspleen DPBS + luc treatment [24]. Increased p130Cas expression in comparison Tumor Biol. to primary tumors was also detected in tumor cells isolated 6. Nikonova AS, Gaponova AV, Kudinov AE, Golemis EA. CAS from pleural effusions of mammary carcinoma patients [25]. proteins in health and disease: an update. IUBMB Life. 2014;66(6):387–95. In addition, a recent study described that 75 % of the patients 7. Dorssers LC, van der Flier S, Brinkman A, van Agthoven T, with triple-negative breast cancer expressed high p130Cas Veldscholte J, Berns EM, et al. Tamoxifen resistance in breast can- levels [26]. Moreover, p130Cas contributes to the progression cer: elucidating mechanisms. Drugs. 2001;61(12):1721–33. of several additional cancers such as gliomas, prostate cancer, 8. Ta HQ, Thomas KS, Schrecengost RS, Bouton AH. A novel asso- ciation between p130Cas and resistance to the chemotherapeutic and leukemias [27, 28]. Therefore, targeting p130Cas may drug adriamycin in human breast cancer cells. Cancer Res. open new avenues for the treatment of certain malignancies. 2008;68(21):8796–804. Initial testing by in vivo downregulation of p130Cas by 9. Kirsch KH, Kensinger M, Hanafusa H, August A. A p130Cas ty- intranipple injection of short-interfering (si)RNA resulted in rosine phosphorylated substrate domain decoy disrupts v-crk signaling. BMC Cell Biol. 2002;3:18. a reduction of tumor growth in BALB/c-HER2/neu mice [29]. 10. Soni S, Lin BT, August A, Nicholson RI, Kirsch KH. Expression of Although speculative, data presented here suggest that a phosphorylated p130(Cas) substrate domain attenuates the phos- therapeutics that specifically prevent p130Cas transloca- phatidylinositol 3-kinase/Akt survival pathway in tamoxifen resis- – tion to membranes and/or block YDxP motif 6–10 tant breast cancer cells. 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