Tumor Biology

, Volume 37, Issue 8, pp 10665–10673 | Cite as

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

  • Joerg Kumbrink
  • Ana de la Cueva
  • Shefali Soni
  • Nadja Sailer
  • Kathrin H. KirschEmail author
Original Article


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


p130Cas Breast cancer Substrate domain Src*/SD Metastasis Bioluminescence imaging Mouse tumor model 


p130 Crk-associated substrate (p130Cas)1 is a scaffold protein that integrates large multi-protein complexes in response to growth factors, hormones, and integrin signaling [1, 2]. p130Cas contains a central substrate domain (SD) where 15 YxxP repeats serve as phosphorylation sites for tyrosine kinases. Through its interaction with Src family members, focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K), it recruits the adaptor proteins Crk and Nck to its phosphorylated SD. These complexes modulate signal transduction pathways that control cell survival, proliferation, adhesion, migration, and invasion [1, 2]. All of these cellular programs are frequently deregulated during tumorigenesis [3]. Recently, functionally distinct amino-terminal variants of p130Cas were identified that exert different binding activities to FAK, thereby suggesting how p130Cas may be involved in the regulation of multiple pathways [4]. Elevated expression and activity of p130Cas promote tumor progression in several cancer types including lung, pancreas, prostate, and mammary cancers [1, 2, 5, 6] and resistance to the anti-tumor drugs adriamycin (Doxorubicin) and tamoxifen in breast cancers [7, 8].

We previously established a decoy approach (Src*/SD) to modulate p130Cas SD downstream signaling [1, 9, 10]. Src*/SD is a chimeric molecule composed of the c-Src kinase domain with reduced activity on cellular substrates [(Src*, tyrosine 416 to phenylalanine (Y416F)] fused to the p130Cas SD. Src* constitutively phosphorylates the SD within Src*/SD independent of upstream signals. Thereby, the phosphorylated SD in Src*/SD competes with endogenous p130Cas for interacting proteins. As a result, the activity of several p130Cas-modulated signal transduction pathways and cellular programs is altered [1, 9, 10].

Earlier, we investigated the effects of the Src*/SD molecule on breast tumor development in vivo by crossing mouse mammary tumor virus (MMTV)-Src*/SD mice and MMTV-polyoma middle T-antigen (PyMT) mice [11]. PyMT mice are a commonly used breast cancer model, because they develop mammary gland tumors with short latency and the tumor progression reflects that of human disease [12]. The double-transgenic PyMT/Src*/SD mice displayed significantly accelerated tumor formation and developed more aggressive lesions compared with single-transgenic PyMT animals [11]. However, during the study period, no metastases were observed with this model system.

Therefore, we aimed to answer the question whether the Src*/SD molecule also drives metastasis formation in vivo and which part of the phosphorylated SD may be important for this process by applying an orthotopic mouse model. Here, we present that the full-length SD and a fragment containing the SD motifs 6–10 [amino acids (aa) 207–315] significantly enhanced tumor progression and metastasis formation. Our studies indirectly show that the phosphorylated SD motifs 6–10 (Y236, Y249, Y267, Y287, and Y306) of endogenous p130Cas may be important for tumor progression.

Material and methods

Expression constructs

The human p130Cas SD fragments (Fig. 1b) were generated by PCR with Pfu polymerase (Stratagene) using p130Cas cDNA as template and subcloned Cla I/Not I into the doxycycline-inducible retroviral expression vector pCXbsr containing HA-tagged Src* or SrcKM. Primer sequences are provided in Table S1. All constructs were verified by sequencing. The rat SD construct has been described [10]. QIAGEN Plasmid Maxi Kit was used for DNA preparation.
Fig. 1

Expression constructs used in this study. a Schematic of the HA-tagged Src/SD fusion proteins. Src* attenuated Src kinase domain, Src KM 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 interaction partners are indicated. The symbols for the motif types and interactors are explained at the bottom of the figure. The number of studies detecting phosphorylation of each motif by proteomics is indicated in the gray box (PhosphoSitePlus, e.g., motif 1 was found phosphorylated in 18 studies). The SD regions present in the generated constructs are displayed and boundaries are marked by aa numbers

Cell culture and retroviral transductions

Cell lines were obtained as follows: TAM-R, estrogen receptor (ER) positive, tamoxifen resistant MCF-7-derivative (Robert Nicholson, Cardiff, UK); LM2, clone 4175, triple-negative, MDA-MB-231-derivative, constitutively expressing luciferase (Joan Massague [13]). Culture conditions: TAM-R (phenol red-free RPMI-1640, 5 % charcoal-stripped FBS, and 4 mM glutamine); LM2 (DMEM, 5 % FBS). Media were supplemented with antibiotics/sodium pyruvate and the cells were maintained as described [14]. Retroviral transductions were carried out as described [14, 15].

Protein analysis and cell fractionation

Whole cell protein extraction, fractionation, and immunoblotting were performed as described [11]. Antibodies: mAbs: β-Actin (Sigma); PARP1 (clone Y17, recognizes 116 kDa full length, Millipore). Polyclonal: HA (12CA5, Roche Applied Science); CasB (directed against the SD of p130Cas, Amy H. Bouton, Charlottesville); phospho-tyrosine (p-Tyr; PY-99, sc-7020, Santa Cruz Biotechnology).

Densitometric analysis of gel bands was performed using Image Studio Lite Version 5.2 (LI-COR Biosciences). Equal adjustments of contrast and brightness of gel pictures and scanned films were applied to all parts of the image using Adobe Photoshop CS2 version 9.0. Densitometric values of the Src*/SD cytoplasmic (S) and membrane/pellet (P) bands were related to the corresponding actin band values. The resulting values were related to the matching S values (set to one).

Mammary fat pad injections and survival surgery

Animal experiments were performed and the mice euthanized according to guidelines of and approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University. At the day of injection, LM2 cells (passaged 1 day earlier) were trypsinized and washed with regular growth medium and then with cold PBS. 1 × 106 cells were diluted in 25 μl cold PBS, mixed with 25 μl growth factor-reduced Matrigel (Fisher Scientific), and kept on ice. The mixture (50 μl) was injected with a 27.5-gauge needle into the fourth inguinal fat pad of anesthetized (3 % isoflurane) 8-week-old NOD/MrkBomTac-Prkdc scid mice (Taconic). Proper injection of the cells into the mammary fat pad (MFP) was confirmed by bioluminescence imaging (BLI). Src*/SD induction in the injected cells was achieved by continuous DOX administration (2 mg/ml) in drinking water. Primary tumor growth and metastases were monitored by bioluminescence imaging (BLI) and caliper measurements as described [11].

Bioluminescence imaging and analysis

The Xenogen IVIS Spectrum Instrument and Living Image Software Version 3.2 (Caliper LifeSciences) were used for imaging, analysis, and quantification of BLI signals. Animals were injected intraperitoneally (IP) with 150 mg/kg d-luciferin (Gold Biotechnology) diluted in Dulbecco’s phosphate-buffered saline (DPBS without magnesium and calcium, Fisher Scientific) 20 min prior to in vivo imaging. Up to five mice at a time were anesthetized (1–3 % isoflurane), placed in a warmed imaging chamber with continuous isoflurane exposure (1–2 %), and imaged for up to 1 min. For ex vivo imaging, 150 mg/kg d-luciferin was injected IP immediately prior to necropsy. Tissues of interest were excised, placed individually into 12-well plates with 300 μg/ml d-luciferin in DPBS, and imaged for up to 2 min.


Identification of regions in the SD of p130Cas with potential function during tumor progression

The rat Src*/SD (Src*/rat SD) decoy molecule (Fig. 1a) has been previously shown by us to promote primary tumor growth and a more severe phenotype in the PyMT breast cancer mouse model in vivo [11]. Based on these studies, we were interested in which tyrosine motifs are especially important for driving tumor development. Previous work from our laboratory and by others has demonstrated that not all of the YxxP SD motifs are phosphorylated by Src or required for Crk binding [16, 17] (Fig. 1b). A PhosphoSitePlus analysis [18] indicates that the central and carboxy-terminal sites 6–15, primarily of the YDVP sequence, are more frequently phosphorylated than the amino-terminal motifs 1 (YLVP), 3, 4, and 5 (YQVP sequences) (Fig. 1b). Motif 2 (YQVP) phosphorylation was reported in >500 studies. This suggests that certain motifs are more important than others for p130Cas function.

To identify the SD region that mediates important p130Cas functions during tumor progression, the Src*/SD approach was used as a tool. For this study, the human full-length SD (construct 1) and the truncated SD fragments 2 to 6 (Fig. 1b) were fused in frame to the attenuated Src kinase domain (Y416F; Src*) or the inactivated Src kinase domain (K295M; SrcKM) as controls. Stable DOX-inducible transductants were established for the Src*/1 to Src*/6 as well as the corresponding SrcKM controls and control (c; empty vector) constructs in metastatic LM2 breast cancer cells. LM2 cells are derived from triple-negative MDA-MB-231 cells [13]. All of the ectopic proteins were expressed, and as expected, the Src*/SD constructs were tyrosine phosphorylated, whereas the corresponding SrcKM/SD were not (Figs. 2a and S1). Because signaling changes by expression of Src*/SD are strongly associated with induction of significant morphologic changes in other breast cancer cell lines [10], the morphology of the LM2 transductants was analyzed.
Fig. 2

Expression of the full-length Src*/1 and the truncated Src*/3, containing an SD fragment (amino acids 207–315 of p130Cas), induces morphological changes in LM2 cells. ad LM2 cells stably transduced 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 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 after 48 h of DOX treatment in three fields from three independent xperiments per transductant (each field contained ∼70–180 cells) and the results were averaged. Percent rounded up cells of all cells and 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 (*P ≤ 0.05)

No alterations in cell morphology were observed in the SrcKM controls (Fig. S1A and not shown) and Src*/4, 5, and 6 transductants (Fig. S1B). In contrast, induced Src*/1 or Src*/3 expression induced cell rounding (Fig. 2b). After 48 h of DOX treatment, 72 % of Src*/1 and 51 % of Src*/3 cells rounded up whereas significantly less (P ≤ 0.01) rounded-up cells were observed in control cells (24 %). The same constructs were also active in a different breast cancer cell line (data not shown). This suggests that YxxP motifs 6–10 within aa 207–315 and present in Src*/3 are important for p130Cas function.

In our PyMT/Src*/SD mouse model, partial localization of Src*/SD to cellular membranes by PyMT accelerated tumor development [11]. Therefore, the cellular localization of the SD proteins was examined. Whole cell extracts of Src*/1 and Src*/3 LM2 transductants were analyzed by cell fractionation and immunoblotting (Fig. 2c). TAM-R cells (rat SD) were included as control because we previously presented that Src*/SD expression is almost entirely restricted to the cytoplasm in these cells [11]. TAM-R cells expressed the phosphorylated SD construct preferentially in the cytoplasm (26 % of cytoplasmic levels were found at membranes) (Fig. 2d). LM2 cells expressed slightly higher Src*/1 and Src*/3 levels in the cytoplasmic than in the membrane fraction. Importantly, a substantial portion of phosphorylated Src*/1 (60 %) and Src*/3 (53 %) was detected at membranes reflecting in part the localization pattern found in PyMT/Src*/SD cells indicated above.

The full-length and a truncated phosphorylated p130Cas SD promote metastasis formation

In the PyMT/Src*/SD mouse model, we observed accelerated primary tumor growth [11]. However, we could not detect metastases within the study period. To investigate whether the full-length and the truncated Src*/SD protein can promote metastasis formation when substantial amounts of these phosphorylated SD molecules are located at membranes (as shown for the LM2 transductants), an orthotopic mouse model was applied. LM2 cells have a high potential to form metastases when injected into the mammary fat pad of mice [13]. LM2 cells expressing the inducible Src*/1, Src*/3, or control constructs were injected into the mammary fat pad of NOD-Scid mice. Src*/1 and Src*/3 were utilized for these in vivo studies because out of all constructs tested in vitro, they were the only ones that induced changes in cell morphology. The rationale for this selection was that previously, we have shown that transductants that displayed these morphological changes also had altered signaling and cell behavior [10]. Primary tumor growth and metastasis formation were monitored by BLI and caliper measurements.

Representative expression and phosphorylation of the SD chimeras in the primary tumors were confirmed by immunoblotting (Fig. 3a). Of note, SD phosphorylation is highly variable but present in all tumors. In all groups, the injected cells established primary tumors and in part formed spleen metastases at later time points (Fig. 3b, BLI quantification in Fig. 3c, d). Compared with control cells, the primary tumor growth in mice injected with Src*/1 cells was significantly accelerated (Fig. 3c). Primary tumors reached a volume of 500 mm3 on average within 29 days (±5.5, P ≤ 0.001) and 37 days (±3.4, P ≤ 0.05) in Src*/1 and Src*/3 animals, respectively, compared with 41 days (±4.3) in control animals (Table 1). While only 36 % of control mice developed metastases by 67 days, metastases were evident in 54 % of Src*/1 and 82 % Src*/3 mice (Fig. 4a and Table 1). Src*/1 animals could not be observed for longer than 50 days because of tumor burden or other pathologies that required euthanasia. Moreover, metastases were formed earlier in Src*/1 (average of 28 ± 8.8 days, P ≤ 0.05) and Src*/3 (42 ± 16.4 days, not statistically significant) than in control animals (47 ± 16.9 days). At the end of the study period or when animals had to be euthanized due to tumor burden, ex vivo imaging of selected organs was performed (Fig. 4b and Table 2) to assay for metastasis formation. In Src*/1 and Src*/3 animals, the tumor cells formed multiple metastases at several organs (spleen, lungs, liver, kidneys) in a single animal, whereas in control animals, majorly smaller single metastases to the spleen or lungs were observed.
Fig. 3

The p130Cas SD drives primary breast cancer growth and metastasis. Mice were injected with LM2 transductants [*/1, */3, or control (empty vector)] into the mammary fat pad and analyzed by bioluminescence imaging (BLI) (b). a Expression (CasB) and phosphorylation (p-Tyr) of the transgene in primary tumor lysates (40 μg) were analyzed by IB (actin, control). Lysates of LM2-*/1 and */3 cells were used as positive control, and in the upper panels, only 20 μg was loaded onto the gel due to the high 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 quantified in c and d. c Time course of primary tumor growth as analyzed by BLI signals of the indicated areas in animals in b. The measurements ended by animal protocol after the first detection of metastases. d Quantification of BLI intensities of metastases at first detection of animals in b. e Comparison of the primary tumor growth in mice injected with the indicated LM2 cells. n number of mice at the beginning of the study. Primary tumor volumes were determined by caliper measurements as described. P values were calculated using Student’s t test (*P ≤ 0.05; **P ≤ 0.01)

Table 1

Tumor growth and metastasis detection after mammary fat pad injections of LM2 transductants in mice



Tumor volume (500 mm3)

Metastasis (within 67 days)

Average metastasis detection



41 days (±4.3)

36 %

47 days (±16.9)



29 days (±5.5, P ≤ 0.001)

54 %

28 days (±8.8, P ≤ 0.05)



37 days (±3.4, P ≤ 0.05)

82 %

42 days (±16.4, n.s.)

n.s. not significant

Fig. 4

The p130Cas SD drives metastasis. Mice were injected with the indicated LM2 cells [*/1, */3, or control (empty vector)] into the mammary fat pad and analyzed by bioluminescence imaging (BLI). a Percent metastases-free animals were calculated based on detections in 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 used as stated. Color bars with diverse signal intensities are displayed. DPBS + luc (luciferin), negative control

Table 2

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


Control (n = 5)

Src*/1 (n = 3)

Src*/3 (n = 4)





















These results show that the Src*/SD approach, including a shorter fragment of the SD, promotes breast cancer progression in this orthotopic in vivo model. Expression of the truncated SD fragment led to a considerably higher number of detected metastases (more than twice of the animals developed metastases compared with control mice). The full-length SD enhanced primary tumor growth and led to a significantly earlier metastasis onset and increased metastasis formation. This suggests that the full-length SD mediates a stronger tumor-promoting activity, including earlier dissemination. Nevertheless, these studies also indirectly indicate that the phosphorylated SD motifs 6–10 (aa 207–315 in wild-type p130Cas) of endogenous p130Cas may be important for tumor progression when a portion of the protein is found at cellular membranes.


Our studies demonstrate that the phosphorylated p130Cas SD is important for p130Cas function during tumor progression in vivo. In an orthotopic mouse model, Src*/SD proteins (full-length SD as well as a SD fragment containing motifs 6–10 of the p130Cas SD) promoted tumor progression. The full-length SD accelerated primary tumor growth and mediated a significantly earlier metastasis onset. Both the full-length and the truncated forms significantly increased metastasis formation.

We recently uncovered that partial localization of Src*/SD to cellular membranes by the polyoma middle T-antigen (PyMT) accelerates PyMT-induced tumor growth and promotes 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 findings that the Src*/SD fusion protein is partially located to membranes in LM2 cells and drives tumor progression in an orthotopic breast cancer model suggest that other mechanisms of membrane targeting exist for this system. Importantly, via the constitutively phosphorylated SD fragment, the chimera might integrate membrane-associated active signaling complexes. This is further supported by our previous studies describing that at membranes, the decoy activates extracellular signal-regulated kinase (ERK) survival signaling [11] whereas in the cytoplasm, it inhibits it [10]. The fact that only a portion of the Src*/SD molecules is located at membranes in the LM2 transductants indicates that the tumor-promoting activity is dominant in this model system.

In the PyMT/Src*/SD mouse model, no metastasis formation was detected during the study period [11] although p130Cas is known to drive metastases [1, 2]. Therefore, in an orthotopic model, we interrogated whether the decoy molecule, when partially located at membranes, also promotes metastases and which part of the phosphorylated SD is important for this process. LM2 transductants, with a substantial portion of the Src*/SD molecules at membranes, were injected into the mammary fat pad of NOD-Scid mice. Similar to the PyMT model, primary tumor growth was enhanced. In addition, 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 formation. 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 control animals developed majorly smaller single metastases only to the spleen or lungs. Furthermore, the full-length chimera greatly accelerated primary tumor growth and metastasis formation, whereas the effects of the truncated form were not as prominent. However, the shorter fragment induced metastases in more animals (82 % of the mice) than the full-length form (54 %). This may in part be explained by the fact that animals expressing the full-length SD developed pathologies within 50 days after tumor cell injection that required euthanasia whereas the animals expressing the truncated protein could be observed for up to 67 days. Taken together, these results suggest that additional sequences in the full-length SD not present in the truncated molecule might be responsible 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 1–5 or 11–15 with the Src*/SD approach did not significantly affect the cellular behavior of two breast cancer 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). Phosphorylation of the SD is essential for the dynamic assembly and disassembly of focal adhesions [19]. Each of these motifs was found to be phosphorylated in more than 190 proteomic 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 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, 20, 21]. In the past few years, p130Cas was also identified as a mechanosensor in response to extracellular matrix-integrin engagement [22, 23]. Upon mechanical 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’ surroundings. 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 treatment [24]. Increased p130Cas expression in comparison to primary tumors was also detected in tumor cells isolated from pleural effusions of mammary carcinoma patients [25]. In addition, a recent study described that 75 % of the patients with triple-negative breast cancer expressed high p130Cas levels [26]. Moreover, p130Cas contributes to the progression of several additional cancers such as gliomas, prostate cancer, and leukemias [27, 28]. Therefore, targeting p130Cas may open new avenues for the treatment of certain malignancies. Initial testing by in vivo downregulation of p130Cas by intranipple injection of short-interfering (si)RNA resulted in a reduction of tumor growth in BALB/c-HER2/neu mice [29]. Although speculative, data presented here suggest that therapeutics that specifically prevent p130Cas translocation to membranes and/or block YDxP motif 6–10 downstream signaling might be considered for future treatment options.

In summary, this study identified a region in the p130Cas substrate domain that when phosphorylated and found at membranes may be essential for promoting tumor progression.


  1. 1.

    The abbreviations used are the following: aa, amino acid; abs, antibodies; BLI, bioluminescence imaging; DOX, doxycycline; FAK, focal adhesion kinase; IB, immunoblotting; KM, kinase inactive; MFP, mammary fat pad; p130Cas, p130 Crk-associated substrate; Src*, attenuated kinase activity; SD, substrate domain; WCE, whole cell extract.



We gratefully acknowledge Joan Massague and Robert I. Nicholson for cell lines and Amy H. Bouton for the CasB antibody. We thank Matthew D. Layne for critical reading of the manuscript. We greatly appreciate the help of Manish Bais and Tom Balon with bioluminescence imaging and of Kim Bayer in establishing the animal procedures. All bioluminescence imaging was performed at the IVIS Imaging Core of Boston University School of Medicine. This work was supported by the National Cancer Institute grant CA106468, by the National Center for Advancing Translational Sciences through the BU-CTSI grant U54TR001012, both from the National Institute of Health, and the Susan G. Komen for the Cure Breast Cancer Foundation grant KG101208.

Compliance with ethical standards

Animal experiments were performed and the mice euthanized according to guidelines of and approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University.

Supplementary material

13277_2016_4902_Fig5_ESM.gif (213 kb)
Fig. S1

Cell morphology of LM2 cells expressing Src*/SD and control constructs that did not induce significant morphological changes. Cells (A and B) Cells (100,000 cells/well in 6-well plates) stably transduced with the indicated inducible constructs were treated for 24 h with DOX or left untreated. KM, SrcKM; *, Src*. c, control (empty vector). Left panels, expression (anti-HA) and phosphorylation (p-Tyr) of the fusion molecules in WCE (30 μg) was confirmed by IB. n.s., non-specific. Right panels, cell morphology was analyzed by microscopy. Scale, 100 μm. (GIF 212 kb)

13277_2016_4902_MOESM1_ESM.eps (22.2 mb)
High Resolution Image (EPS 22773 kb)
13277_2016_4902_MOESM2_ESM.doc (32 kb)
Table S1 (DOC 32 kb)


  1. 1.
    Kumbrink J, Kirsch KH. Targeting Cas family proteins as a novel treatment for breast cancer, breast cancer—current and alternative therapeutic modalities. Esra Gunduz and Mehmet Gunduz (Ed), InTech. 2011;ISBN: 978-953-307-776-5:37-62.Google Scholar
  2. 2.
    Tikhmyanova N, Little JL, Golemis EA. CAS proteins in normal and pathological cell growth control. Cell Mol Life Sci. 2010;67(7):1025–48.CrossRefPubMedGoogle Scholar
  3. 3.
    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74.CrossRefPubMedGoogle Scholar
  4. 4.
    Kumbrink J, Soni S, Laumbacher B, Loesch B, Kirsch KH. Identification of novel Crk-associated substrate (p130Cas) variants with functionally distinct focal adhesion kinase binding activities. J Biol Chem. 2015;290(19):12247–55.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Cabodi S, Pilar Camacho-Leal M, Di Stefano P, Defilippi P. Integrin signalling adaptors: not only figurants in the cancer story. Nat Rev Cancer. 2010;10(12):858–70.CrossRefPubMedGoogle Scholar
  6. 6.
    Nikonova AS, Gaponova AV, Kudinov AE, Golemis EA. CAS proteins in health and disease: an update. IUBMB Life. 2014;66(6):387–95.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Dorssers LC, van der Flier S, Brinkman A, van Agthoven T, Veldscholte J, Berns EM, et al. Tamoxifen resistance in breast cancer: elucidating mechanisms. Drugs. 2001;61(12):1721–33.CrossRefPubMedGoogle Scholar
  8. 8.
    Ta HQ, Thomas KS, Schrecengost RS, Bouton AH. A novel association between p130Cas and resistance to the chemotherapeutic drug adriamycin in human breast cancer cells. Cancer Res. 2008;68(21):8796–804.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kirsch KH, Kensinger M, Hanafusa H, August A. A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-crk signaling. BMC Cell Biol. 2002;3:18.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Soni S, Lin BT, August A, Nicholson RI, Kirsch KH. Expression of a phosphorylated p130(Cas) substrate domain attenuates the phosphatidylinositol 3-kinase/Akt survival pathway in tamoxifen resistant breast cancer cells. J Cell Biochem. 2009;107(2):364–75.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhao Y, Kumbrink J, Lin BT, Bouton AH, Yang S, Toselli PA, et al. Expression of a phosphorylated substrate domain of p130Cas promotes PyMT-induced c-Src-dependent murine breast cancer progression. Carcinogenesis. 2013;34(12):2880–90.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, et al. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am J Pathol. 2003;163(5):2113–26.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, et al. Genes that mediate breast cancer metastasis to lung. Nature. 2005;436(7050):518–24.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kumbrink J, Kirsch KH. Regulation of p130(Cas)/BCAR1 expression in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells by EGR1 and NAB2. Neoplasia. 2012;14(2):108–20.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kumbrink J, Kirsch KH. p130Cas acts as survival factor during PMA-induced apoptosis in HL-60 promyelocytic leukemia cells. Int J Biochem Cell Biol. 2013;45(3):531–5.CrossRefPubMedGoogle Scholar
  16. 16.
    Huang J, Hamasaki H, Nakamoto T, Honda H, Hirai H, Saito M, et al. Differential regulation of cell migration, actin stress fiber organization, and cell transformation by functional domains of Crk-associated substrate. J Biol Chem. 2002;277(30):27265–72.CrossRefPubMedGoogle Scholar
  17. 17.
    Shin NY, Dise RS, Schneider-Mergener J, Ritchie MD, Kilkenny DM, Hanks SK. Subsets of the major tyrosine phosphorylation sites in Crk-associated substrate (CAS) are sufficient to promote cell migration. J Biol Chem. 2004;279(37):38331–7.CrossRefPubMedGoogle Scholar
  18. 18.
    Hornbeck PV, Chabra I, Kornhauser JM, Skrzypek E, Zhang B. PhosphoSite: a bioinformatics resource dedicated to physiological protein phosphorylation. Proteomics. 2004;4(6):1551–61.CrossRefPubMedGoogle Scholar
  19. 19.
    Machiyama H, Hirata H, Loh XK, Kanchi MM, Fujita H, Tan SH, et al. Displacement of p130Cas from focal adhesions links actomyosin contraction to cell migration. J Cell Sci. 2014;127(Pt 16):3440–50.CrossRefPubMedGoogle Scholar
  20. 20.
    Cunningham-Edmondson AC, Hanks SK. p130Cas substrate domain signaling promotes migration, invasion, and survival of estrogen receptor-negative breast cancer cells. Breast Cancer (Dove Med Press). 2009;1:39–52.Google Scholar
  21. 21.
    Donato DM, Ryzhova LM, Meenderink LM, Kaverina I, Hanks SK. Dynamics and mechanism of p130Cas localization to focal adhesions. J Biol Chem. 2010;285(27):20769–79.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Camacho Leal MP, Sciortino M, Tornillo G, Colombo S, Defilippi P, Cabodi S. p130Cas/BCAR1 scaffold protein in tissue homeostasis and pathogenesis. Gene. 2015;562(1):1–7.CrossRefGoogle Scholar
  23. 23.
    Janostiak R, Pataki AC, Brabek J, Rosel D. Mechanosensors in integrin signaling: the emerging role of p130Cas. Eur J Cell Biol. 2014;93(10-12):445–54.CrossRefPubMedGoogle Scholar
  24. 24.
    van der Flier S, Brinkman A, Look MP, Kok EM, Meijer-van Gelder ME, Klijn JG, et al. Bcar1/p130Cas protein and primary breast cancer: prognosis and response to tamoxifen treatment. J Natl Cancer Inst. 2000;92(2):120–7.CrossRefGoogle Scholar
  25. 25.
    Konstantinovsky S, Smith Y, Zilber S, Tuft SH, Becker AM, Nesland JM, et al. Breast carcinoma cells in primary tumors and effusions have different gene array profiles. J Oncol. 2010;2010:969084.CrossRefPubMedGoogle Scholar
  26. 26.
    Tornillo G, Elia AR, Castellano I, Spadaro M, Bernabei P, Bisaro B, et al. p130Cas alters the differentiation potential of mammary luminal progenitors by deregulating c-Kit activity. Stem Cells. 2013;31(7):1422–33.CrossRefPubMedGoogle Scholar
  27. 27.
    Barrett A, Evans IM, Frolov A, Britton G, Pellet-Many C, Yamaji M, et al. A crucial role for DOK1 in PDGF-BB-stimulated glioma cell invasion through p130Cas and Rap1 signalling. J Cell Sci. 2014;127(Pt 12):2647–58.CrossRefPubMedGoogle Scholar
  28. 28.
    Zheng Y, Asara JM, Tyner AL. Protein-tyrosine kinase 6 promotes peripheral adhesion complex formation and cell migration by phosphorylating p130 CRK-associated substrate. J Biol Chem. 2012;287(1):148–58.CrossRefPubMedGoogle Scholar
  29. 29.
    Cabodi S, Tinnirello A, Bisaro B, Tornillo G, Pilar Camacho-Leal M, Forni G, et al. p130Cas is an essential transducer element in ErbB2 transformation. FASEB J. 2010;24(10):3796–808.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Joerg Kumbrink
    • 1
    • 2
    • 3
  • Ana de la Cueva
    • 1
  • Shefali Soni
    • 1
    • 4
  • Nadja Sailer
    • 1
    • 5
  • Kathrin H. Kirsch
    • 1
    Email author
  1. 1.Department of BiochemistryBoston University School of MedicineBostonUSA
  2. 2.Department of Medicine III, University Hospital GrosshadernUniversity of MunichMunichGermany
  3. 3.Institute of PathologyUniversity of MunichMunichGermany
  4. 4.The Leona M. and Harry B. Helmsley Charitable TrustNew YorkUSA
  5. 5.Department of BiochemistryUniversity of MunichMunichGermany

Personalised recommendations