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Crk adaptor protein promotes PD-L1 expression, EMT and immune evasion in a murine model of triple-negative breast cancer

ABSTRACT The tumor infiltration of immune cells in solid cancers can profoundly influence host antitumor responses. In recent years, immunotherapeutic regimens, that target immune checkpoints, demonstrated significant antitumor response by increasing intra-tumoral immune cell populations, including CD8+ effector T cells. However, administration of such immune checkpoint inhibitors is largely inefficacious in inducing immunogenicity and treating breast cancer. Currently, there is a great need to better understand cell autonomous mechanisms of immune evasion in breast cancer to identify upstream therapeutic targets that increase the efficacy of immunotherapy. Here we show that Crk, an SH2 and SH3 domain-containing adaptor protein implicated in focal adhesion signaling, cell migration, and invasion, and frequently up-regulated in human cancers, has an important role in regulating the tumor immune microenvironment. Using a murine 4T1 breast adenocarcinoma model of spontaneous metastasis in immune-competent BALB/C mice, we show that genetic ablation of Crk by CRISPR-Cas9 leads to enhanced anti-tumor immune cell populations, cytotoxic effector and immune surveillance cytokines in primary tumor. Pathologically, this leads to a significant reduction in tumor growth and lung metastasis. Mechanistically, Crk KO suppresses EMT and PD-L1 expression on tumor cells and acts additively with anti-PD1 therapy to suppress tumor growth and metastasis outcomes. Taken together, these data reveal a previously un-described function of Crk adaptor protein expression in tumor cells for cell autonomous regulation of tumor immune microenvironment.


Introduction
Crk adaptor proteins, comprised of two alternatively spliced isoforms that contain SH2 and SH3 modular domains, have critical functions in signal transduction downstream from cell adhesion molecules and growth factor receptors by assembling protein-protein interactions downstream of tyrosine kinases. [1][2][3][4][5][6][7] By binding to tyrosine phosphorylated focal adhesion proteins that include paxillin and p130cas via its SH2 domain, and Guanine Nucletoide Exchange Factors (GNEFs) for GTPases such as Rap1, Rac1 and Ras via its SH3 domain, Crk regulates evolutionarily conserved cell biological processes that impinge on cell adhesion and motility in metazoan cells. [8][9][10][11] Pathophysiologically, Crk is overexpressed in a wide array of human cancers, including but not limited to ovarian cancer, glioblastoma multiformae (GBM), lung cancer, breast cancer, and sarcoma; whereby the level of expression often correlates with tumor grade and inversely segregates with overall patient survival. 8 Although the effects of Crk overexpression are complex and multifactorial, a recurring hallmark of Crk overexpression in solid cancers is ability to induce cell transformation, promote epithelial-to-mesenchymal transition (EMT), and increase metastatic activity in mouse models. 12 More recent studies showed that Crk synergizes with TGF-b in an autocrine activation loop to promote EMT in lung cancer 13 which is consistent with previous studies showing that Crk can facilitate HGF-induced EMT-like phenotypes in canine MDCK cells and T47D human breast cancer cells. 14 Although the roles of Crk in driving transformation and cell migration is reasonably well understood at the biochemical and molecular level, the role of Crk in regulation of cell extrinsic functions with respect to tumor immune microenvironment has not been thoroughly investigated. Recent data showing that FAK, which like Crk regulates focal adhesion signaling and tyrosine phosphorylation, is an important regulator of an immunosuppressive tumor microenvironment suggests that key oncogenic networks that regulate cell adhesion and EMT may also impinge on the cell extrinsic tumor microenvironment. 15 In the present study, using an orthotopic syngeneic immune-competent murine model, we show that knockout of Crk in the poorly immunogenic breast adenocarcinoma 4T1 cells results in an increase of intra-tumoral immune cell infiltrations associated with favorable host anti-tumor outcomes. Crk depleted tumors exhibited increased intra-tumoral CD8C and CD3C T-cells, NK cells, and neutrophils, as well as a decrease in PDL-1 expression and reversal of Zeb-1 induced EMT program. Cytokine profiling of the primary tumors from Crk knockout revealed an elevation of tumor suppressing and immune-surveillance suggesting that infiltrating cells are functional to create an inflamed tumor microenvironment. Additionally, combinatorial in vivo results indicate that Crk KO acts additively with anti-PD1 in suppressing tumorigenesis and lung metastasis. In effect, the reprogramming of immune microenvironment by Crk KO results in reduction of both tumor growth and lung metastasis. These results establish a previously undescribed role of Crk in tumor immunology and immune evasion in an immunecompetent mouse model.

Materials and methods
Generation of Crk knockout 4T1 murine breast cancer cells 4T1 murine adenocarcinoma cell lines were grown in RPMI supplemented with 10% FBS. Two individual guide RNAs targeting exon 1 and exon 2 of cellular murine Crk were synthesized and cloned into All-in-one plasmid vector (U6-gRNA/ CMV-Cas9-RFP) (Sigma). Both vectors containing the guide RNAs were individually transfected into 4T1 cells and sorted for RFP expression after 48 hrs. Cas9-2A peptide-RFP fusion protein expression enabled monitoring of transfection efficiency and FACS based single cell sorting. Single cell clones were grown and screened by western blotting for Crk expression. A total of four individual clones were tested in in vitro experiments and representative data were shown.

In vivo experiments
For the in vivo studies, 6-week-old, female BALB/c from Jackson laboratory were used. All the procedures involving animal care and use were approved by IACUC of Rutgers University. 100,000 Wild-type or Crk KO cells were injected in the mammary fat pad of each mice. The tumors were palpated every 3 days, and body weight and tumor volumes were measured. At the end of the 6 weeks, the mice were sacrificed and tumors and lungs were harvested for immune phenotyping, IHC, western blotting or RNA extraction. Anti-PD1 or isotype antibodies were administered (i.p.) every 3 days at 200 mg/kg/day dosage in the combination experiments with total 4 administrations per group/study.

Cancer Inflammation & Immunity Crosstalk RT 2 Profiler PCR Array
Total RNA was isolated from WT and Crk KO primary tumors (3 tumors/group) using the RNeasy Plus TM total RNA Isolation kit. cDNA was prepared from respective samples using QIAGEN RT 2 First Strand Kit that were subsequently used to perform gene expression analysis using the SABiosciences Mouse Cancer Inflammation & Immunity Crosstalk RT 2 Profiler PCR Array (Cat. PAMM-181Z) and RT 2 SYBR Ò Green qPCR mastermix on Bio-Rad CFX-96 Ò per manufacturer's instructions. The data were analyzed using the RT 2 Profiler PCR array data analysis software (SABiosciences) and normalized to GAPDH, b-actin and b2-microglobulin expression levels.
Cell culture, flow cytometry and MTT assay 4T1 cell line was purchased from Perkin-Elmer and maintained at 70% confluence in RPMI-1640 medium supplemented with 10% FBS. Cell were harvested by Accutase and stained with anti-human PD-L1-PE (Biolegend). BD FACS Calibur was used for data acquisition, and the resulting data was analyzed by FlowJo Software (TreeStar). MTT assay to test the cell proliferation of WT or Crk KO cells was performed using MTT cell growth assay kit purchased from EMD-Millipore using manufacturer's protocol.

Statistical analysis
Differences between groups in all in vivo experiments were examined for statistical significance using a two-tailed Student's t-test. P < 0.05 was considered significant.

Crk adaptor protein promotes tumor growth and metastatic colonization
Previously, xenograft experiments transplanting Crk knockdown human cells into non-syngeneic immune-compromised nude mice demonstrated an intrinsic role for Crk in breast cancer growth and metastasis. 17 However, to investigate the role of Crk on tumor growth, metastasis, and immune evasion in a syngeneic model, Crk KO 4T1 cells were generated using CRISPR-Cas9 and validated Crk KO at genetic and protein level (Fig. 1a). Subsequently, when WT or Crk KO 4T1 cells were injected into the mammary fat pad of immunocompetent virgin BALB/c mice, transplantation of Crk knockout cells led to » 80% decrease in tumor growth (Fig. 1b) and tumor weights ( Fig. 1c-d). Using FACS, GFPC tumor cells in tumors were also found to be suppressed by more than 20% in Crk KO tumors group, indicating decreased in vivo tumor cell proliferation (Fig. 1e). To further test differences in cell proliferation of tumor cells from both groups, we performed Ki67 staining and in vitro MTT assay. We found that Crk KO decreases tumor cell proliferation both in vivo ( Fig. 1f) and in vitro (Fig. 1g). Additionally, metastasis of 4T1 KO cells to lung was reduced to about 70% as compared to the WT cells ( Fig. 1h-i). While all metastatic nodules in WT tumor-bearing mice were macrometastatic (>1 mm), most metastatic nodules found in lungs of Crk KO tumor bearing mice were micro-metastatic (<0.2mm). Tumor growth and metastasis in 4T1/BALB/c model was also associated with significant splenomegaly, consistent with previous reports. 18 Consistent with overall decreased lung metastatic burden, Crk KO tumor-bearing mice showed concomitant decrease in splenomegaly KO (Fig. 1j-k). Together, these results identify an important role for Crk, noted to be frequently overexpressed in human breast cancers, in controlling tumor growth and metastatic colonization in a triple negative breast model of adenocarcinoma.

Genetic ablation of Crk increases tumor infiltration in murine orthotopic breast cancer model
The above-mentioned decrease in splenomegaly in the spontaneous syngeneic tumor model suggested that Crk KO may regulate a tumor-specific host immune response. To investigate this experimentally, we analyzed the molecular landscape of tumor infiltrating immune cells by Nanostring analysis using the whole RNA extracted from the primary tumors. Interestingly, in the Crk KO tumors, we observed that CD8C, and total T cells, DC, NK cells, neutrophils and macrophages were elevated, while CD4C activated T cells and T helper cells were concomitantly down-regulated ( Fig. 2a-b). Additionally, Crk KO tumors showed increased gene expression status of genes frequently associated with dendritic cell functions such as CD86, CD40, CD40lg, and CCL5 (Suppl. Fig. 2). Cumulatively, in Crk KO bearing tumor associated tumor suppressive Th1 cells populations were increased while tumor-promoting cell populations were decreased by Crk KO in tumor cells. Using IHC analysis of formalin-fixed, paraffin-embedded (FFPE) sections of primary tumors as an unbiased alternative strategy, we validated major findings from Nanostring based RNA analysis, whereby a decrease in tumor suppressive populations of T cells (CD3C, CD8C T cells) (Fig. 2c-d) and a marked increase in GranzBC immune cell populations were noted in Crk KO tumors. F4/80C macrophages and Ly6GC neutrophils populations were also upregulated in tumors from Crk knockout 4T1 cells. While we did not detect any changes in CD31C endothelial cells (tumor angiogenesis) in Crk KO tumors, there was a significant reduction in vascularity of the tumors by visual inspections (not shown). Finally, by flow cytometric analysis of the primary tumors, we found that CD45C myeloid cell and CD11bC macrophage populations were potentiated in KO group (Fig. 2e). Together, these results suggest that Crk expression in 4T1-derived primary murine breast adenocarcinomas contributes to a poorly immunogenic tumor microenvironment that, in part, leads to reduced host-anti-tumor immune responsivity and suppressed tumor cell killing of Crk-expressing tumors.
Crk knockout alters intra-tumoral cytokine and chemokine profiles in favor of anti-tumor immunity Tumor associated inflammation, associated with specific regulation of cytokine and chemokine profiles in the tumor microenvironment (TME), are predictive features of anti-tumor immune responses. 19,20 To better access functional outcomes associated with the immune infiltrated cell types (Fig. 2), we next investigated the cytokine profiles using total RNA from whole primary WT and Crk KO tumors (analyzed by Nanostring and shown as dot plots) and by RT 2 PCR profiling using cDNA prepared from replicate total RNA (shown as bar graphs). Consistent with overall immune subsets noted above, we observed that immunoregulatory and cytotoxic cytokines, granzyme A and B, TNF-a, IFN-g, Fas-L, and TRAIL, that are implicated to enhance tumor antigen presentation, cytotoxicity, and induce tumor cell apoptosis were markedly upregulated in Crk KO tumor microenvironment as compared with native WT tumors (Fig. 3a). Moreover, tumors from Crk KO 4T1 cells showed increased expression of CCL5, CXCL9 and CXCL10, chemokines associated with tumor infiltrating lymphocytes (Fig. 3b), as well as subsets of interleukins (IL1b, IL6, IL12b, and IL15) that enhance in T-cell activation, B-cell function, T H 1 immunity and tumor cell cytotoxicity (Fig. 3c). In contrast, tumor-promoting cytokines, IL17a, IL23 and TGF-b were downregulated in Crk KO tumors (Fig. 3d). Cumulatively, these results suggest that Crk KO, in a poorly immunogenic 4T1 model, reprograms a non-inflamed immunosuppressive cytokine microenvironment into an inflamed immunogenic cytokine profile characterized by infiltration of effector T-cells (summarized in Fig. 3e).

Crk promotes Zeb-1 mediated EMT and PD-L1 expression
To better understand the biochemical events of Crk in the tumor microenvironment, we examined intrinsic biomarkers for Crk associated with epithelial-mesenchymal transition (EMT). Indeed, in recent years, a relationship between EMT and tumor immune evasion has been identified, including models of bi-directional cross-talk between the transcription factor Zeb-1 and EMT, and the regulation of PD-L1 and tumor immune evasion. 21,22 While previous studies have implicated Crk in the regulation of HGF/MET-mediated EMT, 14   reports of other cancer cell types, Crk KO in the 4T1 cells under investigation in this study showed a contrasting morphological change associated with a loss of mesenchymal-like spindle shaped cells with cell scattering morphology (Fig. 4a). Notably, in the Crk KO cells, cell-to cell adhesion increased whereby cells grew in sheet-like structures resembling epithelial morphology, while reconstitution of WT Crk II in Crk KO cells reverted this phenotype (Fig. 4a, right panels). Interestingly, Western blot analysis of whole-cell detergent lysates from WT/ KO cells showed that expression of N-cadherin was suppressed while E-cadherin was enhanced following Crk KO (Fig. 4b), while reconstitution of CrkII reversed the N-and E-cadherin switching. Similarly, expression of Zeb-1 (a potent EMT-inducing transcription factor) was also suppressed by Crk KO and rescued by Crk reconstitution in Crk KO cells (Fig. 4b). As EMT has been shown to modulate PD-L1 expression on cancer cells, 23,24 we investigated the effect of Crk ablation (that suppresses EMT) on PD-L1 expression. We found that PD-L1 (total and surface) expression were ablated by Crk KO and rescued by Crk II reconstitution (Fig. 4c-e). Consistently, in Crk KO primary tumors expression of PD-L1 was downregulated ( Fig. 4f), while population of PD-1C immune cells was upregulated (Fig. 4g). as observed by IHC (n D 4), Finally, Nanostring and RT2 PCR profiler data showed that PD1 expression was upregulated in Crk KO tumors (n D 4) (Fig. 4h). These results support a role for Crk in direct regulation of EMT by controlling Zeb-1 expression and in EMT induced PD-L1 expression on 4T1 breast cancer cells.

Crk knockout acts additively with anti-PD1 in suppression of primary tumor growth
To more formally implicate a role for EMT-inducible PD-L1 up-regulation, we experimentally tested WT and Crk KO cohorts (n D 8) in synergy with anti-PD1 checkpoint therapeutics. As shown in Fig. 5, mice were administered either 200mg/ mice (i.p.) isotype control or anti-PD1 twice a week for 4 times after palpable tumors were formed (7 days post injection). Under these conditions, both anti-PD1 and Crk KO independently and significantly decreased tumor growth and weight, while Crk KO cohort administered together with anti-PD1 showed an additive effect on decrease in tumor growth or tumor weight, due to the robust effects of Crk ablation on this phenotype (Fig. 5a-c). Likewise, both anti-PD1 monotherapy and Crk KO also decreased lung metastasis, spleen weight and size (Fig. 5-d, f-h). No significant change in body weight was observed in mice cohorts treated with isotype or anti-PD1 (Fig. 5e). Together, the results suggest that Crk KO increases tumor-suppressive immune cell populations, at least in part, by regulation of the PD-L1 and PD-1 axis.

Discussion
Over the past decade, immune-oncology and immunotherapy have emerged into frontline cancer therapeutics with clear demonstrated clinical benefit in some cancer patients.
Unlike classical radiotherapy, chemotherapy, and oncogenedirected inhibitors that target the growth and survival of tumor cells, immune-therapies typically target negative regulatory immune checkpoint molecules that break tolerance, and when successful, can promote immune mediated tumor destruction by T cell effector functions or NK cells and enhance disease-free survival in multiple cancers. 25,26 However, by reasons that are not completely understood, immune checkpoint inhibitors (ICI) in some cases can also have poor experimental and clinical efficacy, due in part to low mutational burden of neo-antigens in certain cancer types, as well as low intrinsic immunogenicity in some cancers (so-called cold tumors) via the expression of checkpoint inhibitory ligands that include PD-L1. Concerning the notion that certain intrinsic oncogenic networks might contribute to suppression of tumor infiltrating immune cell population outcomes in the tumor microenvironment, several oncogenic pathways that can crosstalk and impinge on expression of checkpoint inhibitors, such as PD-L1, have been recently uncovered. For example, Myc, AKT-mTOR Ras-MAPK, HIF-1a, c-MET, and EGFR signaling have been shown to regulate PD-L1 gene expression, 27,28 as well as surface expression on tumor cells. 21,22,[29][30][31][32][33] In breast cancer, including models of basal type and triple negative, PD-L1 expression has a poor prognostic value with respect to therapeutic outcomes. 34 Therapeutic interventions that consider oncogenic pathways that regulate PD-L1 are predicted to synergize with ICI-based therapeutics. 35 In the present study, by developing a syngeneic mouse model to study Crk tumorigenicity in a poorly immunogenic 4T1 triple-negative breast cancer model, we identified an unrealized role for Crk in regulating an immuno-suppressive TME that inhibits host anti-tumor immunity. Previously, Crk has been intensely studied in the context of cancer cell transformation and shown to be an important regulator of proliferation, cell migration, and invasive phenotypes, and in human cancers expression correlates with metastasis and poor survival outcomes. 8 Mechanistically, Crk binds to tyrosine phosphorylated focal adhesion proteins, i.e., paxillin and p130cas, and complexes of Crk and focal adhesion proteins (FAK and p130cas) are elevated in oncogenically transformed cells by Src and Ras. 1 The implication that Crk may impinge on the activation of focal adhesion kinase (FAK) and downstream signaling is potentially noteworthy, given the recent evidence that FAK (activation) also appears to promote an immunosuppressive TME in pancreatic ductal adenocarcinoma (PDAC). 15 Consequently, FAK inhibitors are shown to enhance the immune-surveillance and increase the efficacy of anti-PD1 immunotherapy in PDAC. The fact that both Crk and Crk-interacting proteins are also part of FAK signaling complexes and participate in cell migration and invasion suggests that Crk may function in parallel pathways to FAK in promoting immunosuppressive signals. However, in addition to FAK, recent reports indicate a role of oncogenes, such as c-Myc, Ras-MAPK, and c-MET in regulation of tumor immunity in different human cancer models, 32,33,36 suggesting that common "oncogenic hubs" may control extrinsic immunogenic components of the TME. By extension, these insights might also imply that Crk by acting as an adaptor protein, via its assemblages of multi-protein complexes, could act coordinately to control both cell intrinsic malignant features of tumor cells, such as EMT and invasion, as well as cell extrinsic tumor microenvironment features that impinge on host antitumor responses.
Increasing evidence also suggests that aberrant activation of EMT, a hallmark for the development and progression of tumors, can also manifest evasion in anti-tumor immunity. 21,23,24,37 While the link between EMT and the immunosuppressed TME is likely to be multi-factorial that may involve down-regulation of MHC class I and other recognition modules utilized by TILs and NK cells, recent evidence also suggests that epithelial versus mesenchymal cancer subtypes have distinct interactions with the TME. Moreover, in global mRNA expression data from TCGA and PROSPECT datasets, mesenchymal type tumors have elevated PD-L1 and PD-L2 that may associate with poorly immunogenic tumors. With respect to Crk and Crk knockout 4T1 cells under investigation in this study, Crk KO cells clearly exhibit more epithelial marker E-cadherin and decreased expression of mesenchymal markers N-cadherin and Zeb1. The fact that Crk KO concomitantly suppressed EMT markers and PD-L1 expression in the poorly immunogenic 4T1 model, is consistent with the idea that Crkmediated alterations in EMT status may be linked to its capacity to regulate the immunosuppressive TME (summarized in Fig. 3e). This is also substantiated by our finding that that tumors harboring Crk knockout 4T1 cells show increased CD3C and CD8C T cells, cytokines associated with infiltrating T cells and their activation status such as IFN-g and interleukins, as well as cytotoxic genes that include granzyme B and TRAIL. Finally, the observations that Crk KO tumors may act additively with anti-PD1 immunotherapy with respect to tumor growth and metastasis outcomes opens the possibility that the development of Crk inhibitors and siRNA strategies may increase the efficacy of anti-PD-1 immunotherapy in breast cancer. Agents, such as EphB4 receptor inhibitors 38 and Cyclophilin A inhibitors, 39 that target the Crk signaling axis, might be considered as promising therapeutic target, possibly akin to the development of FAK inhibitors mentioned above. 15 While the aforementioned discussion has exclusively focused on Crk in cancer cells, it is important to note that Crk, and its related gene product CrkL, are also expressed in infiltrating T cells (including TILs) and myeloid-derived NK cells, where they are likely to be affirmatively involved in host tumor immunity. While in T cells, Crk plays a role in T cell extravasation and motility, it is also enhances activation of NK cells. 40,41 Such studies suggest Crk may have a dual role, first on tumor cells as signals for EMT and immune escape, and second on immune cells that control anti-tumor immune functions. The fact that we tested Crk knockout activity in an orthotopic tumor model clearly supports a tumor-specific role of Crk in controlling the immunosuppressive TME; however further studies, using conditional KO's of Crk in T-cells and NK cells will be of equal importance to assess the role of lymphocytic and monocyte-specific functions of Crk in syngeneic mouse models. In summary, while previous studies have shown that Crk can drive cell intrinsic cancer phenotypic changes, such as increased invasion and EMT that are associated with malignant transformation and metastasis, our present results indicate that Crk has an extrinsic immunosuppressive role in poorly immunogenic tumors; thus, implicating Crk as the first reported adaptor protein that promotes immune evasion in cancer.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed by the authors.