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Background Despite overall improvement in breast cancer patient outcomes from earlier diagnosis and personalised treatment approaches, some patients continue to experience recurrence and incurable metastases. It is therefore imperative to understand the molecular changes that allow transition from a non-aggressive state to a more aggressive phenotype. This transition is governed by a number of factors. Methods As crosstalk with extracellular matrix (ECM) is critical for tumour cell growth and survival, we applied high throughput shRNA screening on a validated ‘3D on-top cellular assay’ to identify novel growth suppressive mechanisms. Results A number of novel candidate genes were identified. We focused on COMMD3, a previously poorly characterised gene that suppressed invasive growth of ER + breast cancer cells in the cellular assay. Analysis of published expression data suggested that COMMD3 is normally expressed in the mammary ducts and lobules, that expression is lost in some tumours and that loss is associated with lower survival probability. We performed immunohistochemical analysis of an independent tumour cohort to investigate relationships between COMMD3 protein expression, phenotypic markers and disease-specific survival. This revealed an association between COMMD3 loss and shorter survival in hormone-dependent breast cancers and in particularly luminal-A-like tumours (ER+/Ki67-low; 10-year survival probability 0.83 vs. 0.73 for COMMD3-positive and -negative cases, respectively). Expression of COMMD3 in luminal-A-like tumours was directly associated with markers of luminal differentiation: c-KIT, ELF5, androgen receptor and tubule formation (the extent of normal glandular architecture; p COMMD3 induced invasive spheroid growth in ER + breast cancer cell lines in vitro, while Commd3 depletion in the relatively indolent 4T07 TNBC mouse cell line promoted tumour expansion in syngeneic Balb/c hosts. Notably, RNA sequencing revealed a role for COMMD3 in copper signalling, via regulation of the Na+/K+-ATPase subunit, ATP1B1. Treatment of COMMD3-depleted cells with the copper chelator, tetrathiomolybdate, significantly reduced invasive spheroid growth via induction of apoptosis. Conclusion Overall, we found that COMMD3 loss promoted aggressive behaviour in breast cancer cells.

Background High-grade serous ovarian carcinomas (HGSCs) are a heterogeneous subtype of epithelial ovarian cancers and include serous cancers arising in the fallopian tube and peritoneum. These cancers are now subdivided into homologous recombination repair (HR)-deficient and proficient subgroups as this classification impacts on management and prognosis. PARP inhibitors (PARPi) have shown significant clinical efficacy, particularly as maintenance therapy following response to platinum-based chemotherapy in BRCA-mutant or homologous recombination (HR)-deficient HGSCs in both the 1st and 2nd line settings. However, PARPi have limited clinical benefit in HR-proficient HGSCs which make up almost 50% of HGSC and improving outcomes in these patients is now a high priority due to the poor prognosis with ineffectiveness of the current standard of care. There are a number of potential lines of investigation including efforts in sensitizing HR-proficient tumors to PARPi. Herein, we aimed to develop a novel combination therapy by targeting SSRP1 using a small molecule inhibitor CBL0137 with PARPi in HR-proficient HGSCs. Experimental design We tested anti-cancer activity of CBL0137 monotherapy using a panel of HGSC cell lines and patient-derived tumor cells in vitro. RNA sequencing was used to map global transcriptomic changes in CBL0137-treated patient-derived HR-proficient HGSC cells. We tested efficacy of CBL0137 in combination with PARPi using HGSC cell lines and patient-derived tumor cells in vitro and in vivo. Results We show that SSRP1 inhibition using a small molecule, CBL0137, that traps SSRP1 onto chromatin, exerts a significant anti-growth activity in vitro against HGSC cell lines and patient-derived tumor cells, and also reduces tumor burden in vivo. CBL0137 induced DNA repair deficiency via inhibition of the HR repair pathway and sensitized SSRP1-high HR-proficient HGSC cell lines and patient-derived tumor cells/xenografts to the PARPi, Olaparib in vitro and in vivo. CBL0137 also enhanced the efficacy of DNA damaging platinum-based chemotherapy in HGSC patient-derived xenografts. Conclusion Our findings strongly suggest that combination of CBL0137 and PARP inhibition represents a novel therapeutic strategy for HR-proficient HGSCs that express high levels of SSRP1 and should be investigated in the clinic.

Background Despite the increasing progress in targeted and immune based-directed therapies for other solid organ malignancies, currently there is no targeted therapy available for TNBCs. A number of mechanisms have been reported both in pre-clinical and clinical settings that involve inherent, acquired and adaptive resistance to small molecule inhibitors. Here, we demonstrated a novel resistance mechanism in TNBC cells mediated by PDGFRβ in response to JAK2 inhibition. Methods Multiple in vitro (subG1, western blotting, immunofluorescence, RT-PCR, Immunoprecipitation), in vivo and publically available datasets were used. Results We showed that TNBC cells exposed to MEK1/2-JAK2 inhibitors exhibit resistant colonies in anchorage-independent growth assays. Moreover, cells treated with various small molecule inhibitors including JAK2 promote PDGFRβ upregulation. Using publically available databases, we showed that patients expressing high PDGFRβ or its ligand PDGFB exhibit poor relapse-free survival upon chemotherapeutic treatment. Mechanistically we found that JAK2 expression controls steady state levels of PDGFRβ. Thus, co-blockade of PDGFRβ with JAK2 and MEK1/2 inhibitors completely eradicated resistant colonies in vitro. We found that triple-combined treatment had a significant impact on CD44+/CD24− stem-cell-like cells. Likewise, we found a significant tumor growth inhibition in vivo through intratumoral CD8+ T cells infiltration in a manner that is reversed by anti-CD8 antibody treatment. Conclusion These findings reveal a novel regulatory role of JAK2-mediated PDGFRβ proteolysis and provide an example of a PDGFRβ-mediated resistance mechanism upon specific target inhibition in TNBC. Electronic supplementary material The online version of this article (10.1186/s13046-019-1075-5) contains supplementary material, which is available to authorized users.

Simple Summary BRF2, a subunit of the RNA polymerase III transcription complex, is upregulated in a wide variety of cancers and is a potential therapeutic target; however, no effective drugs are available to target BRF2. The upregulation of BRF2 in cancer cells confers survival via the prevention of oxidative stress-induced apoptosis. In this manuscript, we report the identification of potential BRF2 inhibitors through in silico drug repurposing screening. We further characterized bexarotene as a hit compound for the development of selective BRF2 inhibitors and provide experimental validation to support the repurposing of this FDA-approved drug as an agent to reduce the cellular levels of ROS and consequent BRF2 expression in cancers with elevated levels of oxidative stress. Abstract The overexpression of BRF2, a selective subunit of RNA polymerase III, has been shown to be crucial in the development of several types of cancers, including breast cancer and lung squamous cell carcinoma. Predominantly, BRF2 acts as a central redox-sensing transcription factor (TF) and is involved in rescuing oxidative stress (OS)-induced apoptosis. Here, we showed a novel link between BRF2 and the DNA damage response. Due to the lack of BRF2-specific inhibitors, through virtual screening and molecular dynamics simulation, we identified potential drug candidates that interfere with BRF2-TATA-binding Protein (TBP)-DNA complex interactions based on binding energy, intermolecular, and torsional energy parameters. We experimentally tested bexarotene as a potential BRF2 inhibitor. We found that bexarotene (Bex) treatment resulted in a dramatic decline in oxidative stress and Tert-butylhydroquinone (tBHQ)-induced levels of BRF2 and consequently led to a decrease in the cellular proliferation of cancer cells which may in part be due to the drug pretreatment-induced reduction of ROS generated by the oxidizing agent. Our data thus provide the first experimental evidence that BRF2 is a novel player in the DNA damage response pathway and that bexarotene can be used as a potential inhibitor to treat cancers with the specific elevation of oxidative stress.

Activating KRAS mutations drive colorectal cancer tumorigenesis and influence response to anti-EGFR-targeted therapy. Despite recent advances in understanding Ras signaling biology and the revolution in therapies for melanoma using BRAF inhibitors, no targeted agents have been effective in KRAS-mutant cancers, mainly due to activation of compensatory pathways. Here, by leveraging the largest synthetic lethal genetic interactome in yeast, we identify that KRAS-mutated colorectal cancer cells have augmented homologous recombination repair (HRR) signaling. We found that KRAS mutation resulted in slowing and stalling of the replication fork and accumulation of DNA damage. Moreover, we found that KRAS-mutant HCT116 cells have an increase in MYC-mediated RAD51 expression with a corresponding increase in RAD51 recruitment to irradiation-induced DNA double-strand breaks (DSBs) compared to genetically complemented isogenic cells. MYC depletion using RNA interference significantly reduced IR-induced RAD51 foci formation and HRR. On the contrary, overexpression of either HA-tagged wild-type (WT) MYC or phospho-mutant S62A increased RAD51 protein levels and hence IR-induced RAD51 foci. Likewise, depletion of RAD51 selectively induced apoptosis in HCT116-mutant cells by increasing DSBs. Pharmacological inhibition targeting HRR signaling combined with PARP inhibition selectivity killed KRAS-mutant cells. Interestingly, these differences were not seen in a second isogenic pair of KRAS WT and mutant cells (DLD-1), likely due to their nondependency on the KRAS mutation for survival. Our data thus highlight a possible mechanism by which KRAS-mutant-dependent cells drive HRR in vitro by upregulating MYC-RAD51 expression. These data may offer a promising therapeutic vulnerability in colorectal cancer cells harboring otherwise nondruggable KRAS mutations, which warrants further investigation in vivo.

Triple-negative breast cancer (TNBCs) is a very aggressive and lethal form of breast cancer with no effective targeted therapy. Neoadjuvant chemotherapies and radiotherapy remains a mainstay of treatment with only 25-30% of TNBC patients responding. Thus, there is an unmet clinical need to develop novel therapeutic strategies for TNBCs. TNBC cells have increased intracellular oxidative stress and suppressed glutathione, a major antioxidant system, but still, are protected against higher oxidative stress. We screened a panel of antioxidant genes using the TCGA and METABRIC databases and found that expression of the thioredoxin pathway genes is significantly upregulated in TNBC patients compared to non-TNBC patients and is correlated with adverse survival outcomes. Treatment with auranofin (AF), an FDA-approved thioredoxin reductase inhibitor caused specific cell death and impaired the growth of TNBC cells grown as spheroids. Furthermore, AF treatment exerted a significant in vivo antitumor activity in multiple TNBC models including the syngeneic 4T1.2 model, MDA-MB-231 xenograft and patient-derived tumor xenograft by inhibiting thioredoxin redox activity. We, for the first time, showed that AF increased CD8+Ve T-cell tumor infiltration in vivo and upregulated immune checkpoint PD-L1 expression in an ERK1/2-MYC-dependent manner. Moreover, combination of AF with anti-PD-L1 antibody synergistically impaired the growth of 4T1.2 primary tumors. Our data provide a novel therapeutic strategy using AF in combination with anti-PD-L1 antibody that warrants further clinical investigation for TNBC patients.

Serrated pathway colorectal cancers (CRCs) are characterised by a BRAF mutation and half display microsatellite instability (MSI). The Wnt pathway is commonly upregulated in conventional CRC through APC mutation. By contrast, serrated cancers do not mutate APC. We investigated mutation of the ubiquitin ligases RNF43 and ZNRF3 as alternate mechanism of altering the Wnt signal in serrated colorectal neoplasia. RNF43 was mutated in 47/54(87%) BRAF mutant/MSI and 8/33(24%) BRAF mutant/microsatellite stable cancers compared to only 3/79(4%) BRAF wildtype cancers (p

Introduction CEP55, discovered first by our laboratory, is a key regulator of cytokinesis, with perturbation of its levels causing cytokinesis failure and multi-nucleation. Since its discovery, CEP55 over-expression has been linked to increased aggressiveness of multiple tumour types. This overexpression has been associated with increased AKT activation, caused by the interaction of CEP55 with p110 and promoting stability of the catalytic subunit of PI3KCA. Further, it has been shown that wild-type p53 suppresses CEP55 expression by negatively regulating PLK1 creating a p53-PLK1-CEP55 axis to directly modulate CEP55 stability and cytokinesis completion. In addition, CEP55 has been shown to be part of the CIN70 gene signature that predicts chromosomal instability and poor prognosis of several tumours. However, despite these studies, it is currently unknown whether elevated Cep55 levels alone are sufficient to promote de novo tumorigenesis. Material and methods We have generated the first novel transgenic mouse model with overexpression of Cep55 from the ubiquitously expressed Rosa26 locus. Results and discussions We observed that Cep55 over-expression in vivo leads to a wide-spectrum of spontaneous tumour formation with a long latency period. In addition, we have generated a bi-transgenic mouse model (Cep55Tg/Tg Trp53+/-) to determine the contribution of p53 loss to Cep55-induced tumorigenesis. Interestingly, we observed a similar latency between Cep55Tg/Tg and Cep55Tg/Tg; Trp53 ± mice. However, we detected a higher incidence of tumours in Cep55Tg/Tg; Trp53 ± compared to Cep55wt/Tg; Trp53 ± and Cep55wt/wt; Trp53 ± mice with the tumours being significantly larger and more penetrant, proliferative and metastatic in nature. In addition, we demonstrated that Cep55 amplification leads to hyper proliferation and favours genomic instability. We also observed an association of Cep55 with polyploidy in a dose-dependent manner and demonstrated that Cep55 overexpression protects a polyploid subpopulation upon perturbed mitosis. Additionally, we provided preliminary mechanistic evidence of Cep55 overexpression leading to upregulation of a Foxm1 and Plk1 axis which in turn might initiate tumorigenesis. Conclusion Collectively, our data proves the oncogenic potential of Cep55 overexpression in vivo and as an important stimulus in the initiation and progression of multiple cancer types.

Background Triple-negative breast cancers (TNBCs) are very aggressive and lethal forms of breast cancer with no effective targeted therapy. TNBCs spontaneously metastasize to distant organs including lungs, bone, and brain. Neo-adjuvant chemotherapies and radiotherapy remains a mainstay of treatment with only 25-30% of TNBC patients responding. Thus, there is an unmet clinical need to develop novel therapeutic strategies for TNBCs. Methods We tested the anti-cancer activity of auranofin monotherapy on primary tumor growth and metastasis in vitro and in vivo. Further, using in vitro analysis we conceptualized the PD-L1 dependent resistance against auranofin which we validated in vivo using syngeneic TNBC model. Results Expression of the thioredoxin pathway genes is significantly upregulated in TNBC patients compared to non-TNBC patients and correlated with adverse survival outcomes. Treatment with auranofin, an FDA-approved thioredoxin reductase inhibitor caused specific cell death and impaired the growth of TNBC grown as spheroids in 3D culture. Further, auranofin treatment exerted a significant in vivo anti-tumor activity in multiple TNBC models including syngeneic 4T1.2 model, human MDA-MB-231 xenograft, and PDX model by inhibiting thioredoxin redox activity. Auranofin also significantly inhibited the invasion potential of TNBC cells in vitro and significantly inhibited lung metastasis in 4T1.2 syngeneic model in vivo by reducing the expression of various EMT markers. We for the first time showed that auranofin increased CD8+Ve T-cells tumor infiltration in vivo and upregulated immune checkpoint PD-L1 expression in ERK1/2-MYC-dependent manner. Moreover, combination of auranofin with anti-PD-L1 monoclonal antibody synergistically impaired the growth of 4T1.2 primary tumor. Conclusions Our data provides a novel therapeutic strategy using auranofin in combination with anti-PD-L1 antibody for TNBCs and warrants further clinical investigation for TNBC patients. Since the success rate of anti-PD-L1 therapy is very low in TNBC patients, our data provides a novel strategy to use auranofin with anti-PD-L1 therapy to that may enhance the efficacy of immune checkpoint therapy in TNBC patients. Legal entity responsible for the study The authors. Funding Cure Cancer Australia & Can Too Foundation Project grant [ID 1147230] to Prahlad Raninga National Health & Medical Research Council (NH&MRC) Program Grant [ID 1017028] to Kum Kum Khanna Perpetual IMPACT Philanthropy project grant [IPAP201602001] to Kum Kum Khanna and Murugan Kalimutho. Disclosure All authors have declared no conflicts of interest.

Despite the increasing progress in breast cancer research, currently there is no targeted therapy available for TNBC. Here, we demonstrated a novel resistance mechanism mediated by PDGFRβ in TNBC cells. We showed that TNBC cells exposed to JAK2 or MEK1/2 inhibitors promote PDGFRβ upregulation. Using publically available databases, we found that basal-like breast cancer patients expressing high PDGFRβ exhibit poor relapse-free survival. We found that JAK2 overexpression controls PDGFRβ protein degradation. Blockade of PDGFRβ with JAK2 and MEK1/2 inhibitors significantly impacted the CD44+/CD24- stem-cell-like cells and the growth of basal-like breast cancer xenografts in vivo through intratumoral CD8+ T cells in a manner that is reversed by anti-CD8 antibody treatment. Together, these findings reveal a novel regulatory role of JAK2-mediated PDGFRβ proteolysis and provide an example of a PDGFRβ-mediated resistance mechanism upon JAK2 and MEK1/2 inhibition in TNBC. Citation Format: Murugan Kalimutho, Kum Kum Khanna. PDGFRβ blockade bypasses resistance to combined MEK1/2-JAK2 inhibition in triple-negative breast cancer via intratumoral CD8+ T-cells infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-256.

Purpose: Despite the use of 5-fluorouracil (5-FU)–based adjuvant treatments, a large proportion of patients with high-risk stage II/III colorectal cancer will relapse. Thus, novel therapeutic strategies are needed for early-stage colorectal cancer. Residual micrometastatic disease from the primary tumor is a major cause of patient relapse. Experimental Design: To model colorectal cancer tumor cell invasion/metastasis, we have generated invasive (KRASMT/KRASWT/+chr3/p53-null) colorectal cancer cell subpopulations. Receptor tyrosine kinase (RTK) screens were used to identify novel proteins that underpin the migratory/invasive phenotype. Migration/invasion was assessed using the XCELLigence system. Tumors from patients with early-stage colorectal cancer (N = 336) were examined for AXL expression. Results: Invasive colorectal cancer cell subpopulations showed a transition from an epithelial-to-mesenchymal like phenotype with significant increases in migration, invasion, colony-forming ability, and an attenuation of EGF receptor (EGFR)/HER2 autocrine signaling. RTK arrays showed significant increases in AXL levels in all invasive sublines. Importantly, 5-FU treatment resulted in significantly increased migration and invasion, and targeting AXL using pharmacologic inhibition or RNA interference (RNAi) approaches suppressed basal and 5-FU–induced migration and invasion. Significantly, high AXL mRNA and protein expression were found to be associated with poor overall survival in early-stage colorectal cancer tissues. Conclusions: We have identified AXL as a poor prognostic marker and important mediator of cell migration/invasiveness in colorectal cancer. These findings provide support for the further investigation of AXL as a novel prognostic biomarker and therapeutic target in colorectal cancer, in particular in the adjuvant disease in which EGFR/VEGF–targeted therapies have failed. Clin Cancer Res; 20(1); 164–75. ©2013 AACR.

Adenosine plays an important role in inflammation and tumor development, progression, and responses to therapy. We show that an adenosine 2B receptor inhibitor (A2BRi) decreases both experimental and spontaneous metastasis and combines with chemotherapy or immune checkpoint inhibitors in mouse models of melanoma and triple-negative breast cancer (TNBC) metastasis. Decreased metastasis upon A2BR inhibition is independent of host A2BR and lymphocytes and myeloid cells. Knockdown of A2BR on mouse and human cancer cells reduces their metastasis in vivo and decreases their viability and colony-forming ability, while transiently delaying cell-cycle arrest in vitro. The prometastatic activity of adenosine is partly tumor A2BR dependent and independent of host A2BR expression. In humans, TNBC cell lines express higher A2BR than luminal and Her2+ breast cancer cell lines, and high expression of A2BR is associated with worse prognosis in TNBC. Collectively, high A2BR on mouse and human tumors promotes cancer metastasis and is an ideal candidate for therapeutic intervention. Cancer Res; 76(15); 4372–82. ©2016 AACR.

Background: Triple negative breast cancers (TNBCs) are the most aggressive and profoundly heterogeneous form of breast cancer (BC), treatment of which is a prevalent challenge faced in clinics. CEP55, discovered first by our laboratory, is a key regulator of cytokinesis, error in which roots to multi-nucleation. Function of CEP55 is critically delimited by ERK2/PLK1 dependent phosphorylation, for accurate cytokinesis. Research has demonstrated connotation of CEP55 with numerous cancers including BC as higher CEP55 mRNA expression is allied to worse prognosis and poor survival. We hypothesised that, CEP55 controls fate of aneuploid cell population among aggressive BC that are heavily reliant on mitotic genes for tumour progression, thus can be targeted for therapy development. Methods: Using in vitro studies we demonstrated that depletion of CEP55 sensitizes TNBC cells to anti-mitotic drugs like PLK1 inhibitor to induce CDK1-Caspase 3-dependent mitotic catastrophe due to unscheduled CDK1/Cyclin B activation. Also we showed ERK1/2 transcriptionally controls CEP55 hence inhibition of MEK1/2 using the small molecule inhibitor Selumetinib, can mimic depletion of CEP55 in vivo. Results: We rationalised the usage of a MEK1/2 inhibitor in combination with a PLK1 inhibitor across a series of BC cell lines. We observed synthetic lethality among the aggressive hormone receptor negative lines with higher CEP55 expression compared to normal like and receptor positive lines with lower CEP55 level. The combination synergistically amplified apoptosis of aneuploid population via premature entry of these cells into mitosis in the presence of antimitotic drugs due to exhaustion of CEP55. We have also validated this synergistic effect of MEK1/2 and PLK1 inhibition using xenograft models, results of which imitated the in vitro findings. Conclusions: We propose a novel treatment tactic of MEK1/2 -PLK1 dual combination for selectively targeting CEP55 over-expressing BC in the clinics.

Triple negative breast cancers (TNBCs), lacking the expression of ER, PR and HER2 amplification, are the most aggressive form of breast cancer (BC). Due to their heterogeneity influenced by high level of genomic instability and aneuploidy, treatment of TNBC patients is one of the biggest challenges faced in the clinics. CEP55, discovered first by our laboratory, is a key regulator of cytokinesis malfunction of which has been shown to cause multinucleation. Furthermore, ERK2/PLK1 critically regulates the functional role of CEP55 via phosphorylation of CEP55 at particular stages of mitosis, allowing it to localize to the midbody for accurate cytokinesis. Research has demonstrated association of CEP55 with various cancers especially BC as over-expression of CEP55 mRNA is associated with worse BC prognosis and poor survival. We hypothesized that, CEP55 regulates the fate of aneuploid cell population, which are highly dependent on mitotic genes for tumor progression among aggressive BC, thus can be targeted for therapy development. We have performed a series of in vitro studies demonstrating that depletion of CEP55 sensitizes cells to anti-mitotic drugs like PLK1 inhibitor (BI2536) and leads to unscheduled CDK1/Cyclin B activation and favor CDK1-Caspase 3-dependent mitotic catastrophe. We also demonstrate that ERK1/2 transcriptionally controls CEP55 mRNA and due to lack of a specific small molecule inhibitor against CEP55, inhibition of MEK1/2 using the small molecule inhibitor Selumetinib, can mimic depletion of CEP55 in vivo. Thus, we rationalized the usage of a MEK1/2 inhibitor in combination with a PLK1 inhibitor across a series of BC cell lines. We observed synthetic lethality among the aggressive hormone receptor negative cell lines, which expressed higher expression of CEP55 compared to normal like and hormone receptor positive BC cell lines with lower CEP55 level. The combination synergistically increased apoptosis of aneuploid population via premature entry of these cells into mitosis in the presence of antimitotic drugs due to exhaustion of CEP55. We have also validated this synergistic effect of MEK1/2 and PLK1 inhibition using xenograft models, results of which imitated the in vitro findings. Therefore, we propose a novel treatment strategy of MEK1/2-PLK1 dual combination for selectively targeting CEP55 over-expressing BC in the clinics. Citation Format: Debottam Sinha, Murugan Kalimutho, J. Alejandro Lopez, Kum Kum Khanna. Dual inhibition of MEK1/2 and PLK1 specifically targets aggressive breast cancer cell population with CEP55 elevated expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1144. doi:10.1158/1538-7445.AM2017-1144

BRAF V600E is a major oncogenic mutation found in approximately 50% of human melanoma that confers constitutive activation of the MAPK pathway and increased melanoma growth. Inhibition of BRAFV600E by oncogene targeting therapy increases overall survival of patients with melanoma, but is unable to produce many durable responses. Adaptive drug resistance remains the main limitation to BRAFV600E inhibitor clinical efficacy and immune-based strategies could be useful to overcome disease relapse. Tumor microenvironment greatly differs between visceral metastasis and primary cutaneous melanoma, and the mechanisms involved in the antimetastatic efficacy of BRAFV600E inhibitors remain to be determined. To address this question, we developed a metastatic BRAFV600E-mutant melanoma cell line and demonstrated that the antimetastatic properties of BRAF inhibitor PLX4720 (a research analogue of vemurafenib) require host natural killer (NK) cells and perforin. Indeed, PLX4720 not only directly limited BRAFV600E-induced tumor cell proliferation, but also affected NK cell functions. We showed that PLX4720 increases the phosphorylation of ERK1/2, CD69 expression, and proliferation of mouse NK cells in vitro. NK cell frequencies were significantly enhanced by PLX4720 specifically in the lungs of mice with BRAFV600E lung metastases. Furthermore, PLX4720 also increased human NK cell pERK1/2, CD69 expression, and IFNγ release in the context of anti-NKp30 and IL2 stimulation. Overall, this study supports the idea that additional NK cell-based immunotherapy (by checkpoint blockade or agonists or cytokines) may combine well with BRAFV600E inhibitor therapy to promote more durable responses in melanoma. Cancer Res; 74(24); 7298–308. ©2014 AACR.

Purpose: To develop effective combination therapy against pancreatic ductal adenocarcinoma (PDAC) with a combination of chemotherapy, CHK1 inhibition, and EGFR-targeted radioimmunotherapy. Experimental Design: Maximum tolerated doses were determined for the combination of gemcitabine, the CHK1 inhibitor PF-477736, and Lutetium-177 (177Lu)–labeled anti-EGFR antibody. This triple combination therapy was investigated using PDAC models from well-established cell lines, recently established patient-derived cell lines, and fresh patient-derived xenografts. Tumors were investigated for the accumulation of 177Lu-anti-EGFR antibody, survival of tumor-initiating cells, induction of DNA damage, cell death, and tumor tissue degeneration. Results: The combination of gemcitabine and CHK1 inhibitor PF-477736 with 177Lu-anti-EGFR antibody was tolerated in mice. This triplet was effective in established tumors and prevented the recurrence of PDAC in four cell line–derived and one patient-derived xenograft model. This exquisite response was associated with the loss of tumor-initiating cells as measured by flow cytometric analysis and secondary implantation of tumors from treated mice into treatment-naïve mice. Extensive DNA damage, apoptosis, and tumor degeneration were detected in the patient-derived xenograft. Mechanistically, we observed CDC25A stabilization as a result of CHK1 inhibition with consequent inhibition of gemcitabine-induced S-phase arrest as well as a decrease in canonical (ERK1/2 phosphorylation) and noncanonical EGFR signaling (RAD51 degradation) as a result of EGFR inhibition. Conclusions: Our study developed an effective combination therapy against PDAC that has potential in the treatment of PDAC. Clin Cancer Res; 20(12); 3187–97. ©2014 AACR.

The WNT and MAPK signaling pathways are central to colorectal homeostasis. Mutation of the APC tumor suppressor gene activates the WNT pathway to initiate most conventional adenomas. By contrast, oncogenic BRAF mutation initiates development of sessile serrated adenomas (SSA) via activation of the MAPK pathway. The purpose of this study was to investigate the frequency, timing and mechanism of WNT pathway activation in serrated colorectal neoplasia. Methods: The WNT pathway was examined in a range of colorectal polyp and cancer subtypes. Aberrant nuclear immuno-localization of β-catenin was used as a surrogate for WNT pathway activation, compared to the normal membranous staining pattern. The entire coding region of the APC gene was sequenced using a custom enrichment panel that included 242 primer pairs (GeneRead DNAseq Targeted Panel, Qiagen) and the constructed library was sequenced using Illumina's HT TruSeq Kit. As another mechanism of activating WNT, mutations were also assessed in the E3 ubiquitin ligase genes RNF43 and ZNRF3 using Sanger sequencing. To test the interaction of the WNT and MAPK pathways in vivo, a conditional BRAF mutant mouse model was crossed with an inducible Cre driven by the murine Villin gene promoter, to restrict Cre expression and induction of the BRAF mutation to the intestine at 2 weeks. The chemical carcinogen azoxymethane was administered weekly for 6 weeks to activate the WNT pathway from 3 weeks of age. The potential therapeutic implication of targeting these pathways was tested in vitro using the porcupine inhibitor LGK974 and MEK inhibitor PD0325901. Results: The majority of conventional pathway polyps and cancers had nuclear localization of β-catenin (22/28, 79% adenomas and 36/42, 86% BRAF wildtype cancers) and mutations in APC (17/20, 85% adenomas and 19/30, 63% BRAF wild type cancers). Sessile serrated adenomas infrequently showed nuclear staining of β-catenin and 0/20 SSA had mutations. By contrast, the WNT signal was increased in the dysplastic component of SSA with a focus of dysplasia (76/137, 55%), however APC mutations were uncommon (3/20, 15%). Similarly, BRAF mutant cancers had nuclear β-catenin in 36/92 (39%) cancers compared to APC mutation in only 9/80 (11%) samples. This did not significantly differ when samples were stratified by microsatellite instability status. RNF43 and ZNRF3 were commonly mutated in BRAF mutant / microsatellite unstable cancers (47/54, 87% and 16/54, 30%, respectively) as well as BRAF mutant / microsatellite stable cancers (8/33, 24% and 5/33, 15%) compared to only 3/79 (4%) RNF43 mutations and 0/27 ZNRF3 mutations in BRAF wildtype cancers. This was accompanied by lower transcript expression for both RNF43 and ZNRF3 in BRAF mutant compared to BRAF wildtype cancers (P Conclusions: The WNT pathway is commonly activated in serrated neoplasia at the transition to dysplasia. This is not due APC mutation but rather mutation of the upstream RNF43 and/or ZNRF43 genes or other WNT pathway targets. The combination of WNT and MAPK pathway inhibition offers potential for improving therapy for patients with serrated pathway cancers. This abstract is also being presented as Poster A21. Citation Format: Vicki Whitehall, Jennifer Borowsky, Catherine Bond, Mark Bettington, Sally-Ann Pearson, Murugan Kalimutho, John Liu, Troy Dumenil, Diane McKeone, Saara Jamieson, Winnie Fernando, Lochlan Fennell, Andrew Clouston, Christophe Rosty, Ian Brown, Neal Walker, Barbara Leggett. Wnt and MAPK pathway activation in conventional and serrated colorectal neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr PR04.

MicroRNAs are tiny non-coding small endogenous RNAs that regulate gene expression by translational repression, mRNA cleavage and mRNA inhibition. The aim of this study was to investigate the hypermethylation of miR-34b/c and miR-148a in colorectal cancer, and correlate this data to clinicopathological features. We also aimed to evaluate the hypermethylation of miR-34b/c in faeces specimens as a novel non-invasive faecal-DNA-based screening marker. The 5-aza-2′-deoxycytidine treatment and methylation-specific PCR were carried out to detect the hypermethylation of miR-34b/c and miR-148a. The miR-34b/c hypermethylation was found in 97.5% (79 out of 82) of primary colorectal tumours, P=0.0110. In 75% (21 out of 28) of faecal specimens we found a hypermethylation of miR-34b/c while only in 16% (2 out of 12) of high-grade dysplasia. In addition, miR-148a was found to be hypermethylated in 65% (51 out of 78) of colorectal tumour tissues with no significant correlation to clinicopathological features. However, a trend with female gender and advanced age was found, P=0.083. We also observed a trend to lower survival rate in patients with miR-148a hypermethylation with 10-year survival probability: 48 vs 65%, P=0.561. These findings show that aberrant hypermethylation of miR-34b/c could be an ideal class of early screening marker, whereas miR-148a could serve as a disease progression follow-up marker.

Centrosomal proteins have long been known to act as a scaffolds, spindle checkpoint regulators as well as key regulating elements of microtubule organizing centre, hence as an essential component of cell cycle progression. CEP55 is a centrosomal protein originally identified as an indispensable regulator of cytokinesis, the final stage of cell division that divides cytoplasm equally amongst two daughter cells. Failure to faithfully complete cytokinesis due to loss of CEP55 resulted in primary genetic lesion leading to multinucleated cells, thus promotes tumourigenesis and aneuploidy. CEP55 has been to shown elevated in wide range of cancers including breast cancer, however the molecular mechanisms on how CEP55 mediated genomic instability in cancers are still not well understood. To decipher the functional role of CEP55 in regulating genomic instability in breast cancer, through a series of in-vitro and in-vivo experiments we found that high levels of CEP55 is significantly associates with basal-like breast cancer subtype with poor overall survival, relapse free survival and distant-metastasis free survival. Furthermore, we found that depletion of CEP55 decreased cell viability due to induction of apoptosis and hamper primary tumour growth due to clearance of aneuploid cells that are particularly important in oncogenic transformation. In addition, depletion of CEP55 hindered premature exit of mitotic arrested cells and this is partly mediated through CEP55-dependent interaction with HSF1 during mitosis to facilitate development of aneuploidy. Strikingly, we found that CEP55 mediates anti-mitotic drug resistance to docetaxel and PLK1 inhibition. Depletion of CEP55 increased sensitivity to anti-mitotic drugs in particular docetaxel or PLK1 inhibitor, providing a rationale to target CEP55 and its dependent-pathways in aggressive and aneuploid breast cancer. Citation Format: Murugan Kalimutho, Nicola Waddell, Jessie Jeffry, Sriganesh Srihari, Kum Kum Khanna. CEP55 is a determinant of genomic instability in aneuploid breast cancer cells and facilitates anti-mitotic drugs resistance by interacting directly with HSF1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3008. doi:10.1158/1538-7445.AM2015-3008

Defects in the number, structure, and function of centrosomes can generate mono- or multipolar mitotic spindles and cytokinesis failure resulting in aneuploidy and chromosome instability, which are common characteristics of tumour cells. Centrosomal proteins thus play central roles in the execution of proper cell division. Cep55 is a centrosomal protein required for proper segregation of two daughter cells during cytokinesis. It often elevated in wide range of cancer types including breast cancer. To ascertain the functional role of Cep55 in breast cancer pathogenesis, we performed publically available data mining as well as IHC staining in a cohort of breast cancer patients and found that elevated levels of Cep55 is significantly associated with p53 mutation, basal like subtype, relapse free and distant-metastasis free survival. Immunoblot analysis of non-malignant and breast cancer cell lines revealed higher expression of Cep55 protein in basal like subtype. RNAi mediated Cep55 depletion in cultured human breast cancer cells resulted in decreased cell proliferation, induction of apoptosis in-vitro and failure to establish xenografts in-vivo. Moreover, these cells failed to establish lung colonisation due to less proliferative, migrative and invasive capacity in experimental metastasis models. In an initial effort to identify causal effective of Cep55 mediated breast cancer pathogenesis, we found that Cep55-depleted cells that survive following long term culture have restored their ploidy compared with aneuploid cells that have constitutively high levels of Cep55. Moreover, these non-tumorigenic cells also have defect in major signalling networks assessed by Kinexus proteomic array. In line with this finding, we found that constitutive overexpression of Cep55 in non-transformed mammary epithelial cells, MCF-10a induced ERK1/2 and STAT3 signalling possibly due to increase in chromosome instability. Taken together, these results indicate that Cep55 promotes breast cancer initiation, progression and lung colonisation through pleiotropic effect and genomic instability that likely contribute to aneuploidy state of breast cancer tumours Citation Format: Murugan Kalimutho, Jacinta Simmons, Winnie Fernando, Jodi Saunus, Kevin Spring, Sunil Lakhani, Fares Al-Ejeh, Kum Kum Khanna. Cep55, a master regulator of cytokinesis in breast cancer pathogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-116. doi:10.1158/1538-7445.AM2014-LB-116

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking expression of estrogen and progesterone receptors (ER/PR) and HER2, thus limiting therapy options. We hypothesized that meta-analysis of TNBC gene expression profiles would illuminate mechanisms underlying the aggressive nature of this disease and identify therapeutic targets. Meta-analysis in the Oncomine database identified 206 genes that were recurrently deregulated in TNBC compared with non-TNBC and in tumors that metastasized or led to death within 5 years. This ‘aggressiveness gene list' was enriched for two core functions/metagenes: chromosomal instability (CIN) and ER signaling metagenes. We calculated an ‘aggressiveness score' as the ratio of the CIN metagene to the ER metagene, which identified aggressive tumors in breast cancer data sets regardless of subtype or other clinico-pathological indicators. A score calculated from six genes from the CIN metagene and two genes from the ER metagene recapitulated the aggressiveness score. By multivariate survival analysis, we show that our aggressiveness scores (from 206 genes or the 8 representative genes) outperformed several published prognostic signatures. Small interfering RNA screen revealed that the CIN metagene holds therapeutic targets against TNBC. Particularly, the inhibition of TTK significantly reduced the survival of TNBC cells and synergized with docetaxel in vitro. Importantly, mitosis-independent expression of TTK protein was associated with aggressive subgroups, poor survival and further stratified outcome within grade 3, lymph node-positive, HER2-positive and TNBC patients. In conclusion, we identified the core components of CIN and ER metagenes that identify aggressive breast tumors and have therapeutic potential in TNBC and aggressive breast tumors. Prognostication from these metagenes at the mRNA level was limited to ER-positive tumors. However, we provide evidence that mitosis-independent expression of TTK protein was prognostic in TNBC and other aggressive breast cancer subgroups, suggesting that protection of CIN/aneuploidy drives aggressiveness and treatment resistance.

Mutations in the Kras proto-oncogene are found in 40%-45% of colorectal cancer (CRC) patients and confer resistance to EGFR monoclonal antibody (mAb) therapies. The identification of druggable targets uniquely required by KrasMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. We carried out microarray profiling of isogenic Kras wild type (WT) and mutant (MT) in vivo CRC models, KrasMT/WT primary colon tumours, pre-treatment metastatic CRC liver biopsies and drug resistant and sensitive CRC cell lines. We have used systems biology approach incorporating pathway analyses of the microarray data followed by siRNA screening to identify novel targets/pathways which are critical for the survival of KrasMT tumours, both basally and following chemotherapy treatment. We found that JAK1/2-STAT3 signaling was essential for the survival of KrasMT CRC cells. Moreover, suppression of JAK1/2-STAT3 activity using RNAi or small molecule inhibitors resulted in more potent increase in apoptosis in KrasMT CRC cells. In addition, treatment with chemotherapy and MEK1/2 inhibition resulted in significant increases in JAK2 and STAT3 activity and this was associated with strong increase in apoptosis when JAK1/2-STAT3 RNAi or small molecule inhibitors were combined with chemotherapy and MEK1/2 inhibition, especially in KrasMT cells. Moreover, we found that these effects were not drug- or cell line-specific. Collectively, using a systems biology approach, we have identified an essential role of JAK1/2-STAT3 signalling in survival of KrasMT CRC models. These findings support the further clinical investigation of JAK/STAT3 inhibitors in conjunction with chemotherapy or MEK inhibition in KrasMT CRC tumours. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 908. doi:1538-7445.AM2012-908

Background Colorectal cancer (CRC) is the third most common cancer in the UK with around 80% of patients undergoing surgery followed by adjuvant chemotherapy treatment. Among patients with clinicopathologically defined poor stage II and stage III CRC, there are subsets of patients who will not benefit from adjuvant 5-FU or 5-FU/oxaliplatin treatment. In fact, there is preclinical evidence to suggest that some of these patients may actually do worse when treated with adjuvant chemotherapy. The aim of this study was to develop in vitro adjuvant colon cancer models with the ultimate goal to identify novel treatment strategies for early stage CRC. Method Progressively invasive populations from parental HCT116 were generated using invasion chambers. Analysis of migration and invasion was carried out using the XCELLigence system, protein activity/expression using receptor tyrosine kinase array (RTK) and Western blotting, colony forming ability was assessed by survival assays and drug sensitivity using MTT assay and Flow cytometry. Results Six sublines of HCT116 cells were initially generated (I1-6) which displayed an increasing invasive/migratory phenotype compared to the parental cell line. At the molecular level, we found increases in basal activity of a number of RTK in the invasive sublines, such as Axl (a phenomenon which is characterized with a more aggressive nature in cancers) and also in other kinases such as ERK1/2, Akt and STAT3. Decreased activation of the EGFR and HER2 receptors was also noted in the invasive cell lines. In addition, exposure to sub-lethal 5-FU(IC20) doses resulted in significant increases in migration in these cell line models. Silencing of Axl resulted in significant decreases in migration of the invasive subpopulations even in the presence of 5-FU. Colony forming assays highlighted an increased ability to form colonies in the invasive sub-populations which also displayed drug resistance to both 5-FU and Oxaliplatin. The generation of invasive cell lines was also carried out in a number of other cell lines which confirmed that an increase in Axl expression could increase the migratory phenotype regardless of the genetic background of the cell and also that the results obtained are not an artefact of the cell line we have used. Conclusion Using our in-house developed in vitro preclinical cell line model we have begun to characterise the changes which tumour cells appear to go through during the transition to an invasive phenotype. We have highlighted Axl as a potential target for inhibition in early stage colorectal cancer as increases in total Axl protein levels correlate well with increases in invasive capacity in our models and silencing of the protein expression by siRNA results in ablation of the invasive potential in our model and also in a panel of CRC cell lines. Combination of an Axl small molecule inhibitor could potentially be a novel treatment strategy for early stage CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4324. doi:1538-7445.AM2012-4324