RAS/MAPK activation is associated with reduced Tumor-infiltrating lymphocytes in Triple-Negative Breast Cancer: Therapeutic Cooperation Between MEK and PD-1/PD-L1 Immune Checkpoint Inhibitors

PURPOSE: Tumor-infiltrating lymphocytes (TIL) in the residual disease (RD) of triple-negative breast cancers (TNBC) after neoadjuvant chemotherapy (NAC) are associated with improved survival, but insight into tumor cell-autonomous molecular pathways affecting these features are lacking. EXPERIMENTAL DESIGN: We analyzed TILs in the RD of clinically and molecularly characterized TNBCs after NAC and explored therapeutic strategies targeting combinations of MEK inhibitors with PD-1/PD-L1-targeted immunotherapy in mouse models of breast cancer. RESULTS: Presence of TILs in the RD was significantly associated with improved prognosis. Genetic or transcriptomic alterations in Ras-MAPK signaling were significantly correlated with lower TILs. MEK inhibition upregulated cell surface MHC expression and PD-L1 in TNBC cells both in vivo and in vitro. Moreover, combined MEK and PD-L1/PD-1 inhibition enhanced antitumor immune responses in mouse models of breast cancer. CONCLUSIONS: These data suggest the possibility that Ras-MAPK pathway activation promotes immune-evasion in TNBC, and support clinical trials combining MEK- and PD-L1-targeted therapies. Furthermore, Ras/MAPK activation and MHC expression may be predictive biomarkers of response to immune checkpoint inhibitors

Metabolic modulation by CDK4/6 inhibitor promotes chemokine-mediated recruitment of T cells into mammary tumors

Inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) delay progression of metastatic breast cancer. However, complete responses are uncommon and tumors eventually relapse. Here, we show that CDK4/6i can enhance efficacy of T cell-based therapies, such as adoptive T cell transfer or T cell-activating antibodies anti-OX40/anti-4-1BB, in murine breast cancer models. This effect is driven by the induction of chemokines CCL5, CXCL9, and CXCL10 in CDK4/6i-treated tumor cells facilitating recruitment of activated CD

Type I interferon/IRF7 axis instigates chemotherapy-induced immunological dormancy in breast cancer

Neoadjuvant and adjuvant chemotherapies provide survival benefits to breast cancer patients, in particular in estrogen receptor negative (ER-) cancers, by reducing rates of recurrences. It is assumed that the benefits of (neo)adjuvant chemotherapy are due to the killing of disseminated, residual cancer cells, however, there is no formal evidence for it. Here, we provide experimental evidence that ER- breast cancer cells that survived high-dose Doxorubicin and Methotrexate based chemotherapies elicit a state of immunological dormancy. Hallmark of this dormant phenotype is the sustained activation of the IRF7/IFN-beta/IFNAR axis subsisting beyond chemotherapy treatment. Upregulation of IRF7 in treated cancer cells promoted resistance to chemotherapy, reduced cell growth and induced switching of the response from a myeloid derived suppressor cell-dominated immune response to a CD4(+)/CD8(+) T cell-dependent anti-tumor response. IRF7 silencing in tumor cells or systemic blocking of IFNAR reversed the state of dormancy, while spontaneous escape from dormancy was associated with loss of IFN-beta production. Presence of IFN-beta in the circulation of ER- breast cancer patients treated with neoadjuvant Epirubicin chemotherapy correlated with a significantly longer distant metastasis-free survival. These findings establish chemotherapy-induced immunological dormancy in ER- breast cancer as a novel concept for (neo)adjuvant chemotherapy activity, and implicate sustained activation of the IRF7/IFN-beta/IFNAR pathway in this effect. Further, IFN-beta emerges as a potential predictive biomarker and therapeutic molecule to improve outcome of ER- breast cancer patients treated with (neo)adjuvant chemotherapy.Peer reviewe

Investigating the role of the Ras/MAPK pathway in increasing immunogenicity of triple negative breast cancers

© 2019 Dr. Sathana DushyanthenThe predictive and prognostic significance of tumour infiltrating lymphocytes (TILs) has been highlighted in various solid cancers in recent years, where TIL infiltration has become a landmark biomarker of survival outcomes and response to therapy. These findings suggest an important role for T cell mediated immunosurveillance in influencing the biology of these cancers. More recently, studies have also demonstrated the prognostic value of TILs in certain breast cancer subtypes such as HER2-positive cancers, and in particular, triple negative breast cancer (TNBC), where the presence of higher levels of TILs in primary tumours was found to correlate with better disease free and overall survival. These associations suggest that immunotherapies may be effective in TNBC, a breast cancer subtype where novel therapies are urgently needed. Studies from our group have shown that oncogenic activation of the Ras/MAPK pathway is associated with significantly decreased levels of TILs and poorer survival in TNBC patients. This observation raises the possibility that Ras/MAPK pathway inhibition may relieve local immunosuppression, thereby enhancing TIL infiltrate and improving patient outcomes. Several studies undertaken through this thesis, in human and mouse TNBC cell lines, revealed that MEK inhibition (MEKi) was efficient at increasing tumour antigenicity, where small disruptions to MAPK signaling were able to enhance expression of immune markers such as MHC-I. Paradoxically, MEK signaling in lymphocytes is critical for CD8+ and CD4+ T cell activation, proliferation, function and survival. Therefore, the studies in this thesis aimed to investigate the effect of MEKi on tumour antigenicity, as well as the long-term effects of MEKi on T cell function. Although MEKi increased tumour antigenicity, interestingly, the data demonstrated that MEKi elicited inhibitory effects in both mouse and human T cells. It was hypothesised that agonist antibodies such as α-4-1BB (CD137) and α-OX-40 (CD134) may rescue T cell function in the presence of MEKi. The studies undertaken in Chapter 3 definitively demonstrated that MEKi significantly inhibits early T cell signaling, where the administration of immune agonists α-4-1BB and α-OX-40 in combination with MEKi, effectively maintained T cell frequency, proliferation and function after TCR stimulation, through the activation of alternative signaling pathways. As such, these results confirm that MEKi can prime tumour immunogenicity, and combination with either α-4-1BB or α-OX-40 agonist immunotherapy results in superior therapeutic efficacy due to protection of early and crucial TIL function in preclinical models of TNBC. Given the inhibitory effect of MEKi on T cells, the work in Chapter 4 endeavoured to develop a model where the effect of MEK1/2 knockdown (MEK KD) could be achieved intrinsically within the tumour, without affecting normal T cell function. This model would therefore allow the investigation into the genes that underpin the relationship between the Ras/MAPK pathway and tumour cell immunogenicity. Using this model, several antigen processing and presentation pathway genes were found to be increased following MEKi and MEK KD in tumours. Notably, immunoproteasome components were found to be upregulated following MEKi or MEK KD, suggesting that MEK suppression promotes the conversion of the proteasome to an immunoproteasome, thereby increasing the number of MHC-I related peptides for presentation to CD8+ T cells. Furthermore, RNAseq analysis of tumours cells with MEKi or MEK KD revealed that several extracellular matrix (ECM) remodelling genes and key chemokines were upregulated following MEK1/2 suppression. In summary, this work has characterised the ability of MEKi to enhance antigenicity of TNBCs, as well as deciphering mechanisms behind the detrimental effects involved with MEKi on T cell functional responses. Moreover, the studies conducted within this thesis highlight the strategies that may be used in order to overcome this inhibitory effect, as well as the alternative MAPK pathways involved in T cell rescue with agonist antibodies; through p38 and JNK signaling. Additionally, important factors involved in antigen processing and presentation and immunoproteasome conversion, associated with MEKi were discovered. This potentially explains the clinical observation that RAS/MAPK activation is associated with poor TIL infiltrate and suggests that novel targets could be validated, and future drugs developed to enhance antigen presentation in cancer, that could salvage or bypass the global effects of MEKi on T cells. Moreover, the principle of exploiting ECM stiffness and degradation to permit greater TIL infiltration and function, or enhanced drug penetration, may be further developed as a potential therapeutic strategy for future treatment approaches in TNBC, particularly those which harbour mutations that activate the RAS/MAPK pathway

The osteoblastic and osteoclastic interactions in spinal metastases secondary to prostate cancer.

Prostate cancer (PC) is one of the most common cancers arising in men and has a high propensity for bone metastasis, particularly to the spine. At this stage, it often causes severe morbidity due to pathological fracture and/or metastatic epidural spinal cord compression which, if untreated, inevitably leads to intractable pain, neurological deficit, and paralysis. Unfortunately, the underlying molecular mechanisms driving growth of secondary PC in the bony vertebral column remain largely unknown. Further investigation is warranted in order to identify therapeutic targets in the future. This review summarizes the current understanding of PC bone metastasis in the spine, highlighting interactions between key tumor and bone-derived factors which influence tumor progression, especially the functional roles of osteoblasts and osteoclasts in the bone microenvironment through their interactions with metastatic PC cells and the critical pathway RANK/RANKL/OPG in bone destruction

An in vivo mouse model of intraosseous spinal cancer causing evolving paraplegia

The spine is the commonest site of skeletal metastatic disease and uncontrolled growth of cancer in the spine will inevitably cause pain and neurologic compromise. Improved understanding of the pathobiology behind this devastating condition is urgently needed. For this reason, the aim of this study was to establish a clinically relevant, animal model of spinal cancer. A percutaneous orthotopic injection of human breast (MDA-MB-231) or human prostate (PC-3) cancer cells was administered into the upper lumbar spine of nude mice (n = 6). Animals were monitored twice daily for general welfare, gait asymmetry or disturbance, and hindlimb weakness. After sacrifice, plain radiographs, micro-CT imaging and histological analysis of the spines were performed on each mouse. All mice recovered fully from the inoculation procedure and displayed normal gait and behaviour patterns for at least 3 weeks post-inoculation. Subsequently, between 3 and 5 weeks post-inoculation, each mouse developed evolving paralysis in their hindlimbs over 48-72 h. All followed the same pattern of decline following onset of neurological dysfunction; from gait asymmetry and unilateral hindlimb weakness, to complete unilateral hindlimb paralysis and finally to complete bilateral hindlimb paralysis. Plain radiographs, micro-CT scanning and histological analysis confirmed local tumour growth and destruction of the spine in all six mice. An in vivo mouse model of human intraosseous spinal cancer has been established forming cancers that grow within the spine and cause epidural spinal cord compression, resulting in a reproducible, evolving neurological deficit and paralysis that closely resembles the human condition

Tumour-infiltrating lymphocytes (TILs) in breast cancer: A predictive or a prognostic marker?

Breast cancer has not been considered as an immunogenic solid cancer type; however, recent studies demonstrate evidence of significant prognostic information that can be derived from immune cell infiltration via tumour-infiltrating lymphocytes (TILs) in patient tumours. The presence of TILs has been associated with increased response rates to cytotoxic chemotherapy as well as targeted therapies, leading to improved disease-free and overall survival in certain breast cancer subtypes. Accordingly, experts have developed a standardized methodology for evaluating TILs within clinical specimens in histopathological practice. An overview of a newly established practical guideline for TIL evaluation in breast cancer is described in this paper. Furthermore, this review discusses the predictive and prognostic significance of TILs, highlighting recent evidence linking TILs to prognosis in breast cancer. In addition, it summarizes the most current understanding of TIL composition as well as mechanisms of immunity generation and suppression. Overall, the current literature demonstrates that TILs are proving to be a promising target for the treatment of immunogenic breast cancers.SCOPUS: re.jinfo:eu-repo/semantics/publishe