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Researchers Discover a New Mechanism Supporting Senescent Cells in the Treatment-damaged Tumor Microenvironment

On October 17, 2018, an original research article titled "Targeting SPINK1 in the damaged tumour microenvironment alleviates therapeutic resistance" was published online at Nature Communications. The research group led by Prof. SUN Yu at Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences reported that the cytotoxicity induced by clinical anticancer regimens including chemotherapy and radiation can generate a large number of senescent cells in the tumor microenvironment (TME). The treatment-damaged TME eventually turns out to be an important source that actively produces a myriad of pro-inflammatory factors, among which is the soluble protein SPINK1. In the local TME, SPINK1 is continuously synthesized by stromal cells and subsequently released into the extracellular space, changing diverse phenotypes of adjacent cancer cells and promoting their multiple drug resistance (MDR). In addition, data output from large scale clinical studies indicate that the dynamics of SPINK1 protein in circulating blood is intimately correlated with the long term survival of cancer patients post-treatment. The amount of SPINK1 in patient serum can thus serve as a novel index biomarker for clinical prognosis and therapeutic appraisal, providing an innovative target and gauge parameter for future clinical oncology.

Despite the rapid advancement of anticancer pipelines and therapeutic strategies, cancer remains the most lethal pathology that claims hundreds of millions of human lives. The concept that progression of cancer is regulated by interactions of cancer cells with their microenvironment was postulated by Dr. Stephen Paget over a century ago. Contemporary TME research focuses on the identification of tumor-interacting microenvironmental constituents, such as resident or infiltrating non-cancer cells, soluble factors and extracellular matrix components, and the large variety of mechanisms by which these constituents regulate and shape the malignant phenotype of cancer cells. In this timeline article, Prof. Sun’s lab illuminated a novel side of the interactions between various microenvironmental components and cancer cells, which is mediated by soluble factors produced by senescent stromal cells emerging after classic chemotherapy, thus providing an new angle for evaluation of treatment outcome and presenting a valuable therapeutic modality by which the TME can be targeted.

In this new study, researchers first chose to perform a large scale deep analysis of human gene expression, and found that stromal cells do not adopt typical apoptosis after genotoxic stress caused by chemotherapy or radiation; instead, they tended to enter another state, cellular senescence. More importantly, the development of a unique feature ensues, which is termed senescence-associated secretory phenotype (SASP). Although the vast majority of the secreted factors encoded by the SASP are well known, their impact on pathological progression such as cancer resistance remains largely unknown. Interestingly, the serine protease inhibitor Kazal-type I (SPINK1, also PSTI or TATI) showed up in the top list of the upregulated genes posttreatment.

In prostate cancer (PCa) patients, SPINK1 expression and ETS gene fusion are mutually exclusive. Former studies reported that SPINK1 as an outlier can serve as an independent predictor for the biochemical recurrence of patients after radical prostatectomy in clinics. Although this attribute was found approximately 10 years ago by the group of Dr. Arul Chinnaiyan, an investigator in Michigan Center for Translational Pathology and Department of Urology, University of Michigan in 2008, the data largely failed to address the influence of TME-derived SPINK1 on disease progression.

In contrast, the study performed by Dr. Sun’s group revealed that SPINK1 expression can be significantly induced in stromal cells by DNA damage events, including those triggered by genotoxic chemotherapy and ionizing radiation. To the contrary, cancer epithelial cells did not follow such an inducible change. What are the functional implications of SPINK1 production in stromal tissue compartments in cancer clinics? The researchers then initiated a series of clinical investigations, which led to the discovery that SPINK1 is indeed dramatically expressed in tumor foci of posttreatment cancer patients, in an astonishing contrast to the pretreatment samples. Through clinical stratification of these patients according to the recently established procedure which allows to assess the expression level or activation status of target proteins in the tissue level, they found a significant correlation between SPINK1 expression and activation of two key enzymes functionally associated with the SASP, namely p38MAPK and mTOR. Further, they noticed that the higher level of SPINK1 expression in stromal cells, the shorter survival of cancer patients post-therapy, implying the significantly implications of SPINK1 in cancer clinics.

In comparison to the conditioned media from control stromal cells, those from SPINK1-positive stromal cells displayed substantially modified gene expression profile. Specifically, both PC3 and DU145, two well-established PCa cell lines, manifested remarkable changes in genes encoding proteins, lncRNAs, miRNAs, miscRNAs and pseudogenes. Systematic analysis of these genes disclosed that there are several key activities modified after SPINK1 stimulation, including immune response, signal transduction and cell communication. Besides the prominent changes such as an epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) development, cancer cells exhibited a new feature, epithelial-endothelial transition (EET), which is supported by the upregulation of CD31 and CD34. Expression site bioinformatics analysis and in vitro tube formation assay demonstrated the angiogenesis capacity of SPINK1-exposed cancer cells, a phenomenon barely caused by a SASP factors.

To delineate the significance of SPINK1 within the TME in vivo, the group performed clinical trials with a self-prepared monoclonal antibody against human SPINK1 protein. In parallel, the FDA-approved therapeutic antibody Cetuximab was employed as experimental control. Interestingly, chemotherapy combined with anti-SPINK1 achieved a treatment efficacy that was even higher than the one generated from combination of chemotherapy plus Cetuximab. The surprising data were supported by not only measurements of the end volumes of xenograft tumors, but bioluminescence imaging of IVIS Xenogen device. The data evidently suggest that there are other receptors on the plasma membrane of recipient cancer cells, in addition to the well-studied EGFR.

Distinctly, new data from the subsequent clinical studies disclosed that SPINK1 is readily detectable in the circulating blood of cancer patients in the post-therapy stage, rather than the pre-treatment period. In particular, SPINK1 displayed a remarkable correlation with IL-8, a typical hallmark factor of the SASP. Immunoblots showed that these two soluble factors emerge concurrently in the patient plasma post-therapy, making a sharp contrast to the pre-treatment samples. These findings suggest that multiple SASP factors released by the damaged TME can indeed enter peripheral blood, whereby they hold the potential to serve as novel noninvasive biomarkers for anticancer treatment outcome and the SASP development in vivo of cancer patients. This has significant implications for advancement of future clinical oncology, by providing a baseline for establishment of a multiplexed evaluation system which can be used to assess both therapeutic outcome and patient prognosis.

This work was supported by the Science and Technology of China, the National Natural Science Foundation of China, Chinese Academy of Sciences, etc.

Illustrative diagram of SPINK1 expression in the treatment-damaged TME, pathological impact of paracrine SPINK1 on intercellular signaling network of cancer cells and its potential as a therapeutic target and novel biomarker for clinical oncology(Image by Prof. Sun's Group)


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