Abstract SY28-03: Targeting tetraploidy for cancer therapy by pharmacological and immunological strategies.
Authors: Kroemer Guido , Senovilla Laura , Zitvogel Laurence , Castedo Maria 
Published: August 2014

The supreme goal of anticancer therapy is the induction of tumor cell death. Physiological cell death, which occurs as a continuous byproduct of cellular turnover, is non-immunogenic or even tolerogenic, thereby avoiding autoimmunity. However, cancer cell death elicited by radiotherapy and some chemotherapeutic agents such as anthracyclines and oxaliplatin can be immunogenic. Immunogenic death involves changes in the composition of the cell surface, as well as the release of soluble immunogenic signals that occur in a defined temporal sequence. This "key" then operates on a series of receptors expressed by dendritic cells (DC, the "lock") to allow for the presentation of tumor antigens to T cells and for the initiation of a productive immune response.

Immunogenic cell death is characterized by the early cell surface exposure of calreticulin, which determines the uptake of tumor antigens by DC. The late release of the protein high mobility group box 1 (HMGB1), which acts on toll-like receptor 4 (TLR4), is required for the presentation of antigens from dying tumor cells. In addition, the release of ATP from dying cells causes the P2Y2 purinergic receptor-mediated attraction, survival and differentiation of DC cell precursors into the tumor bed and the P2RX7 purinergic receptor-dependent activation of the NLRP3 inflammasome in DC, thereby allowing them to release interleukin-1ß and to polarize tumor antigen-specific CD8 T cells towards a Tc1 cytokine pattern.

We postulate that the immune system determines the long-term success of anti-cancer therapies, and that this immune response is dictated by immunogenic tumor cell death. Thus, therapeutic failure can result from failure to undergo immunogenic cell death (rather than cell death as such). Agents that fail to induce immunogenic cell death cannot yield a long-term success in cancer therapy. Moreover, tumors that are intrinsically unable to undergo immunogenic cell death are incurable. Importantly, it appears that mitochondrial events determine whether cancer cells die or not in response to chemotherapy, while an endoplasmic reticulum stress (ER) response combined with autophagy determines whether this cell death is perceived as immunogenic. Indeed, we found that ER stress is required for calreticulin exposure to occur while a premortem autophagic response is required for optimal ATP release.

In a screen designed to identify pharmacological agents that induce calreticulin exposure on the cell surface, we discovered that several agents that induced tetraploidization through distinct mechanisms, either by inhibition of karyokinesis (epothilones, taxanes, vinca alkaloids) or by inhibition of cytokinesis (cytochalasin D) were able to stimulate calreticulin exposure on the cell surface. Similarly, we fond that spontaneous tetraploidization of primary epithelial cells from p53-knockout mice as well as different genetic manipulations to induce tetraploidization (such as knockdown of Mad2, BubR1 and Aurora kinase 1) also led to calreticulin exposure. Thus, CRT exposure did not depend on the agent that induced tetraploidization but rather could be attributed to the tetraploidization process itself. Indeed, all conditions of tetraploidization were associated with an endoplasmic reticulum (ER) stress response manifesting with the inactivating phosphorylation of the eukaryotic initiation factor 2α (eIF2α). This ER stress response was required for the optimal survival of tetraploid cells as well as for the surface exposure of calreticulin. Moreover, dying tetraploid cells could elicit anticancer immune responses in vaccination experiments.

At least in part owing to the increased calreticulin exposure, hyperploid cancer cells inoculated into immunocompetent hosts form tumors with a reduced incidence as compared to their parental counterparts. Conversely, near-to-diploid and hyperploid cancer cells generate tumors with the same incidence when inoculated into immunocompromised mice. Of note, hyperploid cell-derived tumors recovered from immunocompetent mice exhibit reduced ploidy, lower degrees of ER stress and fewer ecto-CRT than tumors generated by the same cells in immunodeficient animals. In addition, carcinogen-induced tumors developing in immunocompetent mice exhibit reduced ploidy and lower degrees of ER than the same lesions developing in immunocompromised (Rag2-/- γc-/-, Stat1-/- or Dnam1-/-) animals. Altogether, our observations indicate that hyperploidy is a cancer-associated trait that is counterselected in vivo by the immune system upon the recognition of increased CRT exposure.

To investigate the translational relevance of our findings, we determined nuclear size (as an indicator of ploidy) and eIF2α phosphorylation (as an indicator of ER stress) in breast carcinoma biopsies from patients who either responded or did not respond to neo-adjuvant chemotherapy. In line with previous reports, patients that underwent clinical responses to chemotherapy (responders), but not subjects with detectable lesions in spite of six cycles of chemotherapy (non-responders), exhibited high amounts of tumor-infiltrating CD8+ T cells over immunosuppressive FOXP3+ cells. In addition, the few breast carcinoma cells that could be detected in responders exhibited a decreased nuclear diameter and lower levels of eIF2α phosphorylation as compared to the neoplastic cells of non-responders. These observations indicate that the clinically relevant activation of the immune system results in the elimination of hyperploid cancer cells.

Our findings reveal an unsuspected mechanism of anticancer immunosurveillance whereby hyperploid malignant cells are recognized and eliminated by the immune system as they exposed increased amounts of calreticulin on their plasma membrane. Moreover, our work suggest that antimitotic agents that induce increases in tumor cell ploidy may stimulate anticancer immune responses as part of their mode of action.

Citation Format: Guido Kroemer, Laura Senovilla, Laurence Zitvogel, Maria Castedo. Targeting tetraploidy for cancer therapy by pharmacological and immunological strategies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY28-03. doi:10.1158/1538-7445.AM2013-SY28-03