As PD-1/PD-L1 immune checkpoint inhibitors exhibited promising clinical outcomes in various types of solid tumors, PD-1/PD-L1 blockades have been explored for the treatment of hepatocellular carcinoma (HCC). However, the association of PD-L1 with antitumor immunoregulation is not clearly defined in HCC. Here, we evaluated the characteristics of PD-L1 expression, CD8 T-cell infiltration and their relationship in HCC. A total of 411 resected tumor specimens from HCC patients were immunostained for PD-L1 and CD8. Only 78 (19%) cases showed ≥5% membranous PD-L1 expression on tumor cells, although a significantly positive correlation was found between PD-L1 expression and CD8 T-cell densities. Moreover, patients with higher tumor PD-L1 expression also showed a higher hepatitis B virus load, which was also related to increased CD8 infiltration. Survival analysis suggested that both tumor and stroma PD-L1 status did not significantly affect overall survival or recurrence-free survival in patients. Although high CD8 T-cell density was overall associated with better overall survival and recurrence-free survival, its favorable prognostic value was eliminated by high tumor PD-L1 expression. Further flow cytometric and enzyme-linked immunosorbent assay (ELISA) results from the coculture of HCC cell lines with specific CD8 cytotoxic T lymphocytes (CTLs) demonstrated that CD8 CTLs remarkably upregulated PD-L1 expression on tumor cell lines by HLA class-I specificity, and the overexpression of tumor PD-L1 impaired interferon-γ secretion by CD8 CTLs in a negative feedback regulation mechanism. In conclusion, our findings reveal an interaction between PD-L1 expression and CD8 T-cell immunity in HCC, although PD-L1 is not a prognostic factor for the patients.

The role of pyruvate kinase M2 isoform (PKM2) in tumor progression has been controversial. Previous studies showed that PKM2 promoted tumor growth in xenograft models; however, depletion of PKM2 in the -loss-driven mammary tumor mouse model accelerates tumor formation. Because oncogenic kinases are frequently activated in tumors and PKM2 phosphorylation promotes tumor growth, we hypothesized that phosphorylation of PKM2 by activated kinases in tumor cells confers PKM2 oncogenic function, whereas nonphosphorylated PKM2 is nononcogenic. Indeed, PKM2 was phosphorylated at tyrosine 105 (Y105) and formed oncogenic dimers in MDA-MB-231 breast cancer cells, whereas PKM2 was largely unphosphorylated and formed nontumorigenic tetramers in nontransformed MCF10A cells. PKM2 knockdown did not affect MCF10A cell growth but significantly decreased proliferation of MDA-MB-231 breast cancer cells with tyrosine kinase activation. Multiple kinases that are frequently activated in different cancer types were identified to phosphorylate PKM2-Y105 in our tyrosine kinase screening. Introduction of the PKM2-Y105D phosphomimetic mutant into MCF10A cells induced colony formation and the CD44/CD24 cancer stem-like cell population by increasing Yes-associated protein (YAP) nuclear localization. ErbB2, a strong inducer of PKM2-Y105 phosphorylation, boosted nuclear localization of YAP and enhanced the cancer stem-like cell population. Treatment with the ErbB2 kinase inhibitor lapatinib decreased PKM2-Y105 phosphorylation and cancer stem-like cells, impeding PKM2 tumor-promoting function. Taken together, phosphorylation of PKM2-Y105 by activated kinases exerts oncogenic functions in part via activation of YAP downstream signaling to increase cancer stem-like cell properties. These findings reveal PKM2 promotes tumorigenesis by inducing cancer stem-like cell properties and clarify the paradox of PKM2's dichotomous functions in tumor progression. .

The functions of immune cells in brain metastases are unclear because the brain has traditionally been considered “immune privileged.” However, we found that a subgroup of immunosuppressive neutrophils is recruited into the brain, enabling brain metastasis development. In brain metastatic cells, enhancer of zeste homolog 2 (EZH2) is highly expressed and phosphorylated at tyrosine-696 (pY696)–EZH2 by nuclear-localized Src tyrosine kinase. Phosphorylation of EZH2 at Y696 changes its binding preference from histone H3 to RNA polymerase II, which consequently switches EZH2’s function from a methyltransferase to a transcription factor that increases c-JUN expression. c-Jun up-regulates protumorigenic inflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF), which recruits Arg1+- and PD-L1+ immunosuppressive neutrophils into the brain to drive metastasis outgrowth. G-CSF–blocking antibodies or immune checkpoint blockade therapies combined with Src inhibitors impeded brain metastasis in multiple mouse models. These findings indicate that pY696-EZH2 can function as a methyltransferase-independent transcription factor to facilitate the brain infiltration of immunosuppressive neutrophils, which could be clinically targeted for brain metastasis treatment.

This report describes an ongoing Phase I clinical trial testing the safety of adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) in patients with primary hepatocellular carcinoma (HCC). Fifteen HCC patients were treated with their activated and expanded TILs following tumor resection. From a total of 17 patients with HCC, TIL were successfully expanded from 15 patients (88%), whereas two patients showed minimal or no expansion of TIL. Transient increase in the frequency of T cells was observed after adoptive transfer who was found only associated with grade I flu-like symptoms and malaise. After a median follow-up of 14 months, 15 patients (100%) were alive; and 12 patients (80%) showed no evidence of disease, 3 patients (patient 1,11,12) had tumor recurrence. The time to the diagnosis of tumor recurrence following therapy ranged from 105 to 261 days. These results indicate that immunotherapy with activated and expanded autologous TIL could be successfully performed with low toxicity, thus would serve as a novel treatment modality for patients with HCC.