Chloroquine (CQ) is an antimalarial drug and late-stage inhibitor of autophagy currently FDA-approved for use in the treatment of rheumatoid arthritis and other autoimmune diseases. Based primarily on its ability to inhibit autophagy, CQ and its derivative, hydroxychloroquine, are currently being investigated as primary or adjuvant therapy in multiple clinical trials for cancer treatment. Oncogenic RAS has previously been shown to regulate autophagic flux, and cancers with high incidence of RAS mutations, such as pancreatic cancer, have been described in the literature as being particularly susceptible to CQ treatment, leading to the hypothesis that oncogenic RAS makes cancer cells dependent on autophagy. This autophagy “addiction” suggests that the mutation status of RAS in tumors could identify patients who would be more likely to benefit from CQ therapy. Here we show that RAS mutation status itself is unlikely to be beneficial in such a patient selection because oncogenic RAS does not always promote autophagy addiction. Moreover, oncogenic RAS can have opposite effects on both autophagic flux and CQ sensitivity in different cells. Finally, for any given cell type, the positive or negative effect of oncogenic RAS on autophagy does not necessarily predict whether RAS will promote or inhibit CQ-mediated toxicity. Thus, although our results confirm that different tumor cell lines display marked differences in how they respond to autophagy inhibition, these differences can occur irrespective of RAS mutation status and, in different contexts, can either promote or reduce chloroquine sensitivity of tumor cells.

Reports regarding the frequency of SMAD4 loss in human head and neck squamous cell carcinoma (HNSCC) vary significantly. We have shown that SMAD4 deletion contributes to HNSCC initiation and progression. Therefore, accurately detecting genetic SMAD4 loss is critical to determine prognosis and therapeutic interventions in personalized medicine. We developed a SMAD4 fluorescence in situ hybridization (FISH) assay to identify chromosomal SMAD4 loss at the single cell level of primary HNSCC specimens and patient derived xenograft (PDX) tumors derived from HNSCCs. SMAD4 heterozygous loss was detected in 35% of primary HNSCCs and 41.3% of PDX tumors. Additionally, 4.3% of PDX tumors had SMAD4 homozygous loss. These frequencies of SMAD4 loss were similar to those in The Cancer Genome Atlas (TCGA). However, we identified significant heterogeneities of SMAD4 loss (partial or complete) among cells within each tumor. We also found that aneuploidy (monosomy and polysomy) contributed greatly to how to define chromosomal SMAD4 deletion. Furthermore, in cultured PDX tumors, SMAD4 mutant cells outcompeted SMAD4 wildtype cells, resulting in establishing homogenous SMAD4 mutant HNSCC cell lines with partial or complete genomic SMAD4 loss, suggesting a survival advantage of SMAD4 mutant cells. Taken together, our study reveals inter- and intra-tumor heterogeneities of SMAD4 chromosomal loss in HNSCCs. Further, SMAD4 FISH assay provides a platform for future clinical diagnosis of SMAD4 chromosomal loss that potentially serves as a molecular marker for prognosis and therapeutic intervention in cancer patients.