The results of a study by researchers at the University of Pittsburgh and the University of Pittsburgh Medical Center suggest that the antimalarial drug hydroxychloroquine (HCQ) might also boost the effectiveness of chemotherapy against some types of cancer by inhibiting pathways that drive drug resistance.

The newly reported work found that among human patients with squamous cell carcinoma of the head and neck (SCCHN), high expression of the ion channel TMEM16A predicts poor survival, and that TMEM16A is involved in the process that promotes cisplatin resistance, by driving the sequestration of cisplatin into lysosomes. Using a murine model of SCCHN, the team then showed that combination therapy using the lysosomal inhibitor HCQ and cisplatin held back the growth of cisplatin-resistant tumors, in vivo.

The investigators, headed by co-senior author Umamaheswar Duvvuri, MD, PhD, head and neck surgeon at UPMC Hillman Cancer Center and professor of otolaryngology at Pitt’s School of Medicine, reported their findings in Proceedings of the National Academy of Sciences (PNAS), in a paper titled, “Lysosomal inhibition sensitizes TMEM16A-expressing cancer cells to chemotherapy,” in which they concluded, “These results uncover a model of treatment for resistance in cancer, its reversal, and a role for TMEM16A … the repurposed use of HCQ may eventually be translated to combat the resistance to cytotoxic therapies.” The researchers are also now designing a Phase II clinical trial to evaluate treatment of head and neck cancer patients using a combination of hydroxychloroquine and cisplatin.

SCCHN is a devastating disease that continues to have low cure rates despite the recent advances in therapies, the authors wrote. Cisplatin is the most used chemotherapy agent against SCCHN, but treatment failure is largely driven by resistance to the drug. “Despite the advances in methods used to administer these treatments, many patients fail cisplatin-based regimens and subsequently experience recurrence and disease-specific mortality,” the investigators continued. “An improved understanding of the mechanisms that affect sensitivity to cisplatin would allow us to define novel treatment strategies to potentially circumvent cisplatin resistance.”

Duvvuri further noted, “When caring for patients with head and neck cancers, I often see chemotherapy fail. Cisplatin is a very important chemotherapy drug, but tumor resistance to cisplatin is a huge problem. My lab is interested in understanding the mechanisms of resistance so that we can find better ways to treat these patients.” And while researchers have attempted to elucidate the molecular mechanisms that impact cisplatin sensitivity and resistance, they still don’t have a comprehensive and definitive understanding of the mechanisms of resistance.

Umamaheswar Duvvuri, MD, PhD, head and neck surgeon at UPMC Hillman Cancer Center and professor of otolaryngology at Pitt’s School of Medicine [Image courtesy of UPMC]

Previous research found that a protein called TMEM16A is linked with cisplatin resistance in patient tumors. Overexpression of this protein, which occurs in about 30% of head and neck cancers, is also associated with decreased survival. TMEM16A is a calcium-activated chloride ion channel. Straddling the cell’s outer envelope, the ion channel protein provides a passageway to chloride ions, which regulate muscle and nerve activation and transport of salt and water. Because impaired chloride transport is typically linked with neurological and kidney diseases such as epilepsy, cystic fibrosis, and kidney stones, Duvvuri was surprised by the link between TMEM16A and cancer. “It’s always been a bit of a puzzle as to why an ion channel is upregulated in cancer,” he said. “This research provides important clues towards solving this puzzle.”

The new study suggests that TMEM16A promotes the expulsion of cisplatin in cellular compartments called lysosomes. In a healthy cell, lysosomes act like a recycling and waste disposal system, breaking down molecules for reuse and expelling cellular detritus.  And interestingly, the authors commented, “Emerging evidence suggests that cancer cells actively regulate their lysosomes to facilitate drug sequestration and expulsion. Thus, it is likely that the lysosomal sequestration and exocytosis of cisplatin are actively regulated by the cancer cells.”

The newly reported results suggest that in tumors that overexpress TMEM16A, the ion channel protein drives a novel signaling pathway, boosting the production of lysosomes, which sequester and expel cisplatin from the cell, according to first author Avani Vyas, PhD, postdoctoral associate at Pitt. “TMEM16A overexpression is associated with an increase in the expression of the lysosomal genes and lysosomal numbers, indicative of increased lysosomal biogenesis,” Vyas and colleagues commented in their paper.

Based on the evidence connecting TMEM16A expression with enhanced exocytosis, the team measured the amount of platinum in cisplatin-treated cells that were engineered to overexpress TMEM16A. “We found that TMEM16A-overexpressing cells retain less intracellular platinum compared to control cells,” they noted. “Taken together, these data strongly suggest that TMEM16A promotes lysosomal exocytosis, leading to reduced cellular cisplatin levels.”

Co-senior author Kirill Kiselyov, PhD, associate professor of biological sciences at Pitt, commented, “We show that cancer cells have an active mechanism to discard chemotherapeutic drugs. After dissecting this process on a fundamental level and identifying TMEM16A as a critical node, the next step was to test whether disrupting this process with hydroxychloroquine could have translational potential.”

Hydroxychloroquine is an antimalarial agent that inhibits lysosomal function. “We chose to take advantage of the anti-lysosomal property of HCQ to explore the potential anticancer functions of this already FDA–approved drug,” the investigators explained. To evaluate its potential to treat cisplatin-resistant cancers, the team first implanted human cancer cells onto the membrane surrounding the embryo in fertilized chicken eggs. They found that eggs treated with both hydroxychloroquine and cisplatin had greater tumor cell death than those treated with cisplatin alone.

Similarly, in mice carrying tumors (patient-derived xenografts; PDX) derived from individuals with cisplatin-resistant cancer, the combination of hydroxychloroquine and cisplatin slowed tumor growth to a greater degree than either compound alone. “We treated mice bearing this PDX with either vehicle control or cisplatin with or without HCQ …” the investigators explained. “HCQ treatment restored sensitivity to cisplatin in this PDX, suggesting that lysosomal inhibition potentiates cisplatin toxicity in vivo.”

“These experiments suggest that hydroxychloroquine has a synergistic effect with cisplatin,” Duvvuri concluded. “This is relevant for patients because repurposing hydroxychloroquine, which is an existing drug, will allow us to translate these findings to the clinic much faster than we could with a novel compound.” And as the authors further pointed out, “From the translational perspective, these data implicate TMEM16A as a potential biomarker that prognosticates resistance to cisplatin.”

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