Abbreviations

NFV: Nelfinavir

PI: HIV Protease Inhibitors

ERS: Endoplasmic reticulum stress

UPR: Unfolded protein response

BCRP: Breast cancer resistance protein

FAS: Fatty Acid Synthase

Introduction

In March 1997, the United States Food and Drug Administration (FDA) approved Nelvinavir (NFV, brand name Viracept) for HIV treatment in humans ¹. NFV is a safe, orally available, and potent drug against HIV-1 and HIV-2 ². This protease inhibitor (PI) was developed by the private pharmaceutical sector and was a big success in the treatment of AIDS in association with other anti-retroviral drugs ³. The introduction of PIs combined with HIV reverse transcriptase inhibitors started the era of HAART (highly active anti-retroviral treatment) and is nowadays the standard of care in HIV-AIDS ⁴.

PIs inhibit HIV-1 and HIV-2 proteases (which are aspartate proteases), impeding virus replication and release of infecting viral particles from diseased cells. The mechanism of action of protease inhibitors involves competitive binding to the enzyme ⁵.

NFV is being progressively displaced from HIV therapeutics by second generation HIV PIs, but has shown interesting off target actions in cancer.

The possible use of anti-HIV drugs against cancer is not new: in the 1990s AZT (zidovudine or azidothymidine) was proposed as anti-neoplastic drug, but clinical trials did not confirm the preliminary good results obtained in vitro ⁶.

That HIV PIs target other molecules besides the HIV protease is quite evident if we examine adverse effects like insulin resistance and lipodystrophy. These and other evidences such as inhibition of tumor cell production of cytokines, anti-angiogenesis, induction of apoptosis and others, suggest off targets effects for PIs, and hints to the concept of a new class of drugs against cancer with multiple anti-cancer effects ⁶.

NFV, the most important anti-cancer drug of the PI family, if repurposed for cancer treatment, would have an important advantage: it has been used for more than 15 years in HIV treatment and its safety, pharmacokinetics, and adverse events are well known. Serious adverse events are not common with the exception of diarrhea when used at high doses.

Research on NFV as a potentially useful drug for cancer treatment ⁶ started in 2009.

In this article, we thoroughly review the literature published in this matter and analyze mainly the anti-cancer mechanisms of action of NFV.

Certain controversies regarding NFV activity in lipid metabolism will be considered in depth.

Evidences of nelfinavir anti-cancer activity

A partial response of Kaposi’s sarcoma patients to PIs was published in 1998 ⁷ and good results with regression (six complete responses out of 10 patients) ⁸. In 1999 Niehues et al. ⁹ published complete regression of Kaposi’s sarcoma in a child treated with highly active anti-retroviral therapy (HAART). Sgadari et al. (2003) described also the inhibition of Kaposi’s sarcoma with protease inhibitors and they also mention that these drugs can antagonize vital properties of tumor cells like growth, invasion, tissue remodelling, angiogenesis and survival. They consider these effects to be a consequence of inhibition of invasion, matrix metalloprotease, proteasome and NF-κB signaling ¹⁰. The possible mechanisms of PIs off target activity on tumor cells were described by pioneering work of Schmidtke et al. in 1999 ¹¹: they observed that ritonavir was a modulator of proteasomal activity, allowed normal proliferation when used at low concentrations, but affected protein degradation when present at higher concentrations, and cell cycle was arrested.

Ikezoe et al. ¹² described that protease inhibitors increased cellular growth inhibition of all transretinoic acid (ATRA) on cell cultures of myelocitytic leukaemia lines. Protease inhibitors also increased differentiation of acute myeloid leukemia cell lines.

In 2004, Ikezoe ¹³ described the mechanisms involved in anti-cancer activity of protease inhibitors in myeloma cells.

The mechanisms involved in PIs anti-cancer activity are summarized in chronological order on Table 1.

Nelfinavir and the ERS-UPR pathway

NFV inhibits the proteases S1P and S2P that are involved in SREBP-1 maturation and other proteases necessary for protein maturation and folding (yet not fully identified) in the endothelial reticulum ⁴⁹.

Activation of the unfolded protein response (UPR) starts in the ER when abnormal accumulation of protein is detected ⁵⁷. This was investigated thoroughly in yeasts where detection of abnormal protein occurs through Ire1p/Ern1p-mediated signaling from the ER (in mammals there are three sensor proteins IRE1α, PERK and ATF6 ⁵⁸). UPR activation leads to the specific removal of 252 nucleotides intron from a precursor mRNA of the transcription factor HAC-1p, and the resulting mature mRNA HAC-1p is translated to produce active HAC-1p. This transcription factor translocates to the nucleus and promotes the transcription of chaperones like GRP78 that facilitates removal of abnormal proteins from the ER through retrotranslocation and final disposal by the ubiquitin-proteasome pathway ⁵⁹.

HAC1 precursor mRNA is constitutively expressed but not translated until Ire1p/Ern1p sensor removes the necessary nucleotides.

Thus the UPR is an intracellular signaling pathway where the ER “informs” the nucleus on the need to increase the levels of molecular chaperones and folding enzymes in order to maintain the ER homeostasis. Therefore UPR keeps unfolded proteins in the ER until they are correctly folded before they can go to their final destination. NFV seems to produce cellular stress by accumulation of misfolded or abnormal proteins in the ER, overwhelming the normal ER protein folding machinery ⁶⁰. Chaperones bound to unfolded proteins in the ER initiate protein kinase cascades that inhibit translation, reverse translocation, activate ubiquitination enzymes, induce autophagia, and when stress is extreme, induce apoptosis.

Mechanism of action of Nelfinavir in cancer

Interactions of NFV and PIs with other drugs

Indinavir and NFV increase anti-malarial action of artemisinin in vitro on Plasmodium falciparum ⁷⁸, but artemisinin has also an off target anti-cancer activity. Thus it is reasonable to raise the question: may the association of artesiminin with NFV increase anti-cancer activity?

Another research team ⁷⁰ has included both, NFV and artesiminin, in a multidrug repurposed protocol (CUSP 9) for the treatment of relapsed glioblastoma.

Celecoxib is an ER stressor that may enhance NFV anti-tumor activity ⁷⁹.

Chloroquine and hydroxicloquine are autophagy inhibitors and may work synergistically with NFV, down-regulating autophagy and increasing apoptosis ^{70, 80}.

Nelfinavir may produce overproduction of mcl1 through upregulation of Erk ½, which would reduce apoptosis. The problem can be solved adding sorafenib ^{81, 82}.

In breast cancer cells, tamoxifen enhances anti-cancer activity of NFV ⁸³. This synergism was independent of the estrogen receptor status so that the authors consider that the association of NFV and tamoxifen may be advantageous even in patients with no hormone responsive tumors.

Saquinavir has an interesting off target effect: it decreases intracellular ATP in adipocytes ⁵⁶. If this effect is similar in tumor cells, an association with metformin and 2-deoxyglucose may produce anti-cancer activity ^{84– 86}. There is growing interest on metabolic perturbators in cancer therapy and saquinavir may play a role in this field.

Clinical trials

A phase I dose escalation trial performed in 2014 ⁸⁷ established a MTD (maximum tolerated dose) of 3125 mg twice daily and described that 45% of patients with solid tumors treated with this dose decreased AKT activity and increased ERS indicators. This indicated a possible benefit in neuroendocrine tumor patients and also established that dose limiting toxicity consists in neutropenia.

The dose (3125 mg bid) is more than twice the dose used in HIV treatment. But lower doses, in the range of those used in HIV treatment have been tested, combining nelfinavir with chemoradiotherapy in pancreatic cancer with evidence of efficacy ⁸⁸. No control group was used in this research, so comparison was established with known data from previous publications, mainly the favourable possibility of tumor resection after treatment.

Even lower doses (625 mg and 1250 mg bid) were tested in a phase I trial of NSCLC in stages IIIA/IIIB combined with chemoradiotherapy ⁸⁹. In nine out of 12 patients a PET scan was available post-treatment with 100% overall response (56% complete response and 44% partial response). Unfortunately, in this trial there was no control group; 50% of the patients (six out of 12) lived for more than 22 months after treatment; 25% (three out of 12) lived without disease for more than 32 months. The results may be considered favourable, even without a control group.

Buijsen et al. ⁷⁵ recommend a dose of 750 mg NFV bid for a phase II trial of this drug in combination with chemoradiotherapy in locally advanced rectal cancer.

Ongoing phase II trials are mainly in myeloma (associated with bortezomib or lenalomide), glioblastoma patients (associated with chemoradiation), pancreas (associated with gemcitabine and radiation) and lung. (See http://www.clinicaltrials.gov).

The first results of clinical trials did not show a meaningful improvement in the outcome of patients with refractory adenoid cystic carcinoma which is a malignant salivary gland tumor which usually has a poor prognosis. In this case NFV was used as monotherapy ⁷⁶.

Why nelfinavir has a role to play in cancer therapy?

Akt activation is an important step in cancer phenotype and is a key player in acquisition and maintenance of cancer hallmarks. Akt is a nodal regulator of cellular survival pathways ⁹⁰. There are no drugs at the present time that can inhibit this protein with a good safety profile. Wortmannin, perifosine and other chemicals designed for PI3K/Akt inhibition were too toxic for clinical use or have shown disappointing results, so they did not enter the medical practice. Insulin stimulation of Akt phosphorylation was reduced by 55% at achievable doses ²⁰. At the same time there is clear evidence that it favours apoptosis and growth inhibition at clinically tolerable and achievable doses.

This anti-Akt activity of NFV can be reinforced by concomitant mTOR inhibition which results in synergistic cytotoxicity ⁶³. This may be due to the fact that mTOR inhibition without Akt inhibition eliminates a negative biofeedback loop on Akt, producing increased phoshorilation of Akt ⁹¹. According to Sarbassov ⁹² this negative feedback is born in the mTORC2 complex.

According to Carracedo ⁹³, this negative feedback loop goes as far as PI3K ( Figure 6).

mTOR inhibitors have become a new and important tool against cancer, for example in renal cell carcinoma. But the negative biofeedback loop on Akt must be solved to achieve really good results. NFV could be the clue.

But the most important anti-tumor activity of NVR is not limited to Akt inhibition but ER stress and UPR which may be one of the pathways leading to apoptosis ⁹⁴.

Additional features of NFV and other protease inhibitors are

• 1)

  the ability to sensitize cancer cells to chemoradiotherapy,

• 2)

  anti-angiogenesis by decreasing VEGF/HIF expression,

• 3)

  decreased expression of FAS (fatty acid synthase),

• 4)

  the combination of radiation and PIs is well tolerated ^{39, 94},

• 5)

  NFV cancer cell killing ability can easily be enhanced with other ER stressors like celecoxib ⁷⁹. Cho et al. found enhanced killing of chemoresistant breast cancer cells after celecoxib treatment that aggravated ER stress; perillyl alcohol is another stress aggravator that has been used with that purpose ⁹⁶,

• 6)

  In head and neck cancer related to HPV, NFV produced down-regulation of Akt and radiosensitization ⁹⁷,

• 7)

  NFV not only down-regulates Akt but also MAPK (in adenoid cystic cancer) ⁹⁸, and retards oral cell proliferation including normal keratinocytes and squamous cell cancer ⁹⁹,

• 8)

  There are evidences, at least in pancreatic cancer, that NFV dependent down-regulation of Akt is independent of the mutational status of K-ras ¹⁰⁰,

• 9)

  There is clear evidence (in glioblastoma) of the relation between NFV and apoptosis through the following pathway ⁴⁶:

• 10)

  Down-regulation of MMP-9 (reduced expression and secretion of MMP-9 by human preadipocytes) ^{64, 101},

• 11)

  Increased apoptosis by NFV when associated with anti-autophagy drugs like chloroquine or hydroxychloroquines, particularly in triple negative breast cancer cells ¹⁰².

Possible controversies

The SREBP pathway for regulation of fat metabolism is initiated through proteolytic cleavage of precursor forms of the SREBPs (125 Kd protein) in ER membranes. When cells are in need of sterol, the precursor SREBPs are hydrolyzed by a 2-step mechanism involving membrane-bound serine protease S1P and a metalloprotease S2P. The N-terminal fragment of SREBP (nSREBP) is a 68 Kd protein that translocates to the nucleus where it works as a promoter-enhancer, binding to sterol regulatory elements located in DNA and activates gene transcription ( Figure 4). The nuclear SREBP can be rapidly degraded by a proteasome-mediated mechanism. This provides regulation of gene transcriptional activities ¹⁰³.

Transgenic mice over-expressing the constitutively active nuclear forms of the SREBPs (nSREBPs) revealed that overexpression of SREBP-1 or SREBP-2 leads to activation of genes involved in the cholesterol and fatty acid biosynthesis cascades. These transgenic mice displayed the classical features of generalized lipodystrophy, similar to those found in patients under PI therapy ¹⁰⁴.

Riddle et al. in 2001 ¹⁰⁵ found that PI therapy (they used ritonavir) induced the accumulation of activated SREBP-1 and SREBP-2 in the nucleus of liver and adipose tissues. As a consequence, fatty acid and cholesterol biosynthesis were increased in these tissues. The authors consider that lipodystrophy, hyperlipidemia, and insulin resistance, are the consequence of activated SREBP-1 and SREBP-2 accumulation in the nucleus of liver and adipose tissues. The possible mechanism for these events, according to their criteria is PI suppression of activated SREBP degradation in the nucleus. In summary, Riddles’s study showed that ritonavir induced lipid metabolism abnormalities through stabilization of activated SREBP-1 and SREBP-2 in the nucleus of liver and adipose tissues.

These findings are in contrast with those of Guan ^{49, 106} where NFV inhibited the nuclear translocation of the sterol regulatory element binding protein-1 (SREBP-1) in castration resistant prostate cancer and liposarcoma through inhibition of S1P. This led to accumulation of unprocessed SREBP-1.

Riddle et al. described accumulation of processed SREBP-1 in the liver and adipose tissue while Guan found accumulation of unprocessed SREBP1 in ER and Golgi with no translocation to nucleus in liposarcoma and castration resistant prostate cancer tissue.

The controversy may be explained in the following way:

• 1)

  There are three different isoforms of SREBP: SREBP-1a, SREBP-1c and SREBP-2.

• 2)

  SREBP-1a and -1c have different expression profiles: SREBP-1a is highly expressed in proliferating cells, such as cancer cells, while SREBP-1c is the predominant form in normal cells, particularly hepatocytes ¹⁰⁴.

• 3)

  The target genes for the three SREBP isoforms are different.

• 4)

  Riddle et al. found increased SREBP-1 and two in the nucleus of liver and adipose tissues; these SREBPs are the active form (they make no difference between SREBP-1a and SREBP-1c).

• 5)

  Guan et al. found increased SREBP in Golgi in the inactive form (precursor) of tumor tissues treated with NFV.

• 6)

  It is possible that tumor tissues that overexpress SREBP-1a behave in a different way than liver and adipose tissue that overexpress SREBP-1c.

• 7)

  Riddle et al. tested ritonavir and Guam et al. tested NFV, so the pharmacological effects between these PIs may differ.

A second controversy that stems from the one described above is on the effect of NFV on FAS:

• 1)

  According to Guan et al. ¹⁰⁶, NFV decreases expression of FAS in liposarcoma cells and castrate resistant prostate cancer as was depicted in Figure 3.

• 2)

  According to Lenhard et al. 2000 ¹⁰⁷, NFV increases expression of FAS in HepG2 cells (which show many of the normal biochemical functions of non tumor liver parenchymal cells).

May this difference be due to tissue-specific effects of NFV? Does NFV have different effects in tumor tissues and normal tissues?

To definitely solve these controversies, it is necessary to proceed with further experimental research, but the findings described above necessarily raise the doubt that mechanisms that work in tumor cells might be slightly different from those working in hepatocytes and adipocytes.

Possible negative aspects of PIs in cancer

Despite the anti-cancer activity of NFV and PIs, these drugs do not reduce the risk of developing cancer in HIV population ¹⁰⁸ and also exert certain depression of immunological functions, interfering with the differentiation program of monocytes into dendritic cells ¹⁰⁹.

PIs increase the expression of P-glycoprotein (ABCB1) in Kaposi’s sarcoma cell lines increasing the multidrug resistance phenotype ¹¹⁰. At the same time ABCB1 expression depends on Akt activation ¹¹¹ and NFV inhibits partially Akt. The final result of the two antagonistic aspects requires further research.

There are well known undesirable side effects with HIV PIs, like hyperlipidemia, insulin resistance and lypodystrophy (peripheral fat wasting and excessive central fat deposition). One of the main responsible mechanisms of these side effects is the suppression of the breakdown of SREBP in the liver and adipose tissues resulting in increased fatty acid and cholesterol biosynthesis. SREBP accumulation in adipose tissue causes lipodystrophy.

PIs suppress proteasome-mediated breakdown of nascent apolipoprotein (apo) B, resulting in the overproduction of triglyceride. Finally, PIs also suppress the inhibition of the glucose transporter GLUT-4 activity in adipose tissue and muscle. This contributes directly to insulin resistance and diabetes ¹¹².

Hepatomegaly and hepatic steatosis are direct consequences of the metabolic alterations explained above ¹⁰⁵.

New PIs with anti-cancer activity

In 2010 You et al. ¹¹³ synthesized a new indinavir analogue with remarkable anti-cancer activity, similar to NFV: CH05-10. This drug achieved similar cytotoxity to NFV but at lower concentrations, against leukaemia, melanoma, ovarian and prostate cancer cell lines.

In 2009 Saquinavir-NO was introduced ¹¹⁴; it showed interesting anti-cancer properties in melanoma xenografts with significantly lower toxicity than saquinavir.

Conclusions

The most relevant mechanisms of PIs anti-cancer activity are Akt inhibition and ER stress.

Following our exhaustive analysis of the current medical literature we conclude that NFV anti-cancer activity is mainly dependent on ER stress-UPR.

Akt inhibition plays also a very important role but is not the unique or main source of anti-cancer effects.

The evidences that support these conclusions are:

• 1)

  Even at very high doses of NFV (3,125 mg bid), Akt achieved a level of inhibition around 55% in cell culture ²⁰.

• 2)

  NFV is at the same time a strong ER stressor and an Akt inhibitor.

• 3)

  Anti-cancer activity can be achieved at much lower doses than those necessary for Akt inhibition.

• 4)

  Increasing ER stress by adding Celecoxib to NFV enhances cytotoxicity.

• 5)

  Autophagy, which is one of the mechanisms cells use to survive increasing ER stress ¹¹⁵, is inhibited by adding chloroquine or hydroxychloroquine to NFV. In this case, apoptosis is significantly enhanced ^{80, 102}.

• 6)

  The PIs with anti-cancer activity like NFV, ritonavir ¹¹⁶, saquinavir ¹¹⁷, and the experimental drug CH05-10 ¹¹³ are strong ER stressors. Amprenavir is a PI that induces no ER stress and its anti-cancer activity is significantly weaker than that of NFV, although it has Akt inhibiting effects.

• 7)

  Ritonavir, which is ER stressor, shows anti-cancer activity although it does not down-regulate Akt at concentrarions usually found in HIV patients ²³.

• 8)

  Inhibition of proteasome with bortezomib has a synergistic effect with NFV apoptotic activity ¹¹⁸

• 9)
  PIs can be classified regarding anti-cancer activity at clinically achievable concentration in patients in

  • A)
    Akt inhibitors only: e.g. amprenavir
  • B)
    ER stressors only: e.g. ritonavir
  • C)
    Akt inhibitors and ER stressors: examples NFV and experimental PI CH05-10. Of course this is the group that shows stronger anti-cancer activity.

There is enough evidence of NFV anti-cancer effects and there is adequate knowledge of how this activity works, so that NFV deserves well designed phase II clinical trials, as adjunct cancer therapy.

Associations with proteasomal inhibitors, celecoxib and other cell stressor should also be investigated in the clinical setting due to possible synergy. Tamoxifen with NFV may show interesting results in breast cancer.

Although a large amount of publications, including reviews, have been written on NFV and other PIs in cancer, none has been dedicated to a thorough examination and analysis of the mode of action of these pharmaceuticals as off target drugs (with the exception of the review by Gantt et al. ²). It is hoped that this review will encourage an increment adequately powered and well-designed clinical trials in the various cancer types, beyond the phase I trials that have been recently performed, and specifically trials where these compounds may be tested in association with other known anti-cancer pharmaceuticals like NFV associated to bortezomib and hydroxychloroquine in myeloma, or mTOR inhibitors with NFV in HNSCC and many other possible combinations where the dual feature of NFV, ER stressor and Akt inhibitor, are required.

In myeloma NFV increases proteasome inhibition by bortezomib and may overcome resistance to proteasomal inhibitors. This is an action exclusive of NFV and not shared with other PIs ⁶⁷, with the additional advantage that NFV shows the highest cytotoxic activity against primary myeloma cells.

If apoptosis is described as a cascade, then apoptosis stimulator drugs like NFV should be viewed as enhancers of this cascade. An initiator of the cascade is still necessary, for example chemoradiotherapy. After this initial step, apoptosis stimulator drugs increase the amount of cells entering this pathway. This might be one of possible reasons why nelfinavir alone has shown poor results in a clinical trial used as monotherapy.

This does not mean that NFV cannot act as an initiator, but the evidences show that it is prone to be an enhancer of apoptosis rather than an initiator.

Future directions

All the evidences presented in this review reinforce the concept that NFV is a useful drug in cancer treatment. It should be considered in association with chemoradiotherapy in the design of new protocols for diseases like multiple myeloma (in association with bortezomib and hydroxychloroquine) and prostate, pancreas and lung cancer where clinical trials are ongoing. New PIs are being developed with better anti-cancer profile like CH05-10 and saquinavir-NO ⁷⁷ and further development of new PIs with stronger anti-cancer activity, will probably go on in the future.

Funding Statement

The author(s) declared that no grants were involved in supporting this work.